Variation of five flowers tea formula

Etymologically, the term Wu Hua Cha can be directly translated into Tea with Five Flowers. The most used formula is based on the combination of five herbs, B. ceiba, P. rubra, C. morifolium, L. japonica and S. japonica. These five herbs possessed the function of heat clearing, dampness draining and detoxifying. The herbs would act on intestinal area and liver. In terms of pharmacological effect, most of the five herbs possess the function of anti-inflammatory, antibacterial, antiviral, antidiarrhoeal and diuretic effect, aligning with the ethnomedical claims.

In this study, we only found eight samples comprised of the five floral herbs. Some herbal shop owners mentioned that some herbs are cut to reduce the cost. Herbal tea is a grassroot remedy in respond to the expensive fee for consulting a clinician. Therefore, there is often a trade-off between the cost and potency of the herbal tea. Prunella vulgari, for example, is a cheaper alternative to other expensive herbs like Pueraria lobata and S. japonica. Over half of the parcels (63.6%) contain it. Prunella vulgari is renowned in the heat clearing function and as a major ingredient in another popular herbal tea Xiasangju [15].

A. capillaris was found in 28 out of 33 parcels (84.8%) (Fig. 3). This herb is not a component in the classic formula. Also, the herb is not in the flowering stage. The harvesting season of A. capillaris is between March and April [16]. It is believed that during this period, the medicinal properties of A. capillaris are at their strongest for clearing heat and draining dampness, surpassing its effectiveness when collected during the flowering period.

A. capillaris was used to substitute S. japonica. According to the principle of Chinese medicine, A. capillaris has a greater dampness draining ability, but S. japonica is better in heat clearing. Some studies suggested that A. capillaris may have hepatoprotective, anticancer and anti-inflammatory properties [17,18,19]. Therefore, herbalists may preferentially select A. capillaris for its superior dampness dispelling capacity.

DNA authentication of herbs

Herbal remedies often face issues of adulteration due to the difficulty of visually identifying authentic herbs from fraudulent substitutes. The visual appearance of closely related herbal species can be highly similar, making organoleptic identification difficult for those without specialized knowledge of the subtle morphological differences. This confusion has led to many instances of adulteration, such as D. styracifolium being used as L. christinae, Potenilla species substituting for A. capillaris and Lonicera confuse being used in place of L. japonica.

NA barcoding method was performed to identify the herbs used in Five Flowers Tea parcel. DNA barcoding is a molecular technique used to identify species based on species-specific differences in a short, standardised fragment of nucleus and organelle DNA from a specific gene or genome region. That DNA fragment is then compared to a reference DNA database to determine the identity [20].

This method can precisely and rapidly identify species from various raw materials. In plant identification, the common barcode regions used are ITS2 and psbA-trnH. The region ITS2 (Internal Transcribed Spacer 2) is located in the nuclear genome, between 5.8S and 28S rRNA genes [21]. The region psbA-trnH is in the non-coding region of the chloroplast genome. Due to their high variability, the region can serve as a distinctive marker for a particular species [22].

The COI region, which is located in the mitochondrial genome of eukaryotic organisms, was used to identify insects due to its significant level of intra-specifically similarity and inter-specifically variation [23].

Using DNA barcoding technique, we detected the substitution of L. japonica, A. capillaris and L. christinae by L. macranthoides, Potentilla species and D. styracifolium, respectively. This may pose risks of having a reduced therapeutic efficacy or unintended side effects.

The pros and cons of using the substitutes and adulterants

The first herb in question was L. japonica. While L. japonica and L. macranthoides show similar pharmacological efficacy as evidenced by their medicinal usage history, the Chinese Pharmacopeia (2005 Edition) lists them separately. Both L. japonica and L. macranthoides exhibit low toxicity [24]. There have been many clinical trials conducted on L. japonica. In contrast, clinical investigations on L. macranthoides are limited. Both L. japonica and L. macranthoides share similar properties, including being categorized as cold in nature, sweet in flavour and having comparable therapeutic functions and applications. L. macranthoides is commonly cultivated and used as a substitute for L. japonica in China. Despite having similar chemical profiles and pharmacological effects, L. macranthoides offers a higher yield and lower production cost. This makes L. macranthoides an economical alternative to L. japonica [25].

The second herb in question was A. capillaris. Studies have shown that A. capillaris has greatly benefited the treatment of many diseases such as liver inflammation, cirrhosis and liver cancer, contributing to the wider development and advancement of traditional Chinese medicine [26]. A. capillaris and its active compounds have antioxidant, anti-inflammatory, antifibrotic, antiviral and anticancer effects. This also supports its traditional uses for treating liver and viral disorders as well as cancer [27,28,29].

We found that Potentilla species was used to replace A. capillaris in Five Flowers Tea. Potentilla species have been noted for their medicinal functions that are akin to those of A. capillaris, although there are variations between the herbs in terms of chemical profiles and effectiveness in treating certain conditions. Potentilla species have been used in traditional medicine, particularly in Chinese medicine, for a long time to treat ailments such as diarrhoea, hepatitis, rheuma, scabies and for detoxification purposes [30]. A. capillaris was found to be adulterated by Potentilla species, in particular P. discolor and P. chinensis. The aerial and underground parts of P. discolor have been used to treat inflammation, wound, cancer, infection caused by bacteria, fungi, and viruses, diarrhoea and diabetes mellitus [31]. There have been no reports of toxic effects during the long history of using Potentilla species and their extracts in traditional medicine [30].

Substituting A. capillaris by P. discolor and P. chinensis may be appropriate due to the pharmacological properties of the two latter herbs. A. capillaris is indicated for dispelling dampness due to its bitter and pungent flavour. P. discolor and P. chinensis, with their stronger cooling properties, are more suitable for resolving excessive heat conditions compared to A. capillaris.

The third herb in question was L. christinae. L. christinae is a traditional herb that possesses heat-clearing, diuretic, detumescent and detoxifying properties [32]. The adulterant D. styracifolium is widely used in traditional Chinese medicine for its diuretic and heat-clearing properties and for the management of urinary calculi, cardiovascular and cerebrovascular disorder and hepatitis [33].

From the Chinese medicine perspective, L. christinae and D. styracifolium have some similarities but also distinct differences in their indications and usage. Both herbs are valued for their diuretic and heat-clearing properties, which help resolve accumulated dampness heat and dissipate pathogenic factors. However, L. christinae focuses on dispelling dampness heat in liver and gallbladder. It is indicated for conditions that involve pathogenic factors affecting the hepatobiliary system. In contrast, D. styracifolium focuses more on dispelling dampness heat in the urinary bladder and kidney for urological conditions.

L. christinae and D. styracifolium therefore should be used interchangeably as they are for treating different types of conditions.

Herbal tea poisoning incidents have occasionally been reported, typically resulting from the use of toxic adulterants [34, 35]. In 2003, a local individual sought help after experiencing symptoms of poisoning. Datura metel, a plant known to contain toxic tropane alkaloids, was discovered in the brewing residue of the Five Flowers Tea [36]. The presence of adulterants in herbal tea can pose significant dangers to public health, ensuring the safety and quality of herbal tea products is therefore crucial.

Instructions on brewing and precautions

Instructions for brewing the tea differed among the different shops. The time, quantity of water, procedure of brewing and heat level vary (Additional file 1).

Furthermore, indications on the contradictions are simple and not mentioned in every parcel. For instance, sample HK10 stated that it is suitable for everyone regardless of their health condition. Sample NT02 suggested that people who are of “cold” constitution can only drink half of a bowl. G6PD deficient patients were suggested to avoid taking L. japonica [37, 38]. Of the 33 samples surveyed, only one parcel provided notice that people with G6PD deficiency should avoid using the product.

Apart from the potential danger of G6PD, a retailer has mentioned that pregnant women should avoid taking the Five Flowers Tea. However, a survey has indicated that more than half of Chinese pregnant women living in Hong Kong have taken Chinese herbal medicine, including Five Flowers Tea and L. japonica, for improving their gastrointestinal and digestive health. The use of Chinese herbal medicine during pregnancy did not appear to affect the condition, incidence or severity of jaundice in both normal and G6PD-deficient infants at birth [39]. The herbs used in Five Flowers Tea are not included in the List of Restricted Chinese Herbal Medicines for Pregnant Women [40].

Lasioderma serricorne infestation and quality control

L. serricorne was first reported and identified on tobacco in France in 1848 [41]. It is currently globally distributed and exceptionally prevalent in tropical and subtropical areas and is the most destructive pest affecting a wide variety of materials derived from both plants and animals [42]. It has long been recognised as the dominant pest species that infests stored Chinese medicinal materials, resulting in significant economic loss [43]. In our study, 12 out of 33 Five Flowers Tea samples were contaminated by L. serricorne.

The larvae of L. serricorne can cause significant damage by feeding on herbal tea leaves, resulting in reduced quality and value. Some conditions give rise to the growth of L. serricorne. Firstly, since herbal tea parcels are stored in compacted form, they provide an ample food source for the beetle to feed on. Secondly, the beetles like to hide away from bright light and low humidity. The best temperature range and relative humidity for rapid development of L. serricorne are 29–35 °C at 75% [44]. All our samples found with insects were moist. Hong Kong is warm and humid, not to mention the hot and humid storage conditions in warehouses and wet markets.

Thirdly, packaging material and styles would be another issue. The adult stage of the beetle is capable of biting through the packaging material [45]. In our study, different packaging styles are found (Fig. 2). Insect infestation is higher when the herbs are wrapped in a piece of paper or put in a transparent plastic bag with opening tied with a nylon. L. serricorne can easily penetrate the paper wrap, and paper wrapping cannot protect the herbs from the humidity. Nylon string tied on the bag opening is easily loosened, creating an entry point for the insect and moisture to enter.

The presence of contaminated and gnawed herbs, as well as the excrement, cast-off skin and other products, can pose a menace to the health of consumers. L. serricorne, in particular, has been linked to canthariasis, a human-enteric infection [46]. Cases of intestinal canthariasis have been reported in China and Malaysia [46, 47].

Effective pest management methods include phosphine fumigation, CO2 treatment and radiation treatments and high temperature. In recent years, pest management has put more emphasis on friendly to the environment and cost-effectiveness [48]. For instance, the package can be treated with natural repellents that prevent L. serricorne from consuming the contents [49]. Examples of such repellents include crude leaf extracts of Tecoma stans and Datura metel, as well as various plant essential oils such as shiso oil and savory oil [50]. For storage, multilayer packaging is an alternative to applying pesticide or temperature control warehouse. L. serricorne was found to get through polyethylene, polypropylene and polyester [51]. Thicker packaging material and multilayer packaging better deterred the spreading of the insect [45].

The interplay of cultural and environmental factors in the composition and evolution of Five Flowers Tea

In our study, we observed a diverse range of ingredients and proportions, with no unique recipe found. Several factors contribute to the variation in herbal tea recipes, including medicinal effects, Chinese philosophy, culture and environments.

Chinese philosophy emphasizes the interaction between human being and outside world and so is the traditional Chinese medicine. Factors such as individual person, time and place should be taken into account. In the holistic approach, each factor influences the others, and any adjustments should be considered in the overall context. We previously mentioned the impact of local climate and environment on the choice of herbs. For instance, locals brew “cold medicine” to get rid of heat-related ailments in Hong Kong’s hot and wet weather. Locals would consume specific herbal teas or Chinese medicine during different solar terms or seasons. For example, illness prevention teas are favoured in spring, Five Flowers Tea is for the hot summer, while soothing herbal teas are popular in autumn [5]. The proportions of ingredients in Five Flower Tea can also be adjusted for year–round consumption, not limited to the summer season.

Individual constitution and nature are factors leading to vast variations in herbal tea recipes. The proportions and the selection of herbs differ based on individual needs. When buying samples, vendors suggested different brewing methods and additives to the herbal tea in order to suit individuals need as well (Additional file 1).

The ingredients of Five Flowers Tea in Hong Kong are influenced and limited by the resources, local culture and habit. Recent studies indicated that not all the five flowers are readily available, leading to the brewing of herbal tea with fewer ingredients, and once the number of the ingredients down to B. ceiba alone [52]. The flexibility in Five Flowers Tea composition reflects its evolution from a specific herbal tea recipe to a cultural symbol, expanding the definition of Five Flowers Tea to a kind of drink which provides similar medicinal effects using various herbs with flowers.

The emerging needs of society, such as the occurrence of pandemics like COVID-19, also shaped the development of herbal tea. While standardisation of Five Flowers Tea can instil the confidence of consumers, certain variations may be beneficial for the medicinal efficacy.

In this study, the Five Flowers Tea samples were from vendors in Hong Kong. As herbal tea is also popular in South China, it will be interesting to expand the area of sample collection, to compare the quality and use of this medicinal product in nearby regions. The identification of herbs in this study is by organoleptic and DNA sequences. These sequences are either from our previous work [53] or GenBank. The matching of these two independent approaches can reveal the herb identity to high accuracy. We are constructing a DNA database of herbal tea material from authenticated samples, which will further enhance the confidence of authentication in the future.