Kermes Vermilio) in the Northwest of Turkey
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DOI: 10.2478/auoc-2019-0001 Ovidius University Annals of Chemistry Volume 30, Number 1, pp. 1-4, 2019 Identification of the main dyestuffs obtained from Kermes (Kermes vermilio) in the Northwest of Turkey Serap AYAZ SEYHAN,*1 Cağlar DEMİRBAĞ,2 and Emre DÖLEN 1 1Marmara University, Faculty of Pharmacy Department of Analytical Chemistry, 34668, Istanbul, Turkey 2Edirne University, Faculty of Pharmacy Department of Analytical Chemistry, 22030, Edirne, Turkey Abstract. Dyer’s kermes (Kermes vermilio) is found only on the kermes oak (Quercus coccifera L). They were used in the past for the preparation of dyes for textiles and as a pharmaceutical. The main constituents of this insect are kermesic acid (ka) and flavokermesic acid (fk). Historically and culturally important dyer’s kermes insect red dyes were investigated by high performance liquid chromatography. Kermes in the Northwest of Turkey were analyzed first time the relative amount of the dyestuffs although their presence has been reported analytical works related to this insect. The relative amount in the acid hydrolyzed extract of Kermes vermilio from the Northwest of Turkey looked very similar to the France. Keywords: flavokermesic acid, HPLC, kermesic acid, kermes insect, natural dyes. 1. Introduction advised medicine during the 8th and 9th centuries was the extract of kermes (Alkermes) and he indicated that the In ancient times, some insects were benefited from color and the aroma of the liqueur have contributed to its various regions of the world especially to obtain the red effectiveness [1, 4, 6]. color. The among of many species of insects containing The main constituent of this insect, kermesic acid (ka) red dyes, who have gained importance: American (CI 75460), which is the aglycone of carminic acid cochineal (Dactylopius coccus), Polish cochineal or (cochineal), was isolated for the first time by R. Heise at Polish kermes (Porphyrophora polonica), Ararat the end of the 19th century. Its chemical structure, cochineal (Porphyrophora hamelii), kermes (Kermes investigated by O. Dimroth and co-workers, was vemilio) and lac insect (Kerria lacca). Kermes was used established definitively in 1964 by J. C. Overeem and G. as the most expensive and the most valued red dye [1, 2]. J. M. van der Kerk. Kermes also contains a small The insect that have been and are still used as sources of proportion of another yellow-orange anthraquinone red textiles dyes, paint, pigment, food and colorant (3 g in 5 kg of insects), the flavokermesic acid pharmaceutical colorants. Kermes dye was prepared (fk) [1, 7]. Verhecken and Wouters have shown that it is from a coccid insect species, Kermococcus vermilis in fact laccaic acid D [8]. These scientists have also Planchon (formerly Kermes ilicis L. or Kermes vermilio) revealed the presence of eight other colorants, as yet (family called the Eriococcidae = Kermidae). The insect unidentified and the relative abundances in the acid producing this dye reproduces and feeds exclusively on hydrolyzed extract were: 15 % fk and 85 % ka the shrub oak Quercus coccifera (Kermes Oak). Kermes (Yugoslavia), 18 % fk and 82 % ka (Spain), 20 % fk and is found in the Mediterranean region of southern Europe 80 % ka (North-Africa), 27 % fk and 73 % ka (France) [9 and Turkey [1, 3]. Alum-mordanted kermes dyeing of - 11]. wool and silk produces bright red hues. On wool, this red Historical objects are mostly dyed derived from has a light-yellow tinge and thus approaches the warm different dye sources with mixtures of natural dyes. Also, color of the madder red. Kermes was known and has been these dye sources generally consist of a mixture of used in Europe from Roman times, or even before. The closely related compounds. The precise composition and most consequential red dye known to the ancient ratio of these compounds can be used for identifying the Mesopotamians was kermes (“girmis” in Arabic). It is specific dye source. High-performance liquid acquired from the dried female insects which grow on the chromatography (HPLC) yields information about Mediterranean oak. Kermes lost its important dyestuff composition. However, it is not to have access significance in the dyeing industry soon after the to standards for some of the compounds separated Spaniards entered the market with the cochineal insect chromatographically. For this reason, the relative from the New World [4, 5]. composition of dyestuff compounds is used as a rule for Kermes was not only utilized by dyers but also for in the identification of insect dye source. The relative ancient times the extract of kermes was utilized as composition (%) is calculated based on the correlation medicine [2]. It was used in the treatment of wounds and between peak area (or height) of each dyestuff and the eye congestion. Abu ben-Masouiach (the famous Arab total peak area (or height). physician) claimed kermes as a medicine to warn the In this paper is presented an analysis of the heart, and it was used as a cardiac drug in Europe until composition of the dyestuffs from kermes (Kermes the 18th century. F. Silvestri confirms that the most * Corresponding author. E-mail address: [email protected] (Serap Ayaz Seyhan) © 2019 Ovidius University Press Ayaz Seyhan et al. / Ovidius University Annals of Chemistry 30 (2019) 1 – 4 vermilio) in the Northwest of Turkey (Çanakkale). ka and fk were detected at 275 nm. ka and fk stock Although the main coloring components (fk, ka, and kvI- solutions were prepared in methanol/water (1/1) and structurally unknown pigment of Kermes vermilio) from stored at 4 oC in darkness. kermes have been characterized, the structures of some It is not to have achieved to standards for some of the minor components remain unknown. For this reason, we compounds separated chromatographically in the presented the relative abundance of three dyestuffs. This analysis of natural dyes. The relative composition of this method of dye analysis may improve the knowledge of component is used as a rule for the identification of a ancient dyeing procedures and may contribute to Scale kermes insect source: kvI, ka and fk. insect chemotaxonomy [10, 12]. 3. Results and discussion 2. Experimental 3.1. Chromatographic determination of dyestuffs 2.1. Reagents and materials Initially, the chromatographic conditions defined by Adult female kermes insects (Kermes vermilio) were Wouters and Verhecken [10] were pursued to separate collected in Canakkale (the Northwest of Turkey) during the pigments. Then different solvent mixtures June and July. All chemicals and solvents were reagent (methanol/water, acetonitrile/water, methanol/water/ or HPLC grade. Kermesic acid and flavokermesic acid orthophosphoric acid) and gradient programs were used were obtained from INCO CT 2005 015406 MED- [13, 14]. The results were better obtained using a mixture COLOUR-TECH of the project. The other chemicals and of orthophosphoric acid (0.05% in water) and methanol. organic solvents were purchased from Merck Detection wavelengths were set at 275 nm and 420 nm (Darmstadt, Germany). Bidistilled deionized water was (yellow pigments) and 500 nm (red pigments) at the same Milli-Q quality. The stock solutions of ka and fk were time. The spectra (from 200 to 700 nm) were recorded for prepared daily. all peaks. To define components separated by chromatography, a wavelength of 275 nm seems 2.2. Sample preparation adequate [14]. Identification of ka and fk was carried out The kermes insects were washed over a griddle to by comparing retention times and their visible spectral eliminate dust and foreign materials. The insects were data with those of standards (Fig. 1). then dried at room temperature. Dried insects were Considering that mobile phase prepared in low pH ground in a ceramic mortar, and in the amber straight value would be effective in the peak separation and peak sided jar, 0.5 g of the powdered insect samples were form correction. The K. vermilio dyestuffs were mixed with 10 ml of methanol/water (9/1, v/v). The separated under the optimal HPLC conditions as mixture was homogenized in orbital shaker (IKA) for 20 demonstrated in Figs. 2 and 3. Samples were injected in min, and the sample was extracted at 27 °C with shaking duplicate. water bath. After 24 hours, the recovery of all dyestuffs considered reached completion in this extracting condition [9, 10]. The extracts were centrifuged at 3600 rpm for 3 minutes. The supernatant was used for the determination of dyestuffs. The composition of an acid hydrolysate was: extract/methanol/37% HCl (1/1/2, v/v/v). Heating was performed at 100 oC for 10 min in boiling tubes. The extracts were dried under N2 and were re-dissolved in water/methanol (1/1, v/v) [9, 10]. The extracts were stored in glass vials at -20 oC in darkness until chromatographic analysis. The extract filtered through a Figure 1. Chromatogram of the standard dyestuff mixture at 275 nm. flavokermesic acid, Rt:21.73; kermesic acid, 0.45 m nylon filter (Biocrom MN 718020, Phonex Rt:22.18. nylon filter 25 mm) prior to injection into the HPLC DAD1 A, Sig=275,4 Ref=off (SEYHAN\SEY00201.D) system. mAU 2.275 2.352 2.523 2.682 2.755 2.928 3.058 3.279 3.688 11.176 15.839 2.3. Chromatographic conditions 200 9.841 All extracts were analyzed on a HPLC-DAD system 8.165 150 1.998 3.957 18.864 (Agilent Technologies, High Performance Liquid 7.525 4.303 100 10.775 4.579 8.014 Chromatography was a combination of a Model G1311A 8.453 7.376 9.585 7.016 5.791 5.968 6.802 5.135 quaternary pump, a Model G1322A vacuum degasser and 6.568 9.300 7.822 7.184 6.220 9.147 13.920 50 21.808 10.268 11.757 8.927 10.414 5.516 12.230 a Model G1315A diode array detector, series 1200 16.359 13.399 19.817 12.618 12.840 13.660 14.669 19.261 21.321 17.963 autosampler).