Quality blues: traditional knowledge used for natural ...

RESEARCH Open Access

Quality blues traditional knowledge usedfor natural indigo identification in southernChinaYuru Shi12 Libin Zhang12 Lu Wang12 Shan Li123 Zuchuan Qiu12 Xiaoyong Ding12 and Yuhua Wang1

Abstract

Background As one of the oldest traditional dyes people worldwide have used natural indigo for centuries Localpeople have unique knowledge about indigo identification which is crucial for indigo quality control anddetermining the dyeing effects However such traditional knowledge is rarely documented and explainedTherefore the aims of this study were to document and assess the traditional knowledge used by local peoplewhen identifying natural indigo paste as well as quantitatively explore the characteristics and material basis of suchtraditional knowledge

Method Three field surveys were conducted between 2019 and 2020 A total of 283 traditional indigo-pasteartisans were interviewed in Guizhou Yunnan and Fujian Provinces The frequency of citation mention index andfidelity level of each indigo-paste quality criterion were calculated to determine the most commonly usedrecognized and important quality criteria To explore the characteristics and material basis of the traditionalknowledge we analyzed 21 indigo-paste samples using high-performance liquid chromatography with diode-arraydetection (HPLC-DAD) pH and particle size analyses

Results Local people possess unique knowledge to identify natural indigo Based on this knowledge accumulatedover thousands of years four criteria (color taste touch and dyeing ability) were chosen by local people and usingthese criteria nature indigo was divided into five quality grades The best quality indigo paste was judgedaccording to the following folk criteria dark blue in color with a purple-red luster smooth and difficult to wipe offhaving a sweet bitter or spicy taste and easy cloth dyeing Additionally the higher the contents of indigo andindirubinmdashespecially indirubinmdashthe better is the quality of the indigo paste Within the pH range of 9ndash12 high-quality indigo-paste was more acidic There was no significant relationship between particle size and quality

Conclusion The ancient methods used by local people for identifying natural indigo are comprehensive andunique By documenting the various folk quality criteria and conducting quantitative analyses this study revealedthe importance of indirubin and pH for assessing the quality of indigo paste These findings differ from existingquality standards for synthetic indigo Amid rapid modernization traditional knowledge remains invaluable as aworld heritage of humanity that warrants preservation

Keywords Ethnobotanical survey Indigo paste Folk quality criteria Quantitative study Indirubin Traditionalknowledge World heritage

copy The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 40 International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original author(s) and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this article are included in the articles Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the articles Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonsorglicensesby40The Creative Commons Public Domain Dedication waiver (httpcreativecommonsorgpublicdomainzero10) applies to thedata made available in this article unless otherwise stated in a credit line to the data

Correspondence wangyuhuamailkibaccn1Department of Economic Plants and Biotechnology Yunnan Key Laboratoryfor Wild Plant Resources Kunming Institute of Botany Chinese Academy ofSciences 132 Lanhei Road Kunming 650201 ChinaFull list of author information is available at the end of the article

Shi et al Journal of Ethnobiology and Ethnomedicine (2021) 1725 httpsdoiorg101186s13002-021-00454-z

BackgroundNatural indigo is considered one of the oldest dyestuffsknown to humanity and one of the most commonlyused natural dyestuffs worldwide particularly in EgyptIndia China and Africa [1 2] It is extracted fromindigo-yielding plant species To date 31 plant speciesacross eight different families have been used as sourcesof indigo (eg Indigofera tinctoria L and I suffruticosaMill) [3] Indigo-yielding plant species are processedusing a variety of methods developed in different re-gions such as sukumo (Persicaria tinctoria [Aiton]Spach) in Japan and woad balls (Isatis tinctoria L) inEurope Indigo paste is traditionally used in China Overthousands of years natural indigo has been integratedinto the cultures of various ethnic groups and used inethnically characteristic ways including tie-dyeing of theBai people batik of the Miao people and in the produc-tion of the bright cloth of the Dong people [4 5] Fur-ther the color of indigo has been endowed with aunique significance and is commonly used as a symbolof independence and individualism whereby it is knownas the king of colors and the color of kings [6] Howeverwith the rise of industrialization synthetic indigo is nowused in almost the entire dyeing industry owing to ad-vantages such as high purity low price and better pro-ducibility [7ndash10] Therefore the production and use ofnatural indigo has declined and gradually disappearedsuch that it currently occurs only in a few remote areasin China India and a few other countries [11 12] How-ever the disadvantages of using synthetic dyes includethe high costs of the raw materials their toxicity andenvironmental pollution [7 8 13ndash17] Therefore in re-cent years consumer interest in natural indigo has beenon the rise which has attracted the attention of dyeingenterprisesIn contrast with synthetic indigo natural indigo is

considered a green dye that has a harmonious and sus-tainable relationship with the environment as it is bio-degradable [11 15 18] In terms of dyeing naturalindigo has many advantages over synthetic indigo Forexample synthetic indigo contains only residual chem-ical impurities apart from the indigo componentwhereas natural indigo is a mixture containing 7ndash45indigo in addition to indirubin dark brown indigo andyellow indigo [1 19] These components give fabricsdyed with natural indigo a richer color and better color-fastness than fabrics dyed with synthetic indigo [1 2021] In addition to its pleasant natural fragrance fabricsdyed with natural indigo have certain health benefits asthey show insect-repellent and disinfectant properties[18 22]Unfortunately the quality of natural indigo currently

found in the market varies considerably and to datethere is no reliable method for evaluating the quality of

this natural dye However indigo identification signifi-cantly influences indigo quality control and the deter-mination of its dyeing effects [23ndash26] While numerousstudies on indigo-yielding plant species and traditionalindigo extraction and dyeing methods have been con-ducted very little is known about the folk criteria usedfor indigo identificationPreviously we found that people still cultivate use

and trade natural indigo (as indigo paste) on a largescale in parts of Guizhou Yunnan and Fujian Provincesin southern China Further we have learned that trad-itional knowledge about indigo paste is passed on fromgeneration to generation and that local people separateindigo paste into different quality grades using folk cri-teria However such traditional knowledge has rarelybeen documented and explained In light of this thepresent study aimed to document and assess the trad-itional knowledge of the local people in identifying thenatural indigo paste Further it sought to as well asquantitatively explore the characteristics and materialbasis of such traditional knowledge

MethodsStudy sitesInformation provided by the local governments togetherwith a preliminary survey played a decisive role inselecting the research sites We identified 15 villages and3 markets in Guizhou Yunnan and Fujian Provinces assuitable survey sites because people in these areas fre-quently used indigo paste and had a good heritage oftraditional knowledge Guizhou and Yunnan Provincesare located in Southwest China whereas Fujian Provinceis located in Southeast China (Fig 1)Congjiang County (25deg16primendash26deg05prime N 108deg05primendash109deg12prime

E) and Zhenfeng County (25deg07primendash25deg44prime N 105deg25primendash105deg56prime E) [27] belong to the Qiandongnan Miao andDong Autonomous Prefecture and Qianxinan Buyi andMiao Autonomous Prefecture respectively CongjiangCounty is located in the middle reaches of the DuliuRiver The climate is classified as humid monsoon of themid-subtropical zone The annual average temperatureand precipitation are 185 degC and 11859 mm respect-ively Ninety-five percent of the population in CongjiangCounty is composed of ethnic minorities such as theMiao and Dong peoples [28] providing an ideal environ-ment for this study because of their strong cultural in-heritance practices The six villages surveyed inCongjiang County were located in mountainous areas onboth sides of the Duliu River and the residents were allMiao people Currently the local area retains a strongindigo-paste culture and the daily dress of Miao womenis still the traditional national costume Every family inthe local area cultivates the indigo-yielding plant Strobi-lanthes cusia (Nees) Kuntze on an annual basis to

Shi et al Journal of Ethnobiology and Ethnomedicine (2021) 1725 Page 2 of 14

prepare indigo paste for dyeing cloth Zhenfeng Countyhas a subtropical humid monsoon climate with an an-nual average temperature and precipitation of 166 degCand 12769 mm respectively [27] In Zhenfeng Countyindigo paste is traded as a commodity and is thus pro-duced on a large scale The local trade in indigo pastetakes place on market day at the farmers market in eachtown Sellers are mostly Han residents from nearby vil-lages and buyers are local ethnic minorities and mer-chants In Zhenfeng County we conducted surveys atthe two main farmers markets and in the three villagesthat produce indigo pasteYuanyang County (22deg49primendash23deg19prime N 102deg27primendash103deg13prime

E) and Jinping County are located in the Honghe Haniand Yi Autonomous Prefecture of Yunnan ProvinceYuanyang County has a subtropical mountain monsoonclimate with an annual average temperature and rainfallof 244 degC and 8995 mm respectively Yuanyang Countyis inhabited by seven ethnic groups including the HaniYi and Han peoples who have lived in this area for gen-erations Ethnic minorities account for 8944 of thetotal population [29] Jinping County has a tropicalmonsoon climate with an annual average temperatureand rainfall of 18 degC and 23586 mm respectivelyNine ethnic groups including the Miao and Yao peo-ples have lived in the area for generations and ethnicminorities account for 876 of the total population[30] In the four villages surveyed in the YunnanProvince (one Yao and three Hani villages) the eld-erly retain and practice traditional natural-indigo cul-ture and artisanship

Xianyou County (25deg11primendash25deg43prime N 118deg27primendash118deg56primeE) is located approximately halfway along the coastlineof Fujian Province across the island of Taiwan The cli-mate is classified as south subtropical maritime mon-soon with an annual average temperature of 206 degC andtotal rainfall ranging from 300 to 2300 mm In thiscounty the town of Shufeng is reputedly the Hometownof Indigo Naturalis because of its long history of makinghigh-quality Indigo Naturalis [31] the powder processedfrom indigo paste Its active ingredients are indigo andindirubin and it is used medicinally to treat oral ulcers[32] ulcerative colitis [33] and psoriasis [34ndash36]

Field survey and data collectionThe first field survey was conducted between Augustand September 2019 over approximately 10 days in eachof Congjiang and Zhenfeng Counties of Guizhou Prov-ince YRS ZCQ and XYD conducted the field surveysand collected the ethnobotanical data and voucher sam-ples The second field survey was conducted over 7 daysin October 2019 in Xianyou County of Fujian ProvinceYRS and LW carried out the field surveys and collectedthe ethnobotanical data The third field survey was con-ducted over 6 days in January 2020 in Yuanyang andJinping Counties of Yunnan Province YRS LBZ andLW carried out the field surveys and collected theethnobotanical data During the field investigation weinvited local people who could speak the local languageand Mandarin to serve as interpreters Methods used fordata collection included purposive sampling [37] snow-ball sampling [38] participatory observation and a

Fig 1 Three markets and 15 villages in Guizhou Yunnan and Fujian were selected as the survey sites

questionnaire survey [39] The interview questionnaire isshown in Table 1 All interviewees possessed traditionalknowledge related to indigo paste Informed consentfrom all interviewees was obtained orally before con-ducting the interviews Once permission had been ob-tained we captured photographs [40] created audio andvideo recordings and collected other materials to assistour researchAs shown in Table 2 a total of 283 informants were

interviewed including 171 from Guizhou Province (139from Congjiang County and 32 from Zhenfeng County)42 from Fujian Province and 70 from Yunnan ProvinceThe age of the interviewees ranged between 31 and 81years with 880 ranging between 30 and 69 years Thenumber of female interviewees (n = 219) was almost 35times that of the male interviewees (n = 64)

Quantitative analysis of the ethnobotanical dataTo screen out the most commonly used most recog-nized and most important quality criteria we used ques-tions 5 6 and 7 (Table 1) to calculate the Frequency ofCitation (FC) Mention Index (QI) [41] and FidelityLevel (FL) [42] of each quality criterion respectivelyThe number of interviewees using each quality criterionwas counted as the FC for the criterion QI used to testknowledge homogeneity was calculated using the fol-lowing formula QI = number of mentionsnumber ofinterviewees In turn FL used to evaluate the import-ance of the different quality criteria was calculated usingthe following formula FL = (total number of inter-viewees providing one quality citationtotal number ofinterviewees providing all quality criteria) times 100

Chemical analysisTo avoid sample interference we obtained 21 indigo-paste samples (all extracted from Strobilanthes cusia)from Guizhou Province which had the largest numberof indigo-paste users among all study sites These 21indigo-paste samples were identified and categorized by3 key informants using 5 quality levels best (3 samples)good (3 samples) general (7 samples) poor (5 samples)and worst (3 samples) We used the values 1 2 3 4 and5 to represent the 5 quality grades of indigo pasterespectively

An Agilent 1260 series equipment (Agilent Technolo-gies USA) was used to quantitatively analyze the activeingredients (ie indigo and indirubin) present in theindigo-paste samples [3] Ground indigo powder samples(05 g measured to 00004 g accuracy) were ultrasoni-cated for 30 min to completely disperse the dye in 50 mlof distilled water at pH 7 [43] A PHS-3C acidity meterand E-201-C composite electrode (Shanghai INESA Sci-entific Instrument Co Ltd Shanghai China) were usedto measure the pH of the 21 indigo-paste samples Anindigo-paste suspension (01 gL) was prepared usingdistilled water and the upper part of the suspension waswithdrawn for particle size analysis after ultrasonic

Table 1 Questionnaire for the interviewees

1 How many indigo-yielding plant species do you 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 2 Sex and age of the interviewees

Number Percentage

Male 64 226

Female 219 774

30ndash49 101 357

50ndash69 148 523

ge 70 34 120

Guizhou

Male 26 (C0 Z26) 152 (C0 Z813)

Female 145 (C139 Z6) 848 (C100 Z187)

30ndash49 79 (C70 Z9) 462 (C504 Z281)

50ndash69 78 (C56 Z22) 456 (C403 Z688)

ge 70 14 (C13 Z1) 82 (C93 Z31)

Yunnan

Male 8 114

Female 62 886

30ndash49 18 257

50ndash69 38 543

ge 70 14 200

Fujian

Male 30 714

Female 12 286

30ndash49 4 95

50ndash69 32 762

ge 70 6 143

Note C Congjiang County Z Zhenfeng County

dispersion for 10 min A Malvern Zetasizer ZEN 3600zeta potential analyzer (Malvern Instruments Ltd Mal-vern UK) was used for particle size testing [44]

Statistical analysisAnalysis of variance (ANOVA) was conducted to deter-mine any significant effects of active ingredient contentand pH (p le 005) on the quality grade of the indigo-paste samples Differences in particle size of the differentquality grades of indigo paste were analyzed using theOrigin Pro learning edition data-analysis software toproduce a line graph of the particle size distribution ofthe 21 indigo-paste samples for comparison

ResultsTraditional knowledge used for indigo identificationAs shown in Table 3 five quality grades and four qualitycriteria for indigo paste were documented The five qual-ity grades were best good general poor and worstwhile the four quality criteria were color taste touchand dyeing ability (Fig 2) All four of these criteria wereused in Yunnan and Guizhou Provinces whereas onlycolor and touch were used in Fujian ProvinceIn Congjiang County of Guizhou Province and Yun-

nan Province people grade indigo paste only in its wetstate and they believe that high-quality indigo pasteshould have dark-blue hue and purple-red metallic luster(Fig 2a) However in Zhenfeng County of GuizhouProvince people also assessed the indigo paste in its drystate by applying wet indigo paste to a small area on theback of their hands or on their arms (Fig 2b) and leav-ing it to dry naturally Thus they combine their assess-ment of the colors of the indigo paste in its wet and drystates to determine its quality believing that high-qualityindigo paste should appear dark-blue and purple-redwhen both wet and dry In Zhenfeng County these as-sessments are known as water color and dry color re-spectively Because of the inconvenience of transportingwet indigo people dry indigo in the sun in XianyouCounty of Fujian Province and they assess the quality by

observing only the color of the dried indigo blocks (Fig2c)People in all the regions under study used the touch

criterion to evaluate indigo quality which involves rub-bing a small amount of moist indigo paste between theindex finger and thumb (Fig 2d) Indigo paste that issmooth and difficult to wipe off is considered of highquality Noticeable graininess indicates slightly lowqualityIn Guizhou and Yunnan Provinces local people think

that high-quality indigo paste has a sweet (gai) spicy(dou) or bitter (aelig) taste and dyes fabrics easily (Fig 2ef) However notably there were differences in the de-scription people provided for the taste criterion and itwas more common in Guizhou Province Although morethan 25 of the informants in Guizhou used this criter-ion different informants offered contrasting descriptionsfor taste Some informants declared that indigo paste isof good quality when it has a sweet taste whereas a spicyor bitter taste indicates poor quality Other informantsheld the opposite view

Quantitative evaluation of the quality criteriaThe color criterion showed the highest FC value (FC =283) QI value (QI = 1) and FL value (100) in all studyareas In contrast to the other criteria the color criterionwas the most commonly used and recognized criterionamong people (Table 3) Although the touch criterionwas used in all study areas its frequency and importancediffered across regions the highest QI and FL valueswere determined in Fujian Province (QI = 021 FL =21) followed by Guizhou Province (QI = 012 FL =12) while Yunnan Province showed the lowest values(QI = 007 FL = 7) The taste and dyeing-ability cri-teria were used only in Guizhou and Yunnan Provincesand there were differences in the frequency and import-ance of these two quality criteria in the two regions InGuizhou Province the taste criterion (FC = 47) wasmore frequently used than the dyeing-ability criterion(FC = 11) whereas people used the dyeing ability criter-ion (FC = 3) more than the taste criterion (FC = 2) in

Table 3 Quality criteria used by folk to assess indigo paste

Criteria Folk quality levels Guizhou Yunnan Fujian

1 2 3 4 5 FCQIFL FCQIFL FCQIFL

Color Dark blue deeppurple-red

Dark bluereddish

Blue Blue-blackblack

Light blue bluish greyturquoise

171100100

70100100

42100100

Touch Exquisite andsmooth

Exquisite Slight granularsensation

Granularsensation

Obvious granularsensation

2001212

50077 902121

Taste ndash ndash ndash ndash ndash 4702727

20033 00000

Dyeingability

Easy Easy General Difficult Hard 110066 30044 00000

Note ndash meaning that the folk description of this criterion in disagreement

Yunnan Province although the quantitative analysisshowed a small difference in the FC value of these twocriteria in this province The QI values of the taste anddyeing-ability criteria in Guizhou Province were 027and 006 respectively and the FL values were 27 and6 respectively However in Yunnan Province the QIvalues of the two criteria were 003 and 004 respect-ively and the FL values were 3 and 4 respectivelyThese findings indicated that the taste and dyeing-abilitycriteria were more important in Guizhou Province thanthose in Yunnan Province and were more frequentlyused by people in Guizhou Province especially the tastecriterionOverall the quantitative analysis revealed that the

most important and recognized evaluation criterionamong informants was color The other three criteria(taste touch and dyeing ability) appear to be accessorycriteria

Verification of traditional knowledge using modernscientific methodsThe main active ingredients identified in the indigo-paste samples were indigo and indirubin In additionsome samples contained minute amounts of indican orindole (Table 4) As the indigo paste contained waterwhen it was sampled we also considered water as a fac-tor Our findings showed that the average indigo andindirubin content decreased with a decrease in qualitygrade (Fig 3) Furthermore there was a positive correl-ation between the active ingredient content and qualitygrade of the indigo paste One-way ANOVA showed

that whether wet or dry there were significant differ-ences in active ingredient content among the differentquality gradesRegarding the pH of the 21 indigo-paste samples the

minimum maximum and average values were 9101164 and 1067 respectively (Table 4) We identified arelationship between pH and quality grade the low-quality indigo-paste samples tended to have high pHvalues and vice versa One-way ANOVA showed thatthere were significant differences in indigo paste pHamong different quality grades (P = 0000) Within a cer-tain range (9 le pH le 12) the pH value of the high-quality indigo paste was relatively low whereas the pHvalue of the poor-quality indigo paste was relativelyhighThe particle size distribution of the indigo-paste sam-

ples ranged from 7882 to 5560 nm with the particle sizeof most samples ranging between 200 and 2600 nm(Table 4) All samples had two or three distribution in-tervals except for three samples (1-11-23-2) whichwere distributed at continuous intervals As indicated inFig 4 the indigo-paste samples in each quality grade didnot have an obvious independent distribution intervaland were randomly distributed across the particle sizerange Evidently there was no correlation between qual-ity grade and particle size

DiscussionSociocultural characteristics of indigo-paste artisansWe documented the traditional knowledge and experi-ence of 283 informants to analyze how they assessed the

Fig 2 Different folk quality criteria andashc Method of color criterion d Method of touch criterion e Method of taste criterion f Method of dyeingability criterion1ndash5 represent the five folk quality grades of indigo paste which are best good general poor and worst respectively

quality of indigo paste As a commodity indigo pasteplays different roles in different regions In ZhenfengCounty of Guizhou Province and Xianyou County of Fu-jian Province the indigo-paste trade provides the mainsource of income for the local Han farmers In contrastin Congjiang County of Guizhou Province the indigo-paste trade serves as a traditional model of national self-sufficiency Both these models exist in Yuanyang andJinping Counties of Yunnan Province

Division of labor sex of indigo-paste artisansThe different social roles of indigo paste lead to differentsocial divisions of labor With regard to the sex ofindigo-paste artisans there were more men (813) en-gaged in indigo-paste production than women (187) inZhenfeng County of Guizhou Province as well as in Xia-nyou County of Fujian Province (714 men versus286 women) However in Congjiang County in

Guizhou Province only women were engaged in theproduction of indigo paste following the traditional self-sufficiency model (Table 2) Although both productionmodels exist in Yunnan Province at the time of our sur-vey the scope and quantity of the trade was relativelysmall and based mainly on the traditional model of na-tional self-sufficiency consequently the number ofwomen (886) engaged in indigo-paste production wasalmost eight times the number of men (114) Theseresults are consistent with the findings of previous stud-ies which reported that traditional dyeing knowledge istransmitted matrilineally and dyeing is mainly masteredand performed by women [45 46] Furthermore activ-ities such as dyeing are considered inappropriate formen [47] However when indigo paste becomes a trad-able commodity and generates economic benefits menreadily become involved in this work [46] indeed theymay even participate as the main labor force

Table 4 Active ingredients pH and particle size of indigo-paste samples from Guizhou Province

Samplenumber

Qualitygrade

Effectivecomponentscontent(ugg)

Percentage of effective ingredients () PH Particle size(d=nm)

Indigo Indirubin Indigo(W)

Indirubin(W)

Indigo(D)

Indirubin(D)

1-1 1 1126855 1022173 037 033 113 102 910 5312ndash825 (100)

1-2 1 1448660 544157 041 015 145 054 947 3961ndash825 (100)

1-3 1 1321867 409602 057 018 132 041 913 9128ndash1281 (874)4145ndash5560 (126)

2-1 2 1046689 317417 021 006 105 032 1003 342ndash825 (909)4801ndash5560 (91)

2-2 2 1058321 418960 041 016 106 042 918 9128ndash1642 (127)3961ndash1106 (873)

2-3 2 1059091 436699 028 012 106 044 1092 1224ndash255 (172)6151minus2669 (828)

3-1 3 1016460 290367 023 007 102 029 1098 1418ndash255(19)5312ndash1718 (81)

3-2 3 884008 235728 020 005 088 024 1083 2202ndash7124 (100)

3-3 3 1067238 202268 011 002 107 020 1066 1057ndash1901 (236)5312ndash1106 (764)

3-4 3 1043008 192120 032 006 104 019 1021 1418ndash255 (297)825ndash2305 (703)

3-5 3 1083298 119315 046 005 108 012 1155 2202ndash9554 (96)4145ndash5560 (4)

3-6 3 1045734 206558 026 005 105 021 1085 9128ndash1642 (174)342ndash825 (826)

3-7 3 1009703 147028 025 004 101 015 1129 7882ndash1418 (132)2202ndash9554 (868)

4-1 4 797697 181884 016 004 080 018 1106 9128ndash1418 (85)4587ndash1106 (889)5560 (27)

4-2 4 923530 113466 037 005 092 011 1039 1224ndash2202 (215)5312ndash1718 (766)5560 (19)

4-3 4 1033490 138650 018 002 103 014 1136 1224ndash2202 (301)5312ndash1281 (672)5560 (28)

4-4 4 925113 148047 020 003 093 015 1134 7882ndash1642 (132)255ndash7124 (868)

4-5 4 969115 131695 029 004 097 013 1153 9128ndash825 (844)3580ndash5560 (156)

5-1 5 783444 110313 010 001 078 011 1122 7882ndash1057 (74)3961ndash9554 (926)

5-2 5 810615 106289 014 002 081 011 1164 9128ndash1418 (75)2953ndash825 (80)4145ndash5560 (125)

5-3 5 1036998 89461 026 002 104 009 1143 1418ndash2953 (249)7124ndash1484 (751)

Significance is indicated by P lt 005 P lt 0001Note W wet weight D dry weight

Division of labor age of indigo-paste artisansRegarding the age of the indigo-paste artisans in Con-gjiang County mainly young women aged 30ndash49 years(504) were engaged in the production and use of in-digo paste 403 were 50ndash69 years old and only 93

were over 70 years old In the local area making indigopaste and dyeing cloth seemed to be the daily work ofminority women In other regions the indigo-paste arti-sans were mainly elderly women aged 50ndash69 years(688 in Zhenfeng 543 in Yunnan and 762 in

Fig 3 The average active ingredient content in each quality grade of indigo paste is shown where different colors represent differentquality grades

Fig 4 A broken line graph of the particle size distribution of 21 indigo-paste samples Different quality grades are indicated by different colors

Fujian) whereas young women aged 30ndash49 years wererelatively rarely involved in indigo-paste production(281 in Zhenfeng 257 in Yunnan and 95 in Fu-jian) Overall young people aged 30ndash49 years (357)and middle-aged and elderly people aged 50ndash69 years(523) showed extensive knowledge and artisanship ofindigo-paste production However previous studies indi-cate that the majority of traditional knowledge is usuallyheld by the elderly whereas young women do not proveto be very knowledgeable about the trade [12 48]

Characteristics and material basis of traditionalknowledge about indigo pasteColor and active ingredientsBy drying the indigo-paste samples in the laboratory weobserved that there were no significant differences incolor or gloss of indigo paste in its wet state except inthe very high- and very low-quality samples Howevercolor differences were noticeable after drying (Fig 5)This indicates a degree of rationality associated with thesimultaneous observation of water color and dry coloramong the people of Zhenfeng County in addition tothe importance of color in assessing indigo-paste qualityOur survey revealed that locals believe a high-quality in-digo paste should have dark-blue hue and purple-redmetallic luster Generally the blue hue in indigo paste isdue to its indigo content whereas its indirubin contentproduces the purple-red luster [49] Thus the ratio ofthe indigo to indirubin content determines the color ofthe indigo paste The HPLC-DAD quantitative analysisconfirmed that the quality of indigo paste was related tothe content of indigo and indirubin The higher the con-tent of indigo and indirubinmdashespecially indirubinmdashthebetter is the quality of the indigo paste All these find-ings confirmed that the color of the indigo paste was anadequate quality criterion However due to the limitednumber of experimental samples the range of indigoand indirubin contents as well as the color distributionrange of the different quality grades of indigo pastecould not be identified in this study Determining theseranges requires further research

Taste and pHIn addition to taste variation among indigo-yieldingplants the taste of indigo paste is strongly related to theamount of lime added during the production processwhere adding too much or too little lime results in alow-quality indigo paste As different alkalinity stimu-lates human taste buds differently local people evaluatewhether the correct amount of lime has been addedbased on a simple taste sensation thus judging the qual-ity of the indigo paste However our survey data re-vealed that the taste descriptions provided by theinformants differed and were even contradictory There

are two possible reasons for these differences one is thattaste description is mainly influenced by personal sub-jectivity and the other is that informants have differentperceptions and descriptions of taste due to cultural di-vergence This phenomenon also occurred with respectto the use of the color criterion For example somepeople might describe high-quality indigo paste as beingred in color despite an apparent purplish-red luster (Fig6)

Touch and particle sizeThe particle size quantitative analysis confirmed thatthere was no correlation between quality grade and par-ticle size However after drying different indigo pasteswe noted differences in appearance of the inner parts ofthe blocks Some indigo-paste blocks had a uniforminternal color no lime particles or impurities wereobserved In contrast there were varying amounts ofwhite or other colored particles in other indigo-pasteblocks (Fig 5) This observation may be related tothe way in which lime is added in the process ofindigo-paste production Some informants describedhow they place lime in a cloth or gauze bag beforeadding it They then rub the bag in the soaking liquidto produce a fine lime slurry that flows out of thebag (Fig 7andashc) This method results in very minutelime particles and a markedly reduced number of im-purities in the lime slurry However other informantsdescribed how they place the lime in a water scoopor bucket add a small amount of soaking liquid mixand then pour this directly into the soaking solution(Fig 7dndashi) This approach ultimately results in the in-clusion of large lime particles and impurities in theindigo paste Given these different approaches to add-ing lime and their potential impact on the final prod-uct the touch criterion is necessary for theassessment of indigo-paste quality The results of theparticle size analysis may have been influenced by thelarge particles in the indigo paste settling in the in-strument and consequently not being recorded Thispotential problem needs to be resolved in future re-search on indigo paste properties

Cultural standards and industrial standards of indigodyestuffThe chemical industry standard of the Peoplersquos Re-public of China stipulates five criteria used to judgethe quality of synthetic indigo These are as followsappearance (dark blue uniform powder or granule)mass fraction of indigo (ge 93) mass fraction ofmoisture (le 10) mass fraction of fineness (le 50beyond 250 μm) residue in the sieve (le 50) andiron content (le 500 mgkg) Although the target ob-jects are different the industrial standards of

appearance mass fraction of indigo and mass frac-tion of fineness were similar to the color and touchcriteria in the cultural standards These industrialand cultural standards are all regulations based oncolor active ingredient contents and particle size

However as organoleptic selection criteria the folkquality criteria for judging indigo paste have aunique cultural imprintFor the indigenous people sensory evaluation based

on odor taste sight and touch plays a critical role in

Fig 5 The status of indigo paste of different quality grades after dryinga b d f Different colors of the indigo paste after drying gndashh Afterdrying the lime particles and impurities in indigo paste can be observed

plant classification and traditional medicine therapy[50ndash53] Such traditional knowledge is a summary of theunderstanding of local people about their surroundings[54] Such knowledge may be independent of modernscientific understanding and not rely on modern scien-tific testing methods For example our survey indicatedthat local people preferred purple-red indigo paste topure blue indigo paste suggesting that in application of

the cultural quality criteria indirubin was judged to bemore important than indigo However in the context ofindustrial dyestuff production indirubin has always beenregarded as a by-product [55 56] This difference maybe related to cultural differences in the same way thatlinks between taste perceptions and medicinal uses ofherbal drugs can be markedly different across diversecultures [57] The color criterion we documented

Fig 6 Different colors of the wetting indigo paste

Fig 7 The way in which lime is added in the process of indigo-paste production andashc put lime into a cloth or gauze bag dndashi Put lime in a waterscoop or bucket

corresponds with the reports of indigo paste quality as-sessment in ancient Chinese books For example thereis a document in Liping Fuzhi(Guizhou) that containsthe following statement ldquo投入生石灰则满地颜色皆收

入灰内以带紫色者为上rdquo which implies that purple in-digo paste is the best Similarly there is a description inDyeing Sutra ldquo明兰宝翠兹浆鲜红至次年春夏可变成

熟红如天青缎rdquo [58] which implies that the best qualityindigo-paste should have a dark blue and red luster Inaddition the Hainan Li and Miao people think that adark blue and reddish indigo paste is of a better quality[48] The Yao Zhuang Dong and other ethnic minor-ities like to dye their fabrics dark blue or black with ared color [59 60] and the presence of indirubin can sat-isfy such preferences The medicinal benefits of indiru-bin help to explain why people in Xianyou County favorthe existence of this substance Therefore in a culturalcontext indirubin in indigo paste is not considered a by-product but a critical determinant of indigo-pastequality

ConclusionsAlthough modernization and urbanization continue tochange the traditional ways in which people producegoods some local people still maintain the traditionalculture and methods used for indigo extraction andindigo-paste preparation This study documented fourfolk criteria and five quality grades of indigo paste andrevealed the importance of indirubin and pH for asses-sing the quality through quantitative analyses Even afterthousands of years the ancient methods used by thelocal people for identifying natural indigo remain com-prehensive and unique The traditional method forindigo-paste quality assessment is seemingly backwardbut it is advantageous not only in its simplicity and easeof use but also in its environment-friendliness and highenergy efficiency Simple traditional knowledge can alsoinspire the development of modern industrial technol-ogy possibly the invention of modern detection equip-ment and the exploitation of novel blue dyesTraditional knowledge remains an invaluable culturalheritage of humanity that we need to actively preserveand transmit to new generationsAcknowledgementsWe are most grateful to all interviewee for their hospi-

tality and willingness to share their traditional know-ledge with us We thank Professor Wenyun Chen YuZhang Yi Gou and Ruyan Fan for their assistance

AbbreviationsFC Frequency of citation QI Mention Index FL Fidelity level

Authorsrsquo contributionsYRS and YHW conceived and designed the research YRS LBZ LW ZCQ andXYD carried out the field surveys collected ethnobotanical data and vouchersamples YRS completed the verification experiments and analyzed the data

and then prepared the manuscript with assistance from SL and LBZ YRSLBZ LW ZCQ and XYD took the photographs and YHW reviewed themanuscript All authors read and approved the final manuscript

FundingThis study was supported by the Strategic Priority Research Program ofChinese Academy of Sciences (nos XDA20050204 XDA19050301 andXDA19050303) National Natural Science Foundation of China (32000261)and the Biodiversity Survey and Assessment Project of the Ministry ofEcology and Environment China (No 2019HJ2096001006)

Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable request

Declarations

Ethics approval and consent to participateThe present study is purely based on filled survey instead of human oranimal trials Ethical guidelines of the International Society of Ethnobiology(httpwwwethnobiologynet) were strictly followed Permissions wereverbally informed by all participants in this study

Consent for publicationThe people interviewed were informed about the studyrsquos objectives and theeventual publication of the information gathered and they were assuredthat the informantsrsquo identities would remain undisclosed Moreover theportraits we used have been agreed by the owner

Competing interestsThe authors declare that they have no competing interests

Author details1Department of Economic Plants and Biotechnology Yunnan Key Laboratoryfor Wild Plant Resources Kunming Institute of Botany Chinese Academy ofSciences 132 Lanhei Road Kunming 650201 China 2University of ChineseAcademy of Sciences Beijing 100049 China 3Key Laboratory for MicrobialResources of the Ministry of Education Yunnan Institute of MicrobiologySchool of Life Sciences Yunnan University Kunming 650091 China

Received 2 December 2020 Accepted 25 March 2021

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3 Xu W Zhang L Cunningham AB Li S Zhuang H Wang Y Blue genomechromosome-scale genome reveals the evolutionary and molecular basis ofindigo biosynthesis in Strobilanthes cusia Plant J 2020104(4)864ndash79httpsdoiorg101111tpj14992 Blue genome chromosome‐scale genomereveals the evolutionary and molecular basis of indigo biosynthesisinStrobilanthes cusia

4 Guo J Between tradition and modernity an art anthropologicalinvestigation of the current situation of Bai peoples tie-dyeing inZhoucheng village Dali Yunnan Ethn Art Stud 201932(5)140ndash50 httpsdoiorg1014003jcnkimzysyj20190515

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6 Błyskal B Indigo dyeing and microorganismndashpolymer interaction JCultHeritage 201622974ndash83 httpsdoiorg101016jculher201605006

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10 Zarkogianni M Mikropoulou E Varella E Tsatsaroni E Colour and fastness ofnatural dyes revival of traditional dyeing techniques Color Technol 2010127(1)18ndash27 httpsdoiorg101111j1478-4408201000273x

11 Dutta S Roychoudhary S Sarangi BK Effect of different physico-chemicalparameters for natural indigo production during fermentation of Indigofera plantbiomass 3 Biotech 20177(5)322 httpsdoiorg101007s13205-017-0923-2

12 Li S Cunningham AB Fan R Wang Y Identity blues the ethnobotany of theindigo dyeing by Landian Yao (Iu Mien) in Yunnan Southwest China JEthnobiol Ethnomed 201915(1)13 httpsdoiorg101186s13002-019-0289-0

13 Hartl A Vogl CR The potential use of organically grown dye plants in theorganic textile industry experiences and results on cultivation and yields ofdyers chamomile (Anthemis tinctoria L) dyers knotweed (Polygonumtinctorium Ait) and weld (Reseda luteola L) J Sustain Agric 200323(2)17ndash40 httpsdoiorg101300J064v23n02_04

14 Tayade PB Adivarekar RV Extraction of indigo dye from Couroupitaguianensisand its application on cotton fabric Fash and Text 20141(1)16httpsdoiorg101186s40691-014-0016-3

15 Hill DJ Is there a future for natural dyes Color Technol 199727(1)18ndash25httpsdoiorg101111j1478-44081997tb03771x

16 Gilbert KG Cooke DT Dyes from plants past usage present understandingand potential Plant Growth Regul 200134(1)57ndash69 httpsdoiorg101023A1013374618870

17 Hossain MD Khan MMR Uddin MZ Fastness properties and color analysisof natural indigo dye and compatibility study of different natural reducingagents J Polym Environ 201725(4)1219ndash30 httpsdoiorg101007s10924-016-0900-6

18 Shen G Yang C Zhang D Research and development of nature colorant(dyestuff) Dyest Color 200946(1)7ndash10 httpsdoiorg103969jissn1672-1179200901002

19 Chanayath N Lhieochaiphant S Phutrakul S Pigment extraction techniquesfrom the leaves of Indigofera tinctoria Linn and Baphicacanthus cusia Bremand chemical structure analysis of their major components Ecol Econ 20021(2)149ndash60

20 Miyoko K Ryoko Y Characteristics of color produced by awa natural indigoand synthetic indigo Mater 20092(2)661ndash73 httpsdoiorg103390ma2020661

21 Miyoko K Urakawa H Mitsuo U Kanji K Color in cloth dyed with naturalindigo and synthetic indigo Fiber 200258(4)122ndash8 httpsdoiorg102115fiber58122

22 Sandoval-Salas F Gschaedler-Mathis A Vilarem G Meacutendez-Carreto C Effectof harvest time on dye production in Indigofera suffruticosa MillAgrociencia 200640(5)585ndash91 httpsdoiorg101016jagee200602012

23 Stoker KG Cooke DT Hill DJ An improved method for the large-scaleprocessing of Woad (Isatis tinctoria) for possible commercial production ofWoad indigo J Agric Eng Res 199871(4)315ndash20 httpsdoiorg101006jaer19980329

24 Perkin F The present condition of the indigo industry Nature 190063(1630)7ndash9 httpsdoiorg101038063302a0

25 Bechtold T Turcanu A Geissler S Ganglberger E Process balance andproduct quality in the production of natural indigo from Polygonumtinctorium Ait applying low-technology methods Bioresour Technol 200281(3)171ndash7 httpsdoiorg101016S0960-8524(01)00146-8

26 Vuorema A John P Keskitalo M Marken F Electrochemical determination ofplant-derived leuco-indigo after chemical reduction by glucose J ApplElectrochem 200838(12)1683ndash90 httpsdoiorg101007s10800-008-9617-0

27 Zhenfeng County peoples government network httpwwwgzzfgovcnyzzf Accessed 13 October 2020

28 Congjiang County peoples government network httpwwwcongjianggovcnzjcj Accessed 13 October 2020

29 Yuanyang County peoples government network httpwwwyyhhgovcnmlyyyygk202009t20200930_473064html Accessed 13 October 2020

30 Jinping County peoples government network httpwwwjphhgovcnbcjpjpgk201909t20190918_365436html Accessed 13 October 2020

31 Xianyou County peoples government network httpwwwxianyougovcnxygk Accessed 13 October 2020

32 Zhao X He X Zhong X Anti-inflammatory and in-vitro antibacterialactivities of traditional Chinese medicine formula Qingdaisan BMC

Complement Altern Med 201616(1)503 httpsdoiorg101186s12906-016-1475-4

33 Suzuki H Kaneko T Mizokami Y Narasaka T Endo S Matsui H et alTherapeutic efficacy of the Qing Dai in patients with intractable ulcerativecolitis World J Gastroenterol 201319(17)2718ndash22 httpsdoiorg103748wjgv19i172718

34 Li J Wang Z Xie Y Zhao W Clinical characteristics and combined use ofmedicine analysis of 2991 hospitalized patients with psoriasis based on realworld database China J Chin mater med 201439(18)3442ndash7 httpsdoiorg104268cjcmm20141806

35 Lin YK See LC Huang YH Chang YC Tsou TC Lin TY et al Efficacy andsafety of indigo naturalis extract in oil (Lindioil) in treating nail psoriasis arandomized observer-blind vehicle-controlled trial Phytomedicine 201421(7)1015ndash20 httpsdoiorg101016jphymed201402013

36 Lin YK Chang YC Hui RC See LC Chang CJ Yang CH et al A Chinese herbindigo naturalis extracted in oil (Lindioil) used topically to treat psoriaticnails a randomized clinical trial JAMA Dermatol 2015151(6)672ndash4 httpsdoiorg101001jamadermatol20145460

37 Almeida CDFCBRD Albuquerque UPD Uso e conservao de plantas eanimais medicinais no Estado de Pernambuco (Nordeste do Brasil) umestudo de caso Interciencia 200227(6)276ndash85

38 Biernacki P Waldorf D Snowball sampling problems and techniques ofchain referral sampling Sociol Methods Res 198110(2)141ndash63 httpsdoiorg101177004912418101000205

39 Devkota S Chaudhary RP Werth S Scheidegger C Indigenous knowledgeand use of lichens by the lichenophilic communities of the Nepal HimalayaJ Ethnobiol Ethnomed 201713(1)15 httpsdoiorg101186s13002-017-0142-2

40 Thomas E Vandebroek I Van Damme P What works in the field Acomparison of different interviewing methods in ethnobotany with specialreference to the use of photographs Econ Bot 200761(4)376ndash84 httpsdoiorg1016630013-0001(2007)615B376WWITFA5D20CO2

41 Liu Y Liu Q Li P Xing D Hu H Li L et al Plants traditionally used to makeCantonese slow-cooked soup in China J Ethnobiol Ethnomed 201814(1)4httpsdoiorg101186s13002-018-0206-y

42 Friedman J Yaniv Z Dafni A Palewitch D A preliminary classification of thehealing potential of medicinal plants based on a rational analysis of anethnopharmacological field survey among Bedouins in the Negev DesertIsrael J Ethnopharmacol 198616(2)275ndash87 httpsdoiorg1010160378-8741(86)90094-2

43 China national standardization administration committee Dyes-Determination of pH Value GBT2390-2013 2013 httpopenstdsamrgovcnbzgkgbstd_listpp1=0amppp90=circulation_dateamppp91=descamppp2=GBT2390-2013 Accessed 13 October 2020

44 Garcia-Macias P John P Formation of natural indigo derived from Woad(Isatis tinctoria L) in relation to product purity J Agric Food Chem 2005527891ndash6 httpsdoiorg101021jf0486803

45 Junsongduang A Sirithip K Inta A Nachai R Onputtha B Tanming W et alDiversity and traditional knowledge of textile dyeing plants in NortheasternThailand Econ Bot 201771(3)241ndash55 httpsdoiorg101007s12231-017-9390-2

46 Mati E De Boer H Contemporary knowledge of dye plant species andnatural dye use in Kurdish Autonomous region Iraq Econ Bot 201064(2)137ndash48 httpsdoiorg101007s12231-010-9118-z

47 Cunningham AB Kadati WD Ximenes J Howe J Maduarta IM Ingram WPlants as the pivot the ethnobotany of Timorese textiles In Hamilton RBarrkmann J editors Textiles of Timor island in the woven sea UCLAUniversity of California Press 2014 p 89ndash103

48 Zhang L Wang L Cunningham AB Shi Y Wang Y Island blues indigenousknowledge of indigo-yielding plant species used by Hainan Miao and Lidyers on Hainan Island China J Ethnobiol Ethnomed 201915(1)31 httpsdoiorg101186s13002-019-0314-3

49 Christie RM Why is indigo blue Biotech Histochem 200782(2)51ndash6httpsdoiorg10108000958970701267276

50 Gollin L The taste and smell of Taban Kenyah (Kenyah medicine) anexploration of chemosensory selection criteria for medicinal plants amongthe Kenyah Leppo` Ke of East Kalimantan Borneo Indonesia University ofHawairsquoi Ann Arbor 2001

51 Jernigan KA The importance of chemosensory clues in Aguaruna treeclassification and identification J Ethnobiol Ethnomed 20084(1)1ndash7httpsdoiorg1011861746-4269-4-12

52 Gollin LX Subtle and profound sensory attributes of medicinal plantsamong the Kenyah Leppo Ke of East Kalimantan Borneo J Ethnobiol 200424(4)173ndash201

53 Leonti M Sticher O Heinrich M Medicinal plants of the Popoluca Meacutexicoorganoleptic properties as indigenous selection criteria J Ethnopharmacol200281(3)307ndash15 httpsdoiorg101016S0378-8741(02)00078-8

54 Zhang Y Yang L LI M Guo Y Li S Wang Y The best choices the diversityand functions of the plants in the home gardens of the Tsang-la (MotuoMenba) communities in Yarlung Tsangpo Grand Canyon Southwest ChinaJ Ethnobiol Ethnomed 202016(1)50 httpsdoiorg101186s13002-020-00395-z

55 Ferreira ESB Hulme AN McNab H Quye A The natural constituents ofhistorical textile dyes Chem Soc Rev 200433(6)329ndash36 httpsdoiorg101039b305697j

56 Maugard T Enaud E Choisy P Legoy MD Identification of an indigoprecursor from leaves of Isatis tinctoria (Woad) Phytochemistry 2001897ndash904

57 Pieroni A Torry B Does the taste matter Taste and medicinal perceptionsassociated with five selected herbal drugs among three ethnic groups inWest Yorkshire Northern England J Ethnobiol Ethnomed 20073(1)21httpsdoiorg1011861746-4269-3-21

58 Liu J Wang YH Guo DH The processing technique of traditional indigodyes J Silk 20091142ndash3 httpsdoiorg103969jissn1001-7003200911014

59 Su S Ma B Huang K Xu Y Ethnobotany study on dye-yielding plants ofZhuang people in the western Guangxi province Chin Agric Sci Bull 201329(11)203ndash7 httpsdoiorg103969jissn1000-6850201311039

60 Liu G She C Zeng H Huang W Ma Z Luo J Ethnobotanical research onthe dying Dong cloth in Tongdao Hunan province Guihaia 201232(3)310ndash4 httpsdoiorg103969jissn1000-3142201203005

Publisherrsquos NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations

Abstract

Background

Method

Results

Conclusion

Background

Methods

Study sites

Field survey and data collection

Quantitative analysis of the ethnobotanical data

Chemical analysis

Statistical analysis

Results

Traditional knowledge used for indigo identification

Quantitative evaluation of the quality criteria

Verification of traditional knowledge using modern scientific methods

Discussion

Sociocultural characteristics of indigo-paste artisans

Division of labor sex of indigo-paste artisans

Division of labor age of indigo-paste artisans

Characteristics and material basis of traditional knowledge about indigo paste

Color and active ingredients

Taste and pH

Touch and particle size

Cultural standards and industrial standards of indigo dyestuff

Conclusions

Abbreviations

Authorsrsquo contributions

Funding

Availability of data and materials

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Author details

References

Publisherrsquos Note