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Abstract Background It has been a long-standing tradition of using herbal tea for preventive and therapeutic healthcare in Hong Kong and South China and Five Flowers Tea is one of the most popular herbal teas. Based on the principle of traditional Chinese medicine, the pharmacological functions are to clear heat and dispel dampness in the body. Heat and dampness are thought to contribute to a range of health problems, especially during the hot and humid season in South China and Hong Kong. The most prevalent herbs in the formula contain bioactive compounds including flavonoids, alkaloids and terpenoids, which have a wide range of pharmacological properties including anti-inflammation, antivirus, antidiarrhoea, antibacteria, and antioxidation. However, with the composition varies widely, the ethnopharmacological benefits described may not be delivered uniformly. This study is to provide a comprehensive analysis on the composition of the Five Flowers Tea sold in Hong Kong and investigate the rationale behind the selection of herbs used in the formula. This study also provides information on the variation and quality of the Five Flowers Tea in the market. Methods Thirty-three Five Flowers Tea samples were collected from various locations in Hong Kong. The size, texture, colour and organoleptic properties were documented. Macroscopic and molecular authentication methods were employed to identify the individual components. Results Macroscopic identification revealed there were 23 herbs belonging to 18 plant families. The most prevalent herb was Bombax ceiba L., followed by Chrysanthemum morifolium. Ten adulterants and the existence of insect Lasioderma serricorne were confirmed by DNA barcoding techniques. Conclusion This study employed a comprehensive approach to authenticate the herbs in Five Flowers Tea samples collected from various locations in Hong Kong. Macroscopic and molecular methods were used to identify the herbs and adulterants. The findings revealed the varied composition in Five Flowers Tea and the occurrence of adulterants in some samples. This shows that quality assurance of Five Flowers Tea is essential for the effective use of this popular folk medicine.

Abstract Background Fritillariae Cirrhosae Bulbus is an antitussive and expectorant Chinese medicinal material derived from the dried bulbs of six Fritillaria species. In the 2015 edition of the Chinese Pharmacopoeia, the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) is the officially listed method for their authenfication. Specifically, the ~ 300-bp ITS1 amplicon of only Fritillariae Cirrhosae Bulbus but not other Fritillaria species can be cleaved into two smaller fragments with restriction enzyme SmaI. Considering repeated reported cases of incomplete digestion of ITS1 amplicon, this study aims to investigate the possibility of heterogeneous ITS1 sequences contained in the Fritillariae Cirrhosae Bulbus. Methods In this study, ITS1 amplicons of Fritillaria Cirrhosae Bulbus and four other Fritillaria species were sequenced on Illumina platform. We utilised high-throughout amplicon sequencing to determine ITS1 haplotypes and their frequencies in Fritillaria genomes. Results Our results showed that all six botanical sources of Fritillariae Cirrhosae Bulbus indeed possess ITS1 haplotypes with no SmaI restriction site, and the average percentages of ITS1 reads containing SmaI restriction site ranged from 63.60% to 91.81%. Conclusion Our findings suggest that the incomplete digestion in PCR–RFLP analysis of Fritillariae Cirrhosae Bulbus is caused by the presence of ITS1 haplotypes without SmaI restriction site due to intragenomic heterogeneity.

Abstract Molecular herbal authentication has gained worldwide popularity in the past decade. DNA-based methods, including DNA barcoding and species-specific amplification, have been adopted for herbal identification by various pharmacopoeias. Development of next-generating sequencing (NGS) drastically increased the throughput of sequencing process and has sped up sequence collection and assembly of organelle genomes, making more and more reference sequences/genomes available. NGS allows simultaneous sequencing of multiple reads, opening up the opportunity of identifying multiple species from one sample in one go. Two major experimental approaches have been applied in recent publications of identification of herbal products by NGS, the PCR-dependent DNA metabarcoding and PCR-free genome skimming/shotgun metagenomics. This review provides a brief introduction of the use of DNA metabarcoding and genome skimming/shotgun metagenomics in authentication of herbal products and discusses some important considerations in experimental design for botanical identification by NGS, with a specific focus on quality control, reference sequence database and different taxon assignment programs. The potential of quantification or abundance estimation by NGS is discussed and new scientific findings that could potentially interfere with accurate taxon assignment and/or quantification is presented.

Abstract Background Pheretima is a minister drug in Naoxintong capsule (NXTC), a well-known traditional Chinese medicine (TCM) formula for the treatment of cardiovascular and cerebrovascular diseases. Owing to the loss of morphological and microscopic characteristics and the lack of recognized chemical marker, it is difficult to identify Pheretima in NXTC. This study aims to evaluate the feasibility of using DNA techniques to authenticate Pheretima, especially when it is processed into NXTC. Methods DNA was extracted from crude drugs of the genuine and adulterant species, as well as nine batches of NXTCs. Based on mitochondrial cytochrome c oxidase subunit I (COI) gene, specific primers were designed for two genera of genuine species, Metaphire and Amynthas, respectively. PCR amplification was performed with the designed primers on crude drugs of Pheretima and NXTCs. The purified PCR products were sequenced and the obtained sequences were identified to species level with top hit of similarity with BLAST against GenBank nucleotide database. Results Primers MF2R2 and AF3R1 could amplify specific DNA fragments with sizes around 230–250 bp, both in crude drugs and NXTC. With sequencing and the BLAST search, identities of the tested samples were found. Conclusion This study indicated that the molecular approach is effective for identifying Pheretima in NXTC. Therefore, DNA identification may contribute to the quality control and assurance of NXTC.