Ethnobotanical survey method
Study site
Information provided by local government and a preliminary survey played a decisive role in the selection of the research site. We selected villages and markets in Guizhou, Yunnan, and Fujian Provinces as survey sites, as the people frequently used indigo paste and had a good heritage of traditional knowledge in these areas (Table 1). Guizhou and Yunnan Provinces are located in southwest China, and Fujian Province is located in southeast China (Fig. 1).
Congjiang County (25°16′–26°05′ N; 108°05′–109°12′ E) and Zhenfeng County (25°07′–25°44′ N;105°25′–105°56′ E) [24] belong to Qiandongnan Miao and Dong Autonomous Prefecture and Qianxinan Buyi and Miao Autonomous Prefecture, respectively. Congjiang County is located in the middle reaches of the Duliu River. The climate is humid monsoon of the mid-subtropical zone. The annual average temperature is 18.5 ℃ and annual average precipitation is 1185.9 mm. Ninety-five percent of Congjiang County’s population are ethnic minorities, such as the Miao and Dong people [25], providing an ideal environment for the study because of its strong cultural inheritance practices. The six villages surveyed in Congjiang County were located in mountainous areas on both sides of the Duliu River, and the residents were all Miao people. Currently, the local area retains a good indigo paste culture, and the daily dress of Miao women is still the traditional national costume. In the local area, every family, except for migrant families, cultivates indigo yielding plants every year to make indigo paste for dyeing cloth. The climate of Zhenfeng County is subtropical humid monsoon climate. The annual average temperature is 16.6 ℃ and average annual precipitation is 1276.9 mm [24]. In Zhenfeng County, indigo paste is produced and traded as a commodity and thus it is produced at scale. The main local trade time is market day and the location is the farmers’ markets in each town. The sellers are mostly Han residents from nearby villages, and the buyers are local ethnic minorities and merchants. In Zhenfeng County, we conducted surveys in two main farmers’ markets and three villages that producing indigo paste.
Yuanyang County (22°49′–23°19′ N; 102°27′–103°13′ E) and Jinping County are located in Honghe Hani and Yi Autonomous Prefecture of Yunnan Province. Yuanyang County has a subtropical mountain monsoon climate. The annual average temperature is 24.4 ℃ and average annual rainfall is 899.5 mm. Yuanyang County is inhabited by seven ethnic groups including the Hani, Yi, and Han people who have lived there for generations. Ethnic minorities account for 89.44% of the total population [26]. Jinping County has a tropical monsoon climate. The annual average rainfall is 2358.6 mm and annual average temperature is 18 °C. Nine ethnic groups including the Miao and Yao groups have lived there for generations, and ethnic minorities account for 87.6% of the total population [27]. In the four villages surveyed in Yunnan Province (one Yao village and three Hani villages), the elderly still retain the traditional natural indigo culture and artisanship.
Xianyou County (25°11′–25°43′ N; 118°27′–118°56′ E) is located in the middle of the southeast coast of Fujian Province. The climate is south subtropical maritime monsoon climate. The annual average temperature is 20.6 ℃ and annual average rainfall is 1300–2300 mm. Among them, the Shufeng Township we investigate has the reputation of being “the Hometown of Indigo Naturalis" because of its long history of making high quality Indigo Naturalis [28]. Indigo Naturalis is the powder processed to make indigo paste. Its active ingredients are indigo and indirubin. It is used to treat oral ulcers [29], ulcerative colitis [30], and psoriasis [31-33].
Table 1 Summary of the survey site.
Survey site
|
Province
|
county
|
national
|
latitude
|
longitude
|
Tingdong Town market
|
Guizhou
|
Congjiang
|
Miao
|
25°49ʹ28.71ʺ
|
108°37ʹ01.22ʺ
|
Jiuyue village
|
|
|
|
25°47ʹ16.32ʺ
|
108°35ʹ25.13ʺ
|
Jiugua village
|
|
|
|
25°46ʹ45.49ʺ
|
108°36ʹ38.97ʺ
|
Miaopeng village
|
|
|
|
25°47ʹ35.90
|
108°36ʹ38.77ʺ
|
Jiali village
|
|
|
|
25°50ʹ15.90ʺ
|
108°38ʹ30.65ʺ
|
Lingwa village
|
|
|
|
25°50ʹ27.15ʺ
|
108°40ʹ32.83ʺ
|
Changzhai village
|
|
|
|
25°50ʹ04.13ʺ
|
108°37ʹ42.98ʺ
|
LongchangTown market
|
|
Zhenfeng
|
Han
|
25°27ʹ43.05ʺ
|
105°30ʹ06.34ʺ
|
Zhenfeng Town market
|
|
|
|
25°23ʹ18.34ʺ
|
105°38ʹ53.50ʺ
|
Weizhai village
|
|
|
|
25°27ʹ16.00ʺ
|
105°33ʹ10.04ʺ
|
Zikong village
|
|
|
|
25°31ʹ49.34ʺ
|
105°34ʹ01.54ʺ
|
Shuiyindong village
|
|
|
|
25°31ʹ29.84ʺ
|
105°33ʹ11.23ʺ
|
Sihuang village
|
Fujian
|
Xianyou
|
Han
|
25°26ʹ52.61ʺ
|
118°39ʹ25.40ʺ
|
Shufeng village
|
|
|
|
25°26ʹ34.12ʺ
|
118°39ʹ16.95ʺ
|
Gama village
|
Yunnan
|
Yuanyang
|
Hani
|
23°01ʹ36.23ʺ
|
103°01ʹ13.84ʺ
|
Zhetai village
|
|
|
|
23°01ʹ59.18ʺ
|
102°59ʹ10.52ʺ
|
Yaorenxiaozhai village
|
|
|
Yao
|
23°00ʹ13.77ʺ
|
103°02ʹ11.23ʺ
|
Gaoxing village
|
|
Jinping
|
Hani
|
22°54ʹ52.74ʺ
|
103°12ʹ11.94ʺ
|
Field survey and data collection
The first field survey was conducted in Congjiang County and Zhenfeng County of Guizhou Province in August and September 2019, for approximately ten days each. The second field survey was conducted in Xianyou County of Fujian Province in October 2019 for seven days. The third field survey was conducted in Yuanyang County and Jinping County of Yunnan Province in January 2020 for six days. During the field investigation, we invited local people who could speak the local language and Mandarin to serve as translators. The intentional sampling method [34], snowball sampling method [35], participatory observation method, and questionnaire survey method [36] were used to collect the data. The interviewee questionnaire is shown in Table 2. All interviewees possessed traditional knowledge relating to indigo paste. Before conducting interviews with them, we obtained informed consent from all interviewees orally. After obtaining permission, we took photographs [37], audio, video, and other forms of material to assist with our research.
As shown in Table 3, a total of 283 informants were interviewed, including 171 from Guizhou Province (139 from Congjiang County and 32 from Zhenfeng County), 42 from Fujian Province, and 70 from Yunnan Province. The age of the informants was between 31 and 81 years. The age of most informants was concentrated between 30 and 69 years (88.0%). The number of female informants (n = 219) was almost 3.5 times that of males (n = 64).
Table 2 Questionnaire for the interviewees.
1 How many types of Indigo-yielding plants in local use?
|
2 What are the local names of these Indigo-yielding plants?
|
3 What do these local names mean?
|
4 How to make Indigo paste after harvesting Indigo-yielding plants?
|
5 How many ways can you judge the quality of Indigo paste?
6 How to judge specifically?
|
7 Which of these methods do you like best?
|
Table 3 Gender and age of the interviewees.
|
Number
|
Percentage
|
All
|
|
|
Gender
|
|
|
Male
|
64
|
22.6%
|
Female
|
219
|
77.4%
|
Age
|
|
|
30-49
|
101
|
35.7%
|
50-69
|
148
|
52.3%
|
≥70
|
34
|
12.0%
|
Guizhou
|
|
|
Gender
|
|
|
Male
|
26 (C:0, Z:26)
|
15.2% (C:0%, Z:81.3%)
|
Female
|
145 (C:139, Z:6)
|
84.8% (C:100%, Z:18.7%)
|
Age
|
|
|
30-49
|
79 (C:70, Z:9)
|
46.2% (C:50.4%, Z:28.1%)
|
50-69
|
78 (C:56, Z:22)
|
45.6% (C:40.3%, Z:68.8%)
|
≥70
|
14 (C:13, Z:1)
|
8.2% (C:9.3%, Z:3.1%)
|
Yunnan
|
|
|
Gender
|
|
|
Male
|
8
|
11.4%
|
Female
|
62
|
88.6%
|
Age
|
|
|
30-49
|
18
|
25.7%
|
50-69
|
38
|
54.3%
|
≥70
|
14
|
20.0%
|
Fujian
|
|
|
Gender
|
|
|
Male
|
30
|
71.4%
|
Female
|
12
|
28.6%
|
Age
|
|
|
30-49
|
4
|
9.5%
|
50-69
|
32
|
76.2%
|
≥70
|
6
|
14.3%
|
Note: C Congjiang county, Z Zhenfeng county.
Survey data analysis
In order to screen out the most commonly used, most recognized, and most important quality judgment indicators, we used questions 5, 6, and 7 (Table 2) to calculate the Frequency of Citation (FC), Mention Index (QI) [38], and Fidelity Level (FL) [39] of each quality judgment index, respectively. For each quality judgment index, the number of informants using the index was counted as the citation frequency of the index (FC). QI was used to test homogeneity of the knowledge, where QI = number of mentions/number of informants. FL was used to evaluate the importance of the different quality judgment indices, where FL = (total number of informants who provide one quality judgment index/ total number of informants who provide all quality judgment indicators) × 100%.
Material collection and pretreatment
In order to avoid sample interference, we obtained 21 indigo paste samples from Guizhou Province, which had the largest number of indigo paste users among all study areas. The 21 indigo paste samples were identified and categorized by 3 key informants using 5 quality levels, i.e., the best (3 samples), good (3 samples), general (7 samples), poor (5 samples), and worst (3 samples). We used the values 1, 2, 3, 4, and 5 to represent the five quality grades of indigo paste, respectively. During the field investigation, indigo paste samples were collected and sealed into 50 ml plastic tubes. At the laboratory, we used a blast-drying oven (Shanghai Chengshun Instruments Co., Ltd. Shanghai, China) to dry the samples to a constant weight at a constant temperature of 60 °C, which were then stored.
High-performance liquid chromatography (HPLC) analysis
Chemicals and reagents
Indigo standard product (≥98%) was purchased from Guangzhou Kehua Commercial and Trading Co. Ltd., Guangzhou, China. Indirubin standard product (≥98%) was purchased from Shanghai Macklin Biochemical Co. Ltd., Shanghai, China. Indole standard product (≥99%) was purchased from Aladdin Reagent (Shanghai) Co. Ltd., Shanghai, China. Indican standard product (≥98%) was purchased from Jiangsu Aikon Biopharmaceutical R&D Co. Ltd., Jiangsu, China. Methanol (≥99.9%) was purchased from Shanghai Xingkegaochun Solvent Co. Ltd., Shanghai, China. N-N dimethylformamide (≥99.5%) was purchased from Yunnan Shusen Biotechnology Co., Ltd., Yunnan, China. All chemical reagents were of analytical grade.
HPLC-DAD
We ground 1–2 g of the dried indigo paste sample in a 5 ml centrifuge tube (60 Hz for 80 s) using a high-throughput tissue grinder (Scientz-48, Ningbo Scientz Biotechnology Co. Ltd., Ningbo, China). This was repeated four times. After grinding, we weighed 100–200 mg of the sample powder into a 10 ml centrifuge tube, added 6 ml of N, N-dimethylformamide (DMF), and performed extraction ultrasonically for 30 min. After cooling to room temperature, the final volume was adjusted to 10 ml using a 10ml volumetric flask. The solution was then passed through a 0.45 µm microporous membrane, ready for use.
The Agilent 1260 series equipment (Agilent Technologies, USA) was used to quantitatively analyze the active ingredients such as indigo and indirubin in the indigo paste samples [40]. Each indigo sample was subjected to three repeat experiments, and the final active ingredient content was the average of the three measured data. The standard curves of indigo, indirubin, indole, and indican were established using five different concentration gradients. The sample was separated using an Agilent ZORBAX Extend-C18 column (5 µm, 4.6 × 250 mm) at 30 °C. The mobile phase consisted of water (solvent A) and methanol (solvent B). The eluent flow rate was 1.0 ml/min, and the injection volume was 10 µl. The mobile phase elution gradient was 0–30 min, 10–80% B; 30–50 min, 80% B; 50–51 min, 80–10% B; and 51–55 min, 10% B. The detection wavelength was 280 nm.
pH analysis
We weighed 0.5 g (accurate to 0.0004 g) of ground indigo powder and ultrasonicated it for 30 min to completely disperse the dye in 50 ml of distilled water with a pH of 7 [41]. A PHS-3C acidity meter and E-201-C composite electrode (Shanghai INESA Scientific Instrument Co. Ltd., Shanghai, China) were used to measure the pH of the 21 indigo paste samples.
Particle size analysis
An indigo paste suspension with a content of 0.1 g/L was prepared with distilled water, and the upper suspension was taken after ultrasonic dispersion for 10 min. A Malvern Zetasizer ZEN 3600 zeta potential analyzer (Malvern Instruments Ltd., Malvern, U.K.) was used for particle size testing [42].
Statistical analysis
In order to analyze whether there were significant differences in the effective ingredient content and pH (95% confidence interval) of the indigo paste samples in different quality grades, we performed an analysis of variance (ANOVA), using the percentage of active ingredient and pH as the variables (p ≤ 0.05). In order to analyze whether there were differences in the particle size of indigo paste with different quality grades, we used Origin Pro learning edition data analysis software to produce a line graph of the particle size distribution of the 21 indigo paste samples for comparison.