Ethnobotanical survey of plant species for herbal tea in a Yao autonomous county (Jianghua, China): results of a 2-year study of traditional medicinal markets on the Dragon Boat Festival

Background Herbal tea is widely consumed in Jianghua, a Yao autonomous county in Hunan Province, China, to prevent and treat diseases. The materials in herbal tea at the traditional medicinal markets at the Dragon Boat Festival remain unknown. The aims of the paper were (1) to specifically investigate the materials of herbal tea used by Yao nationalities in Hunan Province, (2) to record the most common and the culturally important medicinal plant species in the markets, and (3) to compare the medical plant tradition both used for herbal tea between the Jianghua and Lingnan regions. Methods During 2016–2017, 215 vendors were interviewed at traditional medicinal markets at the Dragon Boat Festival in Jianghua to record plants used for herbal tea and to document traditional knowledge of their medicinal function, habitat, and conservation status. Bunches of medicinal plants were purchased to identify the species and to prepare voucher specimens. Cognitive salience (CS) based on free-lists and use value (UV) were calculated to analyze the cultural importance of medical plants; other quantitative methods (coefficient of similarity and chi-square analysis) were applied for comparison of herbal tea tradition between the Jianghua and Lingnan regions. Results A total of 169 species belonging to 66 families and 142 genera were recorded in herbal tea to treat health conditions in the study area. There were 30 health conditions that were recorded, with heat-clearing and detoxifying being the most common medicinal function, followed by treating rheumatism and promoting blood circulation. Of the 169 species, 97 were herbs. The whole plant was the most commonly used plant part in the preparation of herbal tea. According to the national evaluation criteria, three of these species are listed on “China’s red list” and registered as vulnerable (VU). By comparing the coefficient of similarity of herbal tea plants and the number of mentions for part(s) used in Jianghua and Lingnan, the medicinal plant tradition is different in two areas. Conclusions Herbal tea in Jianghua reflects the cultural diversity of the Yao people and the plant diversity of the region. Future research on the safety, efficacy, and the adulterants of herbal tea are needed for sustainable utilization.


Background
The practice of drinking herbal tea is an ancient custom for Yao people. Herbal tea is produced from water infusions of a range of plant species other than Camellia sinensis (L.) O. Ktze. [1]. Plant material may consist of fresh or dried parts from a single species or from multiple species. For millennia, the Yao people have been famous for being good at identifying herbs [2]. However, no documentary records have survived from when Yao medicine originated.
The Yao nationality of China is mainly distributed in Guangxi, Hunan, Guangdong, Yunnan, Guizhou, and Jiangxi Provinces. The Yao people from Hunan Province are the birthplace of the Yao nationality; Jianghua Yao Autonomous County has the largest Yao population in Hunan Province, accounting for 53% of the population there [2]. Thus, Jianghua Yao Autonomous County plays an important part in the medicine and culture of the Yao people. For historical reasons, the Yao people live long in adverse circumstances, and in the long struggle against disease, the local people had to collect herbs from surrounding mountains and valleys, and they made herbal tea to treat associated health conditions. This tradition formed different, plentiful, and special medical customs, especially herbal tea and medicated baths.
The traditional medical market is a unique custom to celebrate the Dragon Boat Festival (May 5 in the Chinese lunar calendar) by Yao, Zhuang, and Han people in Jianghua (mostly Yao people). At every Dragon Boat Festival, people collect herbs from surrounding mountains and valleys and sell them at the medical market, which is a large-scale market, with more variety and larger trades. The traditional medical market has become a unique spectacle of Yao medicinal culture customs. In addition to buying and selling various herbs, people take this opportunity to exchange their experiences in the recognition and usage of herbs. Since the Dragon Boat Festival is at the end of spring and the beginning of summer, weather conditions are volatile and moist, which probably contribute to the disease rate. During this time, many Chinese herbal medicines are in the periods of harvesting or barking, so the timing forms the unique medicinal market of Yao nationality in Jianghua.
The traditional knowledge of herbs is the result of the accumulated experience by the Yao people's long-term struggle against disease; thus, many aspects of these treatments are probably scientific. However, like the loss of biodiversity, due to the influence of foreign culture, and not having their own written languages, with descendants inheriting their knowledge just by dictation, the traditional knowledge and culture of Yao medicine is also in danger of being lost. In fact, the vanishing of traditional knowledge has been a common phenomenon in the undeveloped country [3].
In order to protect the traditional knowledge of Yao medicine, guarantee food safety, and meet the increasingly globalized health supplement market, we started to document, explore, and research the herb materials for the preparation of herbal tea in Jianghua in 2016.
The study aims to not only document plant species used and commercialized as herbal tea in Jianghua but also make a comparison of herbal tea tradition between the Jianghua and Lingnan regions. This is the first study to document the plant species used as herbal tea in Jianghua; the medicinal plant tradition was recorded for future investigations and policy-making. As well as, if these plant materials are classified and used correctly, the opportunity to develop Yao medicine and expand the herbal tea culture will emerge.

Study area
The study was conducted in Jianghua, where herbal tea has a significant cultural value and it is traditionally consumed. This region is located in Yongzhou City, which borders Guangdong and Guangxi Provinces, between 110°25′ S-112°10′ S and 24°38′ W-25°15′ W (Fig. 1). It covers an area of 3248 km 2 . The total population of Jianghua was 510,000 inhabitants in 2013. It is the only Yao autonomous county in Hunan Province, with the largest population of Yao nationality in the 13 Yao autonomous counties throughout the country. This area features a subtropical monsoon climate, and the weather is relatively moderate, with an annual average temperature of 18-18.5°C, and plenty of rainfall. It owns the biggest and most famous medicinal market in Hunan Province and the surrounding region, that is, the traditional medicinal markets at the Dragon Boat Festival.

Traditional medicinal markets at the Dragon Boat Festival
The Dragon Boat Festival, or known as the Duanwu Festival, is a traditional Chinese cultural holiday. The festival occurs on the 5th day of May in the traditional Chinese calendar. There are three most well-known and widespread activities conducted to celebrate the Dragon Boat Festival, preparing and eating zongzi, drinking realgar wine, and dragon boat racing. These customs could be dated back to over 2500 years ago [4]. The Dragon Boat Festival was held at the summer solstice which is a period of high incidence of disease. Many Chinese folklorists pointed out that the Dragon Boat Festival originated from the concept of people fighting diseases and exterminating evils [5,6]. So, during the Dragon Boat Festival, some indigenous persons, country doctors, and herbalists collect various kinds of plant and sell them to customers, retailers, or formal vendors at the traditional medicinal market.

Ethnobotanical methods
Field surveys including informant interview, structured investigation, free-listing tasks, and voucher specimen collection were conducted during the Dragon Boat Festival in 2016 and 2017. A total of 215 vendors between 22 and 83 years of age were interviewed at the traditional medicinal markets at the Dragon Boat Festival in Jianghua, Hunan Province, to record plants used for herbal tea and to document traditional knowledge on their medicinal function, habitat, and conservation status. Of the vendors, 70% were over 50 years of age, and these vendors were almost equally male and female. The study was carried out following the International Society of Ethnobiology Code of Ethics [7], and all of the participants were informed of our intent prior to the start of the interviews. In addition, every vendor signed a benefit-sharing agreement. The majority of the vendors worked independently or in small groups, and when the vendors spoke only the Yao language, translation was required by an individual that we had hired. Vendors were asked to complete structured ethnobotanical questionnaires, which were answered willingly without payment, the questions included (1) Which species are used for herbal tea? (2) Where do you gather this plant? (3) What plant parts can be used for herbal tea? (4) What is the function of this plant in herbal tea? and (5) What plants do Yao people here use for herbal tea? Bunches of medicinal plants were purchased to identify the species and to prepare the voucher specimens followed by the Flora of China (http://frps.eflora.cn/) and the collections in PE (the Herbarium, Institute of Botany, Chinese Academy of Sciences), and KUN (the Herbarium, Kunming Institute of Botany, Chinese Academy of Sciences). We then deposited them in the Ethnobotanical Lab, Minzu University of China. Photographs were taken to record all of the plant species.
The conservation status of each plant was revised by the Information System of Chinese Rare and Endangered Plants (http://rep.iplant.cn/protlist/7) (Table 1).

Statistical analysis
Cognitive salience (CS) [8] and use value (UV) [9] were applied to determine the greatest cognitive and cultural importance of these medical plants in Jianghua.
Free-listing is a method to obtain cognitive salience from relatively large samples [10,11]. Interviewers collected traditional knowledge from large samples of free-lists which reveal cognitive salience from individuals' local knowledge. The measure of cognitive salience includes both list position and list frequency irrespective of list length or number of respondents [8,12]. We interviewed 215 informants and recorded 215 free-lists; here, we calculated the mean cognitive salience (CS) for each listed species,  VU vulnerable, LC least concern, NE not evaluated F is the number of lists where the particular species is mentioned in all lists while Z is the number of informants. B determines how one plant precedes other plants mentioned in a respondent's list. K is the number of listed species in one informant, and r (i) is the ith order of each plant's list position.
The closer to the first position (or rank) the item(s) are, the greater the cognitive salience of item(s) is deemed to be.
The use value (UV) is to quantitatively evaluate the relative importance of species [13][14][15] used by Yao people, where Ui refers to the number of medical use cited by an informant for per species and N is the total number of all informants. When there are many use reports mentioned for one plant, it indicates the use value of this plant is high. The coefficient of similarity (S) of herbal tea plants between Jianghua and Lingnan regions was calculated by the following formula: S = 2c/(a + b) (a and b are species used by Jianghua and Lingnan regions, respectively; c are species in common use) [16].
Chi-square analysis was applied to find whether the traditional knowledge of herbal tea such as plant life form and plant part(s) used varied considerably between Jianghua and Lingnan.

Medicinal plant species sold for herbal tea at the traditional medicinal markets Plant species and life form
According to the results of the taxonomical identification, the medicinal plants used for herbal tea belong to 169 species, grouped into 142 genera and 66 families. In alphabetical order of the family, they are presented in Table 1. Further analyses on the plant families show that Compositae has 18 species, making it the dominant family. Liliaceae, Leguminosae, Orchidaceae, Labiatae, and Myrsinaceae are represented by 11,9,7,6, and 5 species, followed by Urticaceae, Umbelliferae, Rubiaceae, and Araliaceae, with 4 species each, and 13 families containing 3 species, 14 families containing 2 species, and 29 families containing 1 species (Fig. 2). Of the 169 species, the most frequent habits of medicinal plants were herbs (97 spp.), followed by shrubs (35 spp.), vines (22 spp.), ferns (7 spp.), trees (6 spp.), phytoparasites (2 spp.), and thalli (1 sp.) (Fig. 3).

Part(s) used
In this study, the analysis revealed that there were 16 kinds of plant parts that were used for herbal tea as medicinal materials. The whole plant was the most commonly used plant part (38.4%), followed by root (14.2%), leaf (9.13%), stem (7.76%), rhizome (7.76%), and tuber (5.02%) (Fig. 4). The study also found that some other plant parts, such as the flower, fruit, bark, pod, seed, pith, branch, shoot thorn, shoot, and fruit cluster, are used less frequently.

Conservation status
According to the evaluation criteria established by the International Union for Conservation of Nature (http:// rep.iplant.cn/protlist), three of these species are listed on "China's red list" and registered as vulnerable (VU), which means that they are at the highest risk for endangerment, namely, Dysosma versipellis, Bulbophyllum pectinatum, and Dendrobium nobile. In addition, five species are categorized under least concern (LC), which is a lower category of risk; they are Bulbophyllum odoratissimum, Galeola lindleyana, Luisia morsei, Spiranthes sinensis, and Fagopyrum dibotrys, and 159 species were not evaluated (NE) while Paris polyphylla is listed as second degree national protective plants and Dendrobium officinale is listed as first degree national protective plants. There is a need to investigate and provide proper management to avoid a shortage.

Medicinal uses
In our study, a total of 30 medicinal uses were recorded, and heat-clearing and detoxifying was the most common medicinal function, followed by treating rheumatism and promoting blood circulation (Fig. 5). In Jianghua, 49.11% of the medicinal plant species (83 spp.) are used for heat-clearing and detoxifying, 30.18% for treating rheumatism, 17.75% for promoting blood circulation, and 15.38% for relieving cough.

Cultural and medical significance of species
The cognitive salience of 169 species ranked from 0.012 to 0.343 (Table 1)

Comparison of medicinal plant tradition in Jianghua and Lingnan
A comparison of plant materials commonly used for herbal tea in Jianghua and Lingnan shows that there are 23 plant species in total used for herbal tea (Table 3), and Compositae is the predominant family in two regions. For part(s) used for herbal tea, no matter whether they are from Lingnan or Jianghua, the vendors like to use whole plants and roots to prepare herbal tea. By comparing, we found that the common functions of the herbal tea produced by the people both in Jianghua and    (Table 3). They are Fallopia multiflora, Parthenocissus tricuspidata, Acorus tatarinowii, Trachelospermum jasminoides, Hypericum japonicum, and Leonurus artemisia.
The coefficient of similarity of herbal tea plants commonly used in Jianghua and Lingnan is 11.2%. Using chi-square analysis, the number of mentions for part(s) used varied significantly between the two culturally distinct communities (p value < 0.05).

Prospective value of herbal tea plants used by Yao people
Herbal tea in Lingnan region is based on the theory of traditional Chinese Medicine (TCM); many recipes used in herbal tea are evolved from prescriptions of TCM [17]. However, Yao people in Jianghua did not record their traditional knowledge of herbal tea with books or scripts instead of folksongs and teaching generations by experience and dictation. We compared herbal tea plant in Jianghua with Drug Standard Database (http://www.drugfuture.com/standard/), including Chinese PharmacopeiaI (2015 and 1977 versions), Tibetan medicineI, Uygur medicine, and Japanese Pharmacopoeia, and 124 species are not listed in Pharmacopeia (Table 1). Among these 124 species, the medicinal use of not all species can be supported by literatures. For example, Yao people in Jianghua indicated that Achyranthes aspera can relieve pain, which was verified by Barua et al. In 2010, they verified the antinociceptive activity of the methanolic extract of leaves of A. aspera in animal models of nociception [18]. Cirsium japonicum stops bleeding, which was verified by Chen Qi et al. in 2012 [19]. However, most of these 124 species cannot be found in the supporting literatures. Yao people in Jianghua generally believed that Clematis henryi is a good medicine for relieving pain, Heteropanax fragrans can treat rheumatism, and Marsdenia sinensis can treat heatstroke. There is a great need to further study these plant species.

The efficacy and safety of species used in Jianghua
In Jianghua, heat-clearing and detoxifying is the most common medicinal function, followed by treating rheumatism, because the Dragon Boat Festival is at the end of spring and the beginning of summer, weather conditions are hot and humid, so the main plant materials used for herbal tea are focused on heat-clearing and detoxifying and treating rheumatism.
In Jianghua, 22 species were involved in eliminating inflammation; however, of the 83 species used for heat-clearing and detoxifying, 14 species were involved in eliminating inflammation; it shows that 63.6% of the medicinal plant species sold to eliminate inflammation are also used for heat-clearing and detoxifying, so it is important to conduct some studies to understand the dual effect and discover the possible relationship, which is useful for the theoretical construction of the traditional Chinese medicine (TCM).
Over the past 20 years, the safety [20] and pharmacological efficacy [21][22][23][24] of herbal drinks have drawn attention. Findings have elucidated that some phytochemicals in herbal tea are beneficial to human health [25][26][27][28], while some are risky to humans [29][30][31][32][33][34]. Therefore, further research is needed to analyze the bioactivity and toxicity of herbal tea. Among 169 species, two of them are forbidden as raw materials for food based on an announcement from The National Health Commission of the People's Republic of China (http://www.nhfpc.gov.cn/). They are Dysosma versipellis (Hance) M. Cheng ex Ying and Tripterygium wilfordii Hook. f. Dysosma versipellis: Podophyllotoxin, a chemical compound isolated from D. versipellis, is recorded to show cytotoxicity resulting emesis, diarrhea, and hepatic and central nerve system lesion [35][36][37][38]. However, due to its chemical function similar to colchicine, podophyllotoxin and its derivatives have been synthesized and utilized as anti-tumor drugs [39]. Besides, it was recorded to be used as an antiviral material for treating condyloma acuminatum caused by human papilloma virus (HPV) [40]. D. versipellis is largely be utilized for clearing heat and detoxification, rheumatism, and promoting blood circulation by Yao people in Jianghua. However, due to excessive consumption, the conservation status of D. versipellis on "China's red list" is registered as vulnerable. At present, D. versipellis is cultivated in Jianghua.
Tripterygium wilfordii: The extract of T. wilfordii, a Chinese herb, has anti-inflammatory and immunosuppressive activities and an established history of use in the treatment of rheumatoid arthritis [41,42]. However, the most common side effects of T. wilfordii are gastrointestinal tract disturbances, such as diarrhea, leukopenia, thrombocytopenia, rash, skin pigmentation, and malfunction of the male and female reproductive system [43].

Comparison of plant materials used for herbal tea in Jianghua and Lingnan
The resurgence of interest in natural products has fueled the global herbal tea market. In 2013, Yujing Liu recorded 241 species used for herbal tea in Lingnan Region (China) [1]. By comparing the herbal tea plants commonly used by Jianghua and Lingnan, there are 23 common plant species, among which, there were 17 species that had consistent function and 6 species have different functions.
By comparing the 6 species having different functions in Jianghua and Lingnan, we cannot confirm that they have various medical functions. Achyranthes aspera, Fagopyrum dibotrys, Lonicera confuse, Lonicera japonica, Dendranthema morifolium, and Juncus effusus are heat-clearing and detoxifying herbs. In Chinese medicine, the lower the fire is equal to clear heat. We found that there may be a relationship between detoxifying and antibacterial or anti-inflammation properties, because most of the plants with detoxifying properties have antibacterial or anti-inflammation effects (Table 3) [44][45][46][47][48][49][50][51][52]. For Gleditsia sinensis, Jianghua people pointed that it can induce diuresis, and the Lingnan people indicated that it can relieve pathological heat and remove dampness through diuresis. This may represent a direction for our study of the activity of Chinese herbs. So it will be necessary to verify the pharmacological activity in the future.
By comparing the herbal tea plants commonly used by Jianghua and Lingnan, the coefficient of similarity of herbal tea plants is 11.2%, which is low. We compared all plant parts used in the Jianghua and Lingnan regions. The common used plant parts are whole plant, root, leaf, stem, rhizome, tuber, flower, fruit, bark, seed, pith, branch, and shoot thorn. In Lingnan region, there are several particular used plant parts. They were aerial part, bulb, kernel, bud, peel, stigma, stem node with horns, and pollen. However, in Jianghua region, the particular used parts are pod, shoot, and fruit cluster. We selected all common used parts to do statistical analysis with chi-square analysis; the results (p value < 0.05, χ 2 = 61.333) show the used plant parts varied significantly between these two different regions. Hence, the variation of used plant part in two regions accounts not only for the particular mentioned used parts but for varied usage rate of each common used part. For example, in Lingnan region, root (20.78%) is the most frequently mentioned used part, while in Lingnan region, it is whole plant (38.36%). The variation of plant part used suggests that the medical plant tradition is far different between the Lingnan and Jianghua regions. The low coefficient of similarity and the variation of plant part used reflect a relatively great difference of herbal tea plant tradition between Jianghua and Lingnan.

The traditional medicinal market is a bit unstructured
In the ethnobotanical surveys, we found that there are 14 poisonous species, which need to be payed attention. They are Pothos chinensis (Raf.) Merr., Typhonium flagelliforme (Lodd.) Blume, Trachelospermum jasminoides (Lindl.) Lem., Asarum sagittarioides C. F.  (Table 1). So, the plants that are nonexistent in the Drug Standard Database need to be scientifically investigated for their efficacy and safety in the future.

Conclusions
The traditional medical market in Jianghua Yao Autonomous County reflects the plant species richness and cultural diversity. Traditional knowledge of herbal tea is the result of the accumulated experience by the Yao people's long-term struggle against disease, so many aspects must be scientific. With the rise of natural product drugs, there is the need to analyze the chemical composition and activity of the materials of herbal tea. Future research is also needed to understand the safety and efficacy of the recorded herbal tea. For sustainable utilization, the production of herbal tea should be monitored.
In addition, uniform standards of practice and licensing of herbal vendors is required to produce a safer herbal tea market. It is very important for them to have the knowledge to select the proper plants since some herbs are hard to identify due to similar morphological characteristics.