Quality Blues: An Assessment of Cultural and Chemical Factors Used to Dene Indigo Quality in China

Background: The appearance of synthetic indigo has caused most people to forget the past brilliance of natural indigo. However, in parts of southern China, the folk still use and trade in natural indigo paste. The aims of this study were to i) document the traditional knowledge and experience of how people identify the quality of indigo paste and ii) explore the rationality of the quality judgment index used by local people. Method: We interviewed 283 traditional indigo paste artisans in 3 markets and 15 villages in Guizhou, Yunnan, and Fujian Provinces. Frequency of citation (FC), Mention index (QI), and Fidelity level (FL) of each indigo paste quality judgment index were calculated to determine the most commonly used, most recognized, and most important quality judgment index. A quantitative study was conducted on 21 indigo paste samples of different quality grades using high-performance liquid chromatography with diode-array detection (HPLC-DAD), an acidity meter, and particle size analyzer. The relationship between the content of the effective components, pH, and particle size of the indigo paste and quality was explored. Results: The people divided indigo paste into ve quality grades i.e., best, good, general, poor, and worst. Four main quality judgment indices were color, taste, touch, and dyeing ability. Among all study areas, color was the most commonly used, most important, and most recognized index. Effective ingredient content and pH differed signicantly with different quality graded indigo pastes, but there was no signicance difference between particle size and quality. In addition to indigo, indirubin played an important role in identifying the quality of indigo paste. Conclusion: Our study conrmed that the quality of indigo paste could be evaluated using the four indices (color, taste, touch, and dyeing ability). The people used indigo and indirubin content together to determine the quality of the indigo paste, while synthetic indigo is determined by indigo content only. The simple knowledge and experience of traditional folk has guiding signicance for the modern development of indigo paste production.


r color and
better colorfastness compared to fabric dyed with a chemically identical synthetic indigo [1,17,18].In addition to its pleasant natural fragrance, dyed fabrics of indigo paste also have certain health bene ts: they can act as an insect-repellent and as a disinfectant [15,19].

However, the quality of indigo paste currently on the market varies and it lacks a reasonable quality judgment method.The plant source and processing methods used in producing indigo paste signi cantly in uence the quality of the paste, which in turn directly affects the quality of the dye [20][21][22][23].Therefore, in the process of use and trade, quality is an unavoidable issue.The chemical industry standard of the People's Republic of China stipulates ve indicators used to judge the quality of synthetic indigo.These are the appearance (dark blue uniform powder or granule), the mass fraction of indigo (≥93%), the mass fraction of moisture (≤1.0%), the mass fraction of neness (≤5.0%beyond 250 µm), the residue in the sieve (≤5.0%), and the iron content (≤500 mg/kg).In contrast, there are no regulations or standards for indigo paste.There are also a limited number of research reports on the standards of quality judgment.

Through our investigations, we found that in parts of Guizhou, Yunnan, and Fujian Provinces in southern China, people still use and trade in indigo paste on a large scale, and the traditional knowledge of indigo paste is passed on from generation to generation.Moreover, the people separate indigo paste into different quality grades for use and trade without the use of laboratory equipment, and we did not understand how they classi ed indigo paste into different quality grades or how they made quality judgments.Based on this, the aims of this study were: i) to record the indigo paste quality grades and quality judgment indices used by the folk in detail; ii) to identify the most commonly used, most recognized, and most important quality judgment indices; and iii) to explore the rationality of the quality judgment indices used by the folk.


Methods


Ethnobota ical 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 1185.9mm Ninety-ve 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 avera e annual precipitation is 1276.9mm [24].In Zhenfeng County, indigo paste is produced a

traded
s 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 Prefe ture 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 ounty 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][32][33].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 i 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 m ke Indigo paste after harvesting Indigo-yielding plants?

5 How many ways can you judge the quality of Indigo paste?

6 How to judge speci cally?

7 Which of these methods do you like best?


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 p ste samples were identi ed 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 ve quality grades of indigo paste, respectively.During the eld investigation, indigo paste samples were collected and sealed into 50 ml plastic tubes.At the laboratory, we used a blast-drying oven (Shanghai Chengshun Instrume ts Co., Lt .Shanghai, China) to dry the samples to a constant weight at a constant temperature of 60 °C, which were then stored.
High-performance liquid chromatogr phy (HPLC) analysis


Chemicals and reagents

Indigo standard produc (≥98%) was purchased from Guangzhou Kehua Commercial and T ading Co. Ltd., Guangzhou, C ina.Indirubin standard product (≥98%) was purchased from Shanghai Macklin Biochemical Co

trifuge tube, added 6 ml of N, N-dime
hylformamide (DMF), and performed extraction ultrasonically for 30 min.After cooling to room temperature, the nal volume was adjusted to 10 ml using a 10ml volumetric ask.The solution was then passed through a 0.45 µm microporous membrane, ready for use.

The


Particle size analysis

An indigo paste suspension with a content of 0.1 g/L was prepared with distilled water, and the u per 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 signi cant differences in the effective ingredient content and pH (95% con dence interval) of the indigo paste samples in different quality grades, we

erformed
n 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.


Results


Quality grades and quality judgment indices

As shown in Table 4, ve quality grades and four quality judgment indices for indigo paste were recorded.The ve quality levels were best, good, general poor, and worst.The four quality judgment indicators included color, taste, touch, and dyeing ability (Fig. 2).All four indicators were used in Yunnan and Guizhou Provinces, but Fujian Province only used two indicators, namely color and touch.

Because we found differences in the use and storage of indigo paste across the different regions, the detailed operation methods were also slightly different.In Congjiang County of Guizhou Province and Yunnan Province, where people use color to judge quality, they only judged indigo paste in its wet state.However, in the Zhenfeng County of Guizhou Province, people also applied the wet indigo paste to a small area on the back of their hands or arms (Fig. 2 a and b) and waited for it to dry naturally.Thus, the colors of the indigo paste in its wet and dry state are combined to assess qualit

They believed that hi
h-quality indigo paste would appear dark blue and purple-red in both its wet and dry states.These are known as "water color" and "dry color" in Zhenfeng County, respectively.Because of the inconvenience of transporting wet indigo, people would dry indigo in the sun in the Xianyou County of Fujian Province.They assessed the quality by observing the color of the dried indigo blocks.

People in all regions use the touch method to judge the quality of the indigo, in which a small amount of moist indigo paste is rubbed between the index nger and thumb.Indigo paste that is smooth and hard to w

e off is
considered better quality.If there is obviou graininess, the quality would be considered slightly worse.

In Guizhou and Yunnan Province, people thought that high-quality indigo paste had a "sweet" or "spicy" taste, and dyed cloth easily.It should be noted that there were differences in the description of the folk's taste index, and this phenomenon was more obvious in Guizhou Province.Although more than a quarter of the informants in Guizhou used this index, different informants had different descriptions of taste.Some people thought that indigo paste was of good quality when it had a "sweet" taste, and a "spicy" or "bitter" taste indicated poor quality, while other people thought the opposite.Note: -Meaning that the folk description of this index is in disagreement.


Quantitative evaluation of the quality judgment indices

The color index had the highest FC value, QI value (QI = 1), and FL value (100%) in all study areas.In contrast, the color index was the most commonly used and recognized index among the people (Table 4).The touch index was used in all study areas, but its frequency and importance differed across the regions.The touch index had the highest QI and FL values in Fujian Province (QI = 0.21/FL = 21%), followed by Guizhou Province (QI = 0.12/FL = 12%), and Yunnan Province had the lowest (QI = 0.07/FL = 7%).The taste index and dyeing ability index were only used in Guizhou and Yunnan Provinces.Similarly, there were dif erences in the frequency and importance of the two quality judgment indices in the two regions.In Guizhou Province, the taste index (FC = 47)

was more frequently used than the dyeing ability index (FC = 11), while in Yunnan Province, people use the dyeing ability index (FC = 3) more than the taste index (FC = 2), despite the ery small difference in the FC value of the two indices.The QI values of the taste index and dyeing ability index in Guizhou Province were 0.27 and 0.06, respectively, and the FL values were 27% and 6%, respectively.However, in Yunnan Province, the QI values of the two indicators were 0.03 and 0.04, respectively and the FL values were 3% and 4%, respectively.This showed that the taste index and dyeing ability index were more important in Guizhou Province than in Yunnan Province, and were frequently used by the people, especially the taste index.

Overall, the most important and recognized evaluation index among the people was color, while the other three indices (tast

touch, and dyeing ability) were auxiliary methods only.
HPLC analysis

The main active ingredients in the indigo paste samples were indigo and indirubin and some samples contained insigni cant amounts of indican or indole (Table 5).Since the indigo paste contained water when it was sampled, we also considered water as a factor.Figure 3 shows the average content of the active ingredients in each quality grade of indigo paste.The content of indigo and indirubin decreased with a decrease in the quality grade.There was a positive correlation between the content of active ingredients and the quality grade of the indigo paste.The one-way ANOVA showed that, whether wet or dry, there were signi cant differences in the content of the active ingredients in the different q

lity grades of
indigo paste.The minimum pH value of the 21 indigo paste samples was 9.10, the maximum value was 11.64, and the average value was 10.67.As the quality of the indigo paste deteriorated, the pH tended to increase.The one-way ANOVA showed that there were signi cant differences in the pH of the indigo paste with different quality levels (P = 0.000).Within a certain range (9 ≤ pH ≤ 12), the pH value of good quality indigo paste was lower, while the pH value of poor quality indigo paste was higher.


Particle size analysis

The particle size distribution of the indigo paste samples ranged from 78.82 nm to 5560 nm, and the particle size of most samples were concentrated between 200 nm to 2600 nm (Table 5).All samples had two or three distribution intervals, except for three samples (1-1/1-2/3-2) distributed at continuous intervals.Figure 4 shows a broken line graph of the particle size distribution of 21 indigo paste samples.Different quality grades are indicated by different colors.As shown in the gure, the indigo paste of each quality level does not have an obvious independent distribution interval, and is randomly distributed across the whole area.

Evidently, there was no correlation between quality level and particle size.


Discussion


Sociocultural characteristics of indigo paste artisans

In this stu

, we recorded the tradi
ional knowledge and experience of 283 people to investigate how they assessed the quality of indigo paste, and then conducted a quantitative analysis to explore the rationality of the quality judgment index used by the people.Here, we must mention that indigo paste played a different role in the different regions, and thus we found some differences across the regions.In Zhenfeng County of Guizhou Province and Xianyou County of Fujian Province, indigo paste was the main source of income for local Han farmers.However, in Congjiang County of Guizhou Province, indigo paste served as a traditional model of national self-su ciency.Both these odels exist in Yuanyang County and Jinping County in Yunnan Province.

The di

roles of indigo paste lead to different social division
of labor.Firstly, considering the gender of the indigo paste artisans, there were more men (81.3%/71.4%)engaged in indigo paste production than women (18.7%/28.6%) in Zhenfeng County of Guizhou Province and Xianyou County of Fujian Province, respectively.However, in the traditional self-su ciency model of Congjiang County in Guizhou Province, only women (100%) were engaged in the production of indigo paste.Men did not understand traditional knowledge relating to indigo paste production (Table 3).Although both production models exist in Yunnan, the scope and quantity of the trade was relativ ly small, which was mainly the traditional model of national self-su ciency.

Therefore, the number of women (88.6%) engaged in indigo paste production was almost eight times that of men (11.4%).These results are consistent with previous ndings.Traditional dyeing knowledge is transmitted matrilineally, and this knowledge is mainly mastered and used by women [43,44].Furt ermore, activities such as dyeing are considered unfavorable to men [45].

However, when indigo paste becomes a tradable commodity and generates economic bene ts, men also become involved in this work [44], even the main labor force will get involved.

Secondly, considering the age of the indigo paste artisans, in the Cong

ang County, young women a
ed 30-49 years (50.4%) were mainly engaged in the production and use of indigo paste, 40.3% were 50-69 years old, and only 9.3% were over 70 years old.In the local area, making indigo paste and dyeing cloth seemed to be the daily work of minority women.In other regions, it was mainly old women aged 50-69 years (Zhenfeng 68.8%/Yunnan 54.3%/Fujian 76.2%), while young women aged 30-49 years (Zhenfeng 28.1%/Yunnan 25.7%/Fujian 9.5%) were relatively rare.Overall, young people aged 30-49 years (35.7%) and middleaged and elderly people aged 50-69 years (52.3%) had extensive knowledge and artisanship of indigo paste production.However, previous published reports indicate that most traditional knowledge was usually held by the elderly, while young people did not understand [9,46].


Quality judgment indices

Generally, the blue hue in indigo paste comes from indigo, and the purple-red luster comes from indirubin [47].Thus, the ratio of the indigo and indirubin content will determine the color of the indigo paste.However, indirubin, which is an isomer of indigo, has always been regarded as a by-product in the production of indigo paste [48,49].Interestingly, in our survey, the people preferred purple-red indigo paste to pure blue indigo paste.In other wor s, the quality judgment indices revealed that the indirubin content was more important than the indigo content.Although these ndings differ from existing synthetic indigo quality judgment standards, they agree with reports of indigo paste quality judgments in ancient Chinese books.For example, there is a record in "Liping Fuzhi"(Guizhou): " ", which means that the purple indigo paste is the best.There is also a description in "Dyeing Sutra": " " [50], which means that the best quality indigo paste should have dark blue and red luster.In addition, the Hainan Li and Miao people think that dark blue and reddish indigo paste is of better quality [46].Therefore, the indirubin content in the indigo paste is not only a by-product, but also has a decisive effect on the quality of the indigo paste.

In the indigo paste production process, many factors affect the quality [8,22,51], one of which is lime.The rst factor is the way in which lime is added.Before adding lime, some folk would put lime into a cloth or gauze bag.The cloth or gauze bag was then rubbed in the soaking liquid to produce a ne lime slurry that owed out of the bag.This method produce very delicate lime particles and greatly reduced the number of impurities in the lime.However, other folk would place the lime in a water scoop or bucket, and a small amount of soaking liquid was added and mixed, and then poured directly into the soaking solution (Fig. 5); thus, they ignored large lime particles and impurities.This might be why folk rubbed their ngers together to check whether the indigo paste was smooth.The second factor is the amount of lime added.In the production process, adding too much or too little lime resulted in

low-quality indigo paste.In addition to the indigo yielding plant
, the taste of indigo paste was mainly related to the amount of lime added during the production process, and the folk judged whether lime was added properly based on the taste.

However, in the interviewing process, the interviewees' descriptions of taste were different and even opposite to one other.There are two possible reasons for this difference: one is that the taste description is mainly in uenced by personal subjectivity, and the other is that interviewees have different perceptions and descriptions of taste due to cultural divergence.This phenomenon also occurred in the use of the color index.Informants described good quality indigo paste as being red in color, even though it was purplish red (Fig. 6).


Veri cation of traditional knowledge by modern scienti c methods

The olk in the study area used indigo and indirubin together to determine the quality of the indigo paste.Compared to pure dark blue color, people preferred indigo paste with purple-red luster.The HPLC-DAD quantitative analysis con rmed that the quality of indigo paste is related to the content of indigo and indirubin.The better the quality the indigo paste, the higher the content of indigo and indirubin, especially indirubin.This con rmed that the color of the indigo paste was a reasonable quality judgment index.However, due to the limitations of the experimental samples, the active ingredients range of the indigo and indirubin components and the color distribution range of different grades of indigo paste cannot be identi ed by this research which need to be further studied.

By drying the samples in the laboratory, we observed that there were no signi cant differences in the color and gloss of indi

paste in i
s wet state, except in the very good and very poor samples.However, the color difference was obvious after drying and removing water (Fig. 7).This indicates a degree of rationality associated with the simultaneous observation of "water color" or "dry color" in Zhenfeng County, and the importance of color in assessing indigo paste.Likewise, after drying different indigo pastes, the inside of the block had a different appearance.Some indigo pastes had a uniform internal color, and no obvious lime particles or impurities were observed.In other indigo pastes, however, there were varying amounts of white or other colored particles (Fig. 7).Thus, from an experimental perspective

the touch index was also necessary
or assessing the quality of the indigo paste.


Conclusion

Although indigo paste has withdrawn from the stage of history, it

still frequently used in parts of China.Th
ee markets and fteen villages in Guizhou, Yunnan and Fujian were selected as the survey sites.Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning t

legal status of any cou
try, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.This map has been provided by the authors.
Figure 1
Three markets and fteen villages in Guizhou, Yunnan and Fujian were selected as the survey sites.Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.This map has been provided by the authors.The average content of the active ingredients in each quality grade of indigo paste.
Figure 3
The averag

content o
the active ingredients in each quality grade of indigo paste.

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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 nal active ingredient content was the average of the three measured data.The standard curves of indigo, indirubin, indole, and indican were established using ve different concentratio gradients.The sample was separated using an Agilent ZORBAX Extend