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Part Ⅰ：The Quality Monitoring of Cistanches Herba (Cistanche deserticola Ma): A Value Chain Perspective

Linlin Jiang, Baochang Zhou, Xiaoqin Wang, Yaqiong Bi, Wenfang Guo2, Jianhua Wang 1 *, Ruyu Yao3 * and Minhui

Cistanche deserticola Ma was used as a medicine food homology, which was mainly produced in the Alexa region of northwest China. In recent years, it has been widely used in various food items. The increasing demand for Cistanches Herba has led to problems such as overexploitation and quality deterioration. The quality and safety of herbal medicines are critical and have been shown to be affected by the value chain (VC). Using the VC framework, the study is embedded in a larger study aiming to investigate the effects of different VCs types on the quality and stakeholders of Cistanches Herba. In this study, 90 Cistanches Herba samples were collected during fieldwork. An additional 40 samples were obtained from the herbal markets and medicine purchasing stations. Semi-structured interviews and key informant interviews were performed to collect data on stakeholders in major production areas. These samples were analyzed using high performance liquid chromatography (HPLC) coupled with the k-means clustering method; a targeted quality assessment strategy based on chemical analysis was adopted to understand the quality of Cistanches Herba. Based on market research, the collected samples were divided into different grades through k-means clustering analysis. Moreover, quality differences of Cistanches Herba in the Alexa region were explored through DNA barcoding and chemical analysis. Accordingly, 10 different types of VCs were determined in the production of Cistanches Herba. The results show that there is a close relationship between the quality of Cistanches Herba and stakeholder benefits. Vertical integration at different levels was found for independent farmer-based VCs, horizontal collaboration was found in the cooperative-based VCs. The vertical coordination has led to a more consistent traceability system and strict regulation of supply chains. At the same time, the Cistanches Herba were divided into three grades. Through DNA barcoding and chemical analysis, we found that the quality differences between Cistanches Herba in the Alexa area were not significant. It was found that geographical suitability and vertical integration could impact the quality and sustainable production of Cistanches Herba. At the same time, the well-developed VCs can provide products with reliable quality, and ensure adequate financial revenue for relevant stakeholders.

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1 INTRODUCTION

Cistanche deserticola Ma (commonly known as Rou Cong Rong in Chinese or Cistanches Herba) is a parasitic plant. The dried fleshy stem of C. deserticola（Cistanche deserticola) is a precious traditional Chinese medicine (TCM) that has been used for centuries (Fu et al., 2018). Several pharmacological studies have shown that Cistanches Herba displays a range of functions, including antioxidative, anti-nociceptive, anti-inflammatory, immunity, neuroprotective, and hepatoprotective functions (Li Z. et al., 2016; You et al., 2016). Cistanches Herba was used as a homology of medicine and food in 2018, which indicates that in addition to curing specific diseases, it can also be consumed daily for health care needs (Li et al., 2021). In recent years, it has been widely used in various food items, tea, drinks, and beverages. Rou Cong Rong tea is becoming increasingly popular as a health food supplement in Asian countries, such as China and Japan (Li et al., 2018; Hou et al., 2020; Lei et al., 2020). Cistanches Herba is known as desert ginseng due to its nutritional value (Wang et al., 2012). Consequently, Cistanches Herba is popularly used in TCM and health care practices.

With increasing interest in the clinical and health care functions of Cistanches Herba, more research is focused on its active ingredients. Phenylethanoid glycosides comprise a significant class of compounds that exhibit a wide range of pharmacological effects (Li Z. et al., 2016). Echinacoside and acteoside are the main active ingredients of Cistanches Herba. These are described as “marker components” in the Chinese Pharmacopeia 2020 edition (ChP., 2020) (Wang X. Y. et al., 2017). Galactitol is the main laxative constituent of Cistanches Herba. At the same time, it also performs a variety of biological activities, such as anti-aging, and regulation of immune function. Galactitol exhibits low toxicity and minimal side effects and has broad prospects in clinical applications and healthy food development (Gao. et al., 2015). Thus, the determination of galactitol confers a certain significance to the development and utilization of Cistanches Herba.

The demand for Cistanches Herba in the global market has grown rapidly, whereby the annual demand for Cistanches Herba is bordering on approximately 4,000 tons (Lu. et al., 2019). However, the number of its hosts has been declining, as they are being cut down to be used as firewood by herdsmen. The populations of high-value plants that grow in the wild have rapidly declined due to the high market demand for medicinal plants, resulting in their scarcity in the market (Ndou et al., 2019). As a result, with the increase in demand year by year, the wild C. deserticola（Cistanche deserticola) populations have been seriously affected, and the wild resources have been gradually exhausted. It has been listed as a “second grade” nationally protected plant in China, the International Union for Conservation of Nature Red List of Threatened Species and Convention on International Trade in Endangered Species of Wild Fauna and Flora (Appendix II) (Fan et al., 2020), resulting in restrictions being imposed on international trade. This has led to the long-term stagnation of C. deserticola-related industries. Thus, the artificial cultivation of C. deserticola is important. Li. et al. (1989) have studied the artificial cultivation of C. deserticola（Cistanche deserticola) since the 1980s. In the 1990s, researchers systematically elucidated the parasitic mechanism of C. deserticola （Cistanche deserticola）, explored the key technologies associated with the artificial cultivation of parasitic plants, and established a sustainable C. deserticola cultivation system that produces high yields. At present, the artificial inoculation technology for C. deserticola（Cistanche deserticola) cultivation is becoming increasingly advanced, and the cultivated area is expanding on a yearly basis. Therefore, artificial cultivation of C. deserticola may be considered to resolve issues arising from a scarcity of medicinal and protected wild resources (Bi et al., 2020). Moreover, the development of a series of health products based on Cistanches Herba has resulted in a substantial expansion in the international influence of Cistanches Herba.

C. deserticola（Cistanche deserticola) has been cultivated in several plantations to meet the increasing demands of international and domestic herbal medicine markets (Hou et al., 2020). The quality of Cistanches Herba is affected by growing conditions, harvest seasons, and processing methods (Li et al., 2019). Due to natural conditions, ecology, and cultivation techniques, the Cistanches Herba in Alexa have larger and fleshier stems, are richer in pectin and tannin, and are of good quality. Therefore, Alexa is considered to be the ideal location for growing and producing top-quality Cistanches Herba (Daodi herbs) (Li et al., 2019). Daodi herbs have self-adapting characteristics to adapt to their environments over long periods of time. Thus, they reportedly display advanced properties, such as superior active ingredients and better quality (Yin et al., 2019). Plants vary greatly in chemical composition; thus, any quality assessment must take the length, diversity, and uncertainty of value chains (VCs), from which these plants originate, into account (Heinrich et al., 2019). Recent studies have indicated that the VCs of medicinal plants have a significant impact on the quality and safety of herbal medicinal products, such as Curcuma longa L., Ginkgo biloba L., and Lycium barbarum L. (Booker et al., 2014; Booker et al., 2016; Yao et al., 2018). Improvements in medicinal quality and safety add value to the final product. Hence, maintaining the supply of prime materials via VCs is considered vital for meeting the increasing demand for Cistanches Herba.

Starting from cultivation to processing and distribution, a medicinal plant product must pass through several levels of stakeholders before it reaches the final consumer (Székács et al., 2018). Besides production, VC research also focuses on other activities related to the supply chain, including distribution and marketing. VCs define the activities involved in different modes of production while emphasizing the relationship between primary producers and other stakeholders in different production systems and their socio-economic impact (Bi et al., 2020). Although VC analyses have been widely used in various products, only a few studies have investigated the VCs of medicinal plants and their derivatives. It was not until 2012 that people became increasingly aware of the broader role of VCs associated with medicinal plants in global markets (Booker et al., 2012; Booker et al., 2015).

Medicinal plants and their derived products appear to have varied VCs. Within a VC, there may be different stakeholders who contribute to different stages of supply. Understanding the relationship between production and supply helps identify the pressure points in different VCs. By comparing different VCs horizontally and vertically, a better understanding of the differences in the quality and price of herbal medicines in different markets may be acquired (Yao et al., 2018; Bi et al., 2020). In a short chain, such as one where the product is grown and used locally as herbal medicine, the quality, and safety of the product are relatively easy to control and manage. In contrast, using a TCM or food supplement in the domestic market or as an export product involves greater risks and challenges (Booker et al., 2015; Heinrich et al., 2019). It is worth noting that the maximum allowable levels of contaminants, authenticity, and purity are usually controlled at a very late stage. Only careful management along VCs allows improvement in the quality and safety of cultivated medicinal plants.

Alexa, being the main production area, accounts for more than 90% of the total output of Inner Mongolia and more than 70% of China’s total output (Siqin et al., 2019). It is mainly cultivated via wild cultivation mode, without using chemical fertilizers or pesticides, and has great economic and ecological value. Due to its high medicinal value and nutritional effects, adulteration of Cistanches Herba often occurs under market conditions, which limits the safety and sustainable development of the products. There is a lack of information on the quality risks arising from these systems and risk management in production systems and VCs. In the current study, we aimed to investigate the quality differences in Cistanches Herba from the perspective of VCs. We investigated its production and circulation in the prestigious area (Alexa) and analyzed specific issues affecting quality and yield. Differences in multiple stakeholders, capital flows, and information were clarified according to VCs. In addition, market research enabled us to develop a Cistanches Herba grading system; a targeted quality assessment strategy based on chemical analysis was adopted to understand the quality of Cistanches Herba, which provided a basis for ensuring and improving quality as well as the economic benefits of production and consumption.

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2 MATERIALS AND METHODS

2.1 Fieldwork

Based on documentary research and on-site visits, starting August 2016, fieldwork was conducted during the cultivation and harvesting periods in Inner Mongolia, Gansu, Ningxia, and Xinjiang, covering the main production areas as well as several trading center associated with Cistanches Herba. To maintain variability within an acceptable range, samples for content determination were collected only during the spring. We collected 90 samples, including 18 from Alxa Left Banner (105°42′E/38° 50′N), 50 from Alxa Right Banner (101°68′E/39°20′N), and 22 from Ejina Banner (100° 88′E/41°90′N). Moreover, we visited three national herbal markets, namely the Yulin Chinese herbal medicine market, Bozhou Chinese herbal medicine market, and An’guo Chinese herbal medicine market. The standard for grading Cistanches Herba was established by measuring the morphological characteristics of the samples. Participatory observations and semi-structured interviews were conducted with different stakeholders. Farmers, wholesalers, and retailers from core cultivation areas or trading centers of the visited areas were randomly selected to collect general information on VCs, current market conditions, and grade classification of Cistanches Herba.

2.2 Plant Material

90 Cistanches Herba samples were collected in Alexa during field work. An additional 40 samples were offered either by the institutions we visited and bought in the above-mentioned herbal markets and seven medicine purchasing stations in Inner Mongolia, Gansu, Ningxia, or Xinjiang. The whole Cistanches Herba plants were identified by Prof. Wang of Baotou Medical College. The voucher specimens were preserved at the Baotou Medical College, University of Inner Mongolia.

2.3 VCs Analysis

Analysis of VCs was carried out via the following steps: 1) identification of the main links in the production of Cistanches Herba;2) identification of stakeholders in the production process and adding them to the corresponding process; 3) calculation of labor and non-labor costs of each transaction link based on collected data and information, and 4) building of a framework for the VCs to analyze VCs in relation to financial performance, production behavior and quality (Yao et al., 2018). In addition, the strengths and weaknesses of the different VCs were analyzed.

2.4 Reagents and Chemicals

An HPLC system was purchased from Thermo Fisher Scientific (Ultimate 3000, United States). A pulverizer (ST-08,1800 W, China), laboratory water purifier (AMFI-5-P, Yiyang Enterprise, China), electronic analytical balance(AR153O, Mettler Toledo), Retsch-MIVMOO ball mill (model MM400, Retsch, Germany), and desk centrifuge(TGL-16G, Shanghai Anting)were also used. Methanol and acetonitrile were of HPLC grade,(Tianjin Kemiou Chemical Reagent Co. Ltd)while all other reagents were of analytical grade. The reference compounds echinacoside, acteoside, and galactitol were purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China).

2.5 Quantitative Analyses of Main Chemical Indexes in Cistanches Herba

Echinacoside and acteoside were detected under the following conditions: analysis was performed using a C18 column at a flow rate of 1.0 ml/min; the mobile phase was 35% methanol-0.1% acetic acid; the column temperature was set at 30C, and the DAD detector wavelength was 334 nm. Conditions for the determination of galactitol were as follows: sample analysis was carried out on a Prevail Carbohydrate ES polymer gel column at a flow rate of 0.7 ml/min, the mobile phase was acetonitrile-water(77:23), and the column temperature was set to 25°C. An Alltech ELSD 2000ES evaporation light scattering detector(Alltech Technology Ltd.) was used, the drift tube temperature was set to 40°C, and the gas pressure was set to 240 kPa.

2.6 Molecular Identification of Cistanches Herba

DNA Extraction, PCR Amplification, and Sequencing: Genomic DNA was extracted from silica gel-dried medicinal materials. Approximately 0.03g of the molecular material was ground for 45s at a frequency of 3,000 times/s in a Retsch-MIVMOO ball mill. DNA extraction was performed using an optimized cetyltrimethylammonium bromide method. Then, rbcL,matK,trnH-psbA, and ITS regions were amplified by polymerase chain reaction（PCR） with primers. A 25 μL PCR amplification was performed. Purified PCR products were sequenced in the forward direction using the primers used for amplification on an ABI3730XL sequencer.

2.7 Clustering Analysis

The k-means algorithm is capable of effectively analyzing large-scale data and is easy to understand. Automatic grouping was carried out according to the degree of natural affinity, which makes the structural characteristics of individuals within the group very similar, and the characteristics of the individuals between the groups different (Wang T. et al., 2017). k-means clustering was performed using SPSS and Rstudio to analyze market grades and the relationship between the contents of the active ingredients of the field sample and its place of origin.

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3 RESULTS AND DISCUSSION

3.1 Industrial Structure and VCs

Cistanches Herba goes through six stages before it reaches the consumer as follows: 1) cultivation, including activities, such as plowing, inoculation, pruning, rodent control, and harvesting; 2) initial processing, including surface treatment, removing impurities, as well as drying; 3) procurement, which involves trading of unprocessed material produced in step 2; 4) secondary processing, including commercial grading, deep-processing, and packaging; 5) wholesale, mainly to nonlocal markets; and 6) retail (Figure 1).

FIGURE 1 | Cistanches Herba goes through six production stages in food and medicinal chain.

On account of its long production and supply lines, Cistanches Herba production involves 10 mature VCs, which may be distinguished by the various composite patterns of stakeholders. Different stakeholders play a role in each of these steps, resulting in various forms of VCs. The 10 primaries VCs identified as being associated with Cistanches Herba as well as the stakeholders involved are shown in Figure 2.

FIGURE 2 | Cistanches Herba production involves 10 mature VCs.

VCs 1–6 are based on independent farmers with small Cistanches Herba fields offering harvested products to medicinal wholesalers, local individual retailers, or other cultivation-processing-trading integrated companies. VC 7 is run by local individual retailers. Harvested products are often sold as local food products. VC 8 initiates with farmers involved in agricultural cooperatives (with relatively large fields). VCs 9 and 10 are characterized by cultivation-processing-acquisition trading firms. Independent farmers with relatively small fields are placed in VCs 1–6. These tend to be traditional, small-scale economies. In these VCs, independent farmers grow and harvest Cistanches Herba. Farmers near local individual retailers, medicinal wholesalers, or processing companies sell their products directly, without the involvement of middlemen, to increase profits earned from the cultivation of this plant. In VC 1, unprocessed Cistanches Herba is sold to processing firms; Cistanches Herba is processed into raw materials and sold to pharmaceutical factories, and then the products are sold to consumers through pharmacies and the like. Due to the particularities of the growing environment and strict storage requirements, many individual retailers, such as those in VCs 2,3 and 6, are able to procure unprocessed Cistanches Herba directly from farmers, and reduce intermediate links, thus improving the quality. It is then further processed into herbal medicines or functional food and sold to consumers, hospitals, or pharmacies. At the same time, some are dried and processed as agricultural by-products for sale. However, farmers directly sell their products without processing, and the product quality is likely to be not stable due to insufficient drying during the transportation process. Additionally, the development of e-commerce makes it possible for a processing firm to expand downstream. VC 5, the trading firm plays a role that extends up to the retailer.

VC7 is a one-stop VC conducted by self-operating individual retailers who hire local farmers or carry out their own production and processing. They usually sell their products through their own offline and online stores, which play an important part in a sale. These products are usually bought directly by consumers in the form of local agricultural products and functional food products.

VC 8 begins with agricultural cooperative-based VCs. Related governmental departments provide farmers with beneficial policies to plant Cistanches Herba. Thus, enterprises are encouraged to develop agricultural cooperatives. These farmers receive more technical support and equipment from cooperatives than independent farmers.

VC 9 and VC 10 are regulated by companies that control cultivation, processing and trading, and are based in large production areas. They further process the products into functional food products, which are then directly sold to retailers or consumers, mainly through chain stores or online stores. Moreover, some products are sold to foreign wholesalers.

3.2 Trading along with VCs of Cistanches Herba

The value of a product is created by the production activities of the stakeholders and is generated during the transaction process. In different VCs, labor input and non-labor input have different effects on value (Yao et al,2018).

Alexa has a vast land area and rich natural resources, which lays the foundation for its ecological construction and the development of the desert culture industry. Herdsmen mostly contract pastures, where there are large areas of natural Haloxylon ammodendron(C. A.Mey.)Bunge ex Ferzl,as well as alarge space sitabe for planting H. ammodendron. Farmers usually plant H. ammodendron in their own finds or grasslands. After H. ammodendron survives, it is grafted onto C. deserticola, and finally, the industrial model is based on selling C deserticola raw materials Therefore, the cultivation of Cistanches Herba is one of the income sources of herdsmen(Zhao and Zhang,2014). However, the cultivation of Cistanches Herba is a long-term investment owing to the long planting code, large capital demand, and slow harvest

During the planting period of H. ammodendron, a large amount of water is needed for irrigation. Owing to the serious shortage of groundwater in Alexa, the land can only be irrigated manually. At the same time, since H.ammodendron forests are more scattered and the area is larger, it is necessary to have the protection of fencing because the new young H.ammodendron seedings planted every year cannot survive the grazing of animals. In the survey, many farmers and herdsmen generally reported serious issues with rats in H. ammodendron forests. Therefore, in the early stage of planting, the cost of watering, fencing, rodent control, machinery, and human labor in H. ammodendron forests are higher, resulting in higher costs for Cistanches Herba production. However, the wild cultivation mode can save a portion of the non-labor costs of chemical fertilizers and pesticides while ensuring high quality. At the same time, the Alexa government pays a certain amount of compensation to the Cistanches Herba cultivators. However, Cistanches Herba cultivated by independent farmers has lower survival rates and yields than that cultivated by cooperative companies, which mainly leads to lower income for independent farmers.

In addition, some stakeholders, such as agricultural cooperatives and integrated cultivation-processing-trading companies, have well-established facilities that help reduce labor costs and lower inputs, thus guaranteeing high returns. By contrast, VCs8-10 involve large-scale agricultural operations that are increasingly popular, including agricultural cooperatives and plantation companies. In addition to providing a traditional medicinal market, further processing into functional food adds to its benefits and efficiency.

Processing and wholesale are also labor-intensive sites (20-25 Chinese yuan(CNY)/kg). Before they move on to the second processing stage, processing companies spend a large number of funds on production and testing equipment, production workshops, and storage rooms. Storage of Cistanches Herba is an important factor affecting quality and price, and therefore a large amount of capital and labor is invested in storage. In addition, these factories must perform quality testing for each batch of TCM. At the same time, part of the product is processed into the food market. In addition, a large part of the input was made in the management of the factory.

Retail is the final stage of the VC. Here, the resources flow to high-quality hospitals. traditional herbal medicine markets. agricultural markets, and online markets. The trends seen in prices indicate that these are related to the dynamic balance between demand and supply. It is thus believed that as the demand for Cistanches Herba products grows, the price (and the economic benefits to farmers)will continue to rise in the short term. However, over the long term, progressively more Cistanches Herba will be harvested, causing prices (and farmers' financial gains) to decline.

The retail price of stanches Herba is generally higher than its wholesale price. The price of Cistanches Herba appreciates with grade The market price of an entire plant is higher than that of its slices. An increase of 10cm in the length of Cistanches Herba is equivalent to an increase of 100 CNY. In addition, the slicing position and processing method cause a difference in the product phase exerting a significant impact on the price. The size of Cistanches Herba shrinks sharply after drying in the shade or in the sun The surface of the slices is obviously wrinkled, the color deeper, and the price is the lowest. After harvesting at the site of production, it is cleaned, peeled, and dried after being sliced. If the surface of the slice is smooth, then the price is intermediate. In pharmacies and hospitals, Cistanches Herba is further processed into decoction pieces or formula granules, and the retail price becomes generally twice the wholesale price.

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3.3 Quality Evaluation of Cistanches Herba Along with VCs

3.3.1 Grades of Cistanches Herba

In Alexa areas, the wild cultivation mode, without the use of chemical fertilizers or pesticides, helps sustainable development Aka has been considered as the daodi area of Cistanches Herba and has a reputation as "the hometown of Cistanches Herba in the world"(Igin et al,2019). Cultivation according to suitable ecology is a factor that guarantees the yield and quality of Cistanches Herba. However, the current planting of Cistanche Herba in Akxa has not been standardized. Therefore, the disordered classification of Cistanches Herba in the market leads to price differences. Currently, the size of obtainable medicinal materials is generally used as the main basis for grading(Li et al。2019). We conducted an in-depth study of all samples collected Based on requirements, the morphology, surface characteristics and size, and parameters, such as diameter and length, of Cistanches Herba, were analyzed(Yan et al.,2019). More precisely, Cistanches Herba was divided into three grades (Figure 3). These results may be used to determine the specifications and grades of Cistanches Herba (Table 1). At the same time, the market survey found that the price of first-grade products was more than 200 CNY/kg; second-grade products cost about 180CNY/kg, and third-grade products cost lower than 150 CNY/kg. By analyzing the quality differences between Cistanches Herba stocks in the market, a more reasonable standard for grading Cistanches Herba was established to guide a more standard production model.

TABLE 1 | The specification and grade of Cistanches Herba.

FIGURE 3 | Different grades of Cistanches Herba ((A): first class, (B): second class, (C): third grade).

3.3.2 Quality Evaluation of Cistanches Herba

Daodi herbs have been widely recognized by the Chinese medicinal industry as a symbol of stable, high-quality TCM for a long time (Yin et al,2019). Along with the VCs, different criteria were considered as being indicative of good quality. For farmers, size is often a measure of quality; the bigger the fleshy stem, the better the quality. Retailers believe that habitat and size are important indices of the quality of Cistanches Herba. In addition, doctors believe that better quality would have a more effective clinical effect. At present, these measures can be used to evaluate the quality of Cistanches Herba that have been established in pharmacopeia The quality of Cistanches Herba has been evaluated using methods, such as source, morphological, and physicochemical identification in the pharmacopeia (ChP.2020). In addition. some planting companies and pharmaceutical factories impose their own regulations to ensure the quality of their herbal medicines. However. independent farmers are technologically not proactive enough to access them.

Along with the VCs, different criteria were considered as being indicative of good quality. Therefore, a quality investigation of Cistanches Herba from different VCs was performed, and the results are shown in Table 2. The quality of Cistanches Herba is related to planting, storage, drying, and processing. In VCs 1-4, farmers directly sell their products without processing, and the product quality is not stable due to insufficient drying during the transportation process. In VCs 5 and 6, Cistanches Herba is usually sold as an agricultural product, making it difficult to trace or supervise effectively. In VCs 8-10, the integrated chain of cultivation, harvesting, and processing may reduce the loss of active ingredients. In addition, deep processing enterprises will have a stricter traceability and quality assurance system for products, thus guaranteeing quality.

TABLE 2 | Quality of the Cistanches Herba for the different VCs and likelihood of risks being made to its quality during its production

Generally, the effective ingredient content of a plant is related to its growth period(LiZ.et al,2016). Therefore, the evaluation of the main chemical components of Cistanche Herba can reflect its quality to a certain extent. Echinacoside and acteoside are the main active ingredients of Cistanches Herba (LiY.et al,2016). These are described as marker components in the Chinese Pharmacopeia 2020edition (Wang X.Y,ct al.2017). Galactitol is the main laxative constituent of Cistanches Herba. Thus, the determination of galactitol confers a certain significance to the development and utilization of Cistanches Herba (Gao et al, 2015). Moreover. a one-way analysis of variance of active ingredient content and characteristics(Table 3), indicated that the active ingredient content was correlated with the characteristics of Cistanches Herba. The higher the oiliness of the sample, the higher the content of its chemical components. At the same time, the study found that galactitol content is related to the weight of the sample, which further illustrates the significance of galactitol content determination.

TABLE 3 | One-way analysis results of variance between the appearance of Cistanches Herba and echinacoside, acteoside, galactitol.

We determined the content of all samples collected from the field. The results showed that the content of active ingredients in Aka was high (Supplementary Table SI), clarifying the high quality of Alexa in the market and the high prices paid k-means cluster analysis was used to analyze 90 samples collected from the field based on three variables: echinacoside, acteoside, and galactitol(Figures 4A, B). These results show that the samples can be divided into three different clusters, but the clusters representing Cistanches Herba from the three regions are relatively disorganized Analysis of the chemical components of Cistanches Herba from different areas of Alexa revealed that there was no significant correlation between chemical composition and geographical distance. We believe that there is no substantial difference in the quality of Cistanches Herba from different areas of Alexa.

FIGURE 4 | (A, B) Results of the constituents of Cistanches Herba based on k-means cluster (sample_E: the sample of Ejin Banner; sample_R: the sample of Alexa

Right Banner; sample_L: the sample of Alxa Left Banner).

Herbal products contain numerous ingredients, and phytochemical analyses used in the quality control of marker compounds are accurate but have limited applicability in the identification of biological constituents. The herbal products, sold worldwide as medicines or foods, are perceived as low risk because they are considered natural and thus safe(Ichim, 2019). The growing demand for herbal products due to their health benefits has resulted in a proportional increase of their accidental contamination or intentional adulteration. To address this problem, DNA barcoding as a powerful strategy has recently attracted considerable attention and, along with chemical methods, has started to enter the regulatory systems for quality control (Ichim et al.2020).DNA barcoding is widely used for molecular identification for the purpose of solving a broad range of issues in taxonomy, molecular phylogenetics, and population genetics, as well as for preventing illegal wildlife collection and product authenticity monitoring (Janjua et al 2017; Radariu et al.2018).In this study, we randomly selected a few samples for DNA barcoding identification using four DNA barcode sequences (rbcL, matK, trnH-PSA, and ITS) to identify Cistanches Herba from Alexa habitats. A trnH-psbA sequence analysis indicated a deletion in Alxa Left Banner at 298-337 bp, compared with Aka Right Banner and Ejina. It remains to be verified whether this can be used as a specific identification site for Cistanches Herba. Based on the NI tree established by the sequence, the sequence clustering of the four fragments of the sample cannot be distinguished(Figures 5-8).In addition, the genetic distance analysis results showed that there was no significant correlation between genetic distance and geographical distance(Table 4-7) A previous study of ours found that site-specific PCR may be used to screen site-specific primers for the purpose of identifying C. deserticola (Cistanche deserticola), (Rupr.)1. Leonard, and Orobanche. Hance is based on ITS sequences (Li et al,2014). However, the identification of Cistanches Herba remains to be further studied because of the existence of other adulterants. By studying the DNA barcoding of Cistanches Herba, we found that the quality differences between Cistanches Herba populations in the Alexa area were not significant. We also quickly and accurately identified Cistanches Herba and its adulterants, which is of vital importance for guaranteeing food and medicine safety and regulating the market circulation.

In conclusion, the genetic distance and chemical composition of Cistanches Herba were not correlated with a geographical location in Alexa. However, this result may be limited by our representative sample and the availability of the sequences. Thus, these results require further verification.