Ethnopharmacology of five flowers herbal tea, a popular traditional beverage in Hong Kong and South China.

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.

Determination of ITS1 haplotypes of Fritillariae Cirrhosae Bulbus by amplicon sequencing.

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.

Comparison of DNA extraction methods on CITES-listed timber species and application in species authentication of commercial products using DNA barcoding.

Quality and quantity of DNA extracted from wood is important for molecular identification of wood species, which can serve for conservation of wood species and law enforcement to combat illegal wood trading. Rosewood (Dalbergia and Pterocarpus) and agarwood (Aquilaria) are the most commonly found hardwood in timber seizure incidents. To monitor international trade of timber and commercial wood products and to protect these endangered wood species from further population decline, in this study, we have compared three extraction protocols for DNA extraction from 12 samples of rosewood and agarwood timber logs, and later applied the best DNA extraction protocol on 10 commercial wood products claimed to be rosewood and agarwood. We also demonstrated the applicability of DNA mini-barcoding with multi-loci combination with reference library for identifying the species of timber and commercial wood products. We found that a silica column-based method with guanidine thiocyanate-containing binding buffer served the best in DNA extraction from different parts of wood in all three genera with good quality and quantity. Single barcode region ITS2 or multi-loci combinations including ITS2 barcode region generally provide better discriminatory power for species identification for both rosewood and agarwood. All 10 products were identified to species-level using multi-loci combination. In terms of accuracy in labelling, 80% of them were labelled correctly. Our work has shown the feasibility of extracting good quality of DNA from authentic wood samples and processed wood products and identifying them to species level based on DNA barcoding technology.

Assessing the reliability of medicinal Dendrobium sequences in GenBank for botanical species identification.

DNA-based method is a promising tool in species identification and is widely used in various fields. DNA barcoding method has already been included in different pharmacopoeias for identification of medicinal materials or botanicals. Accuracy and validity of DNA-based methods rely on the accuracy and taxonomic reliability of the DNA sequences in the database to be compared against. Here we evaluated the annotation quality and taxonomic reliability of selected barcode loci (rbcL, matK, psbA-trnH, trnL-trnF and ITS) of 41 medicinal Dendrobium species downloaded from GenBank. Annotations of most accessions are incomplete. Only 53.06% of the 2041 accessions downloaded contain a reference to a voucher specimen. Only 31.60% and 4.8% of the entries are annotated with country of origin and collector or assessor, respectively. Taxonomic reliability of the sequences was evaluated by a Megablast search based on similarity to sequences submitted by other research groups. A small number of sequences (211, 7.14%) was regarded as highly doubted. Moreover, 10 out of 60 complete chloroplast genomes contain highly doubted sequences. Our findings suggest that sequences of GenBank should be used with caution for species-level identification. The scientific community should provide more important information regarding identity and traceability of the sample when they deposit sequences to public databases.