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Identification and partial characterization of C-glycosylflavone markers in Asian plant dyes using liquid chromatography–tandem mass spectrometry
Journal of Chromatography A, 1218 (2011) 7325–7330
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Identification and partial characterization of C-glycosylflavone markers in Asian plant dyes using liquid chromatography–tandem mass spectrometry Chika Mouri, Richard Laursen ∗ Department of Chemistry, Boston University, Boston, MA 02215, USA
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 10 August 2011 Accepted 15 August 2011 Available online 19 August 2011 Keywords: C-glycosylflavones Mass-spectrometry Miscanthus tinctorius Arthraxon hispidus Kihachijo yellow dye Nara and Heian periods
a b s t r a c t Flavonoids in the grasses (Poaceae family), Arthraxon hispidus (Thunb.) Makino and Miscanthus tinctorius (Steudel) Hackel have long histories of use for producing yellow dyes in Japan and China, but up to now there have been no analytical procedures for characterizing the dye components in textiles dyed with these materials. LC–MS analysis of plant material and of silk dyed with extracts of these plants shows the presence, primarily, of flavonoid C-glycosides, three of which have been tentatively identified as luteolin 8-C-rhamnoside, apigenin 8-C-rhamnoside and luteolin 8-C-(4-ketorhamnoside). Two of these compounds, luteolin 8-C-rhamnoside (M = 432), apigenin 8-C-rhamnoside (M = 416), along with the previously known tricin (M = 330) and several other flavonoids that appear in varying amounts, serve as unique markers for identifying A. hispidus and M. tinctorius as the source of yellow dyes in textiles. Using this information, we have been able to identify grass-derived dyes in Japanese textiles dated to the Nara and Heian periods. However, due to the high variability in the amounts of various flavonoid components, our goal of distinguishing between the two plant sources remains elusive. © 2011 Elsevier B.V. All rights reserved.
1. 1Introduction For centuries, in China and Japan, and continuing even to the present day, extracts from members of the Poaceae (= Gramineae) family have been used to dye silk yellow. Specifically, the common grass, Arthraxon hispidus (Thunb.) Makino, which is regarded as a weed in many places, was mentioned as a dyestuff as early as the Warring States Period (475–221 BC) in China [1], and today is the source of the distinctive kihachijo (yellow of Hachijo island) dye used to color silk (usually for kimonos) on Hachijo Island, Japan [2]. In addition, extracts of Miscanthus tinctorius (Steudel) Hackel (Chinese grass) have also been used for centuries as a source for a yellow dye. Although these two plants are quite distinct anatomically, there is some confusion in the ancient literature about which plant is being referred to. Despite the long use of A. hispidus and M. tinctorius, there has been little study of the yellow dye components. Kaneta and Sugiyama [3] reported, based on material isolated from extracts, finding the flavonoids, luteolin 7-glucoside, luteolin, tricin and arthraxin (see structures in Fig. 2 below), the latter being a new, previously unreported compound, in extracts of both A. hispidus and M. tinctorius. Furthermore, Wouters [4] observed a large number of acid-stable, flavonoid-like HPLC peaks, in addition to luteolin,
∗ Corresponding author. Tel.: +1 617 353 2491; fax: +1 617 353 6466. E-mail address: [email protected] (R. Laursen). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.08.048
in acid extracts of wool dyed with M. tinctorius. However, the nature of those compounds was not determined. HPLC in combination with various forms of mass spectrometry has become one of the most useful tools for characterization of flavonoids [5–8], although, as in the case previously unreported compounds, purification followed by NMR analysis may also be necessary for complete characterization. In the present study our aim was to characterize at least some of the flavonoids in A. hispidus and M. tinctorius with the goal of identifying marker compounds that could be used to identify these dyestuffs in extracts of textiles. Since the majority of flavonoids in these plants turned out to be flavone C-glycosides, MS–MS and MS3 methods proved to be particularly helpful. Using the information obtained from these studies we have been able to demonstrate that the yellow dyes in certain ancient Japanese textiles were derived from A. hispidus or M. tinctorius. 2. Experimental 2.1. Materials Specimens of M. tinctorius were collected throughout the 2010 growing season at Mt. Takao, Kanazawa, Ishikawa (native plant, in a sunny location); at Asahi, Echizen-cho, Nyu-gun, Fukui (native plant, in a somewhat shady location) in west, central Japan; and Mukoterayama, Hachimancho, Tokushima, on Shikoku Island (cultivated plant, sunny location) in southern Japan. This plant was also
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purchased from Tanaka-nao in Tokyo, where it is sold as a dyestuff. Specimens of A. hispidus were collected at various times during 2009 and 2010 in Kanazawa and were also obtained from Hachijo Island in 2009 and 2010. For reference to the C-glycoside, vitexin, we analyzed samples of Passiflora incarnata and Crataegus monogyna (purchased from Mountain Rose Herbs), both of which contain large quantities of vitexin (as well as orientin and isoorientin) [9,10].
Specimens of A. hispidus and M. tinctorius and other plants were extracted by heating about 10 mg of the ground, dry plant with 1.0 mL of methanol/water (1:1) at 65 ◦ C for 1 h. The supernatant liquid was removed by pipet and was centrifuged at 12,000 rpm for about 5 min, after which the clear supernatant was subjected to analysis by LC–MS. This solution was diluted with methanol/water (1:1) if necessary. In some cases, the plant extracts were heated in 2 M HCl at 95 ◦ C for 60 min to hydrolyze O-glycosides.
O 448
V 432
2.4. Extraction of dyed silk Extraction of either laboratory-dyed silk or fabric from Hachijo Island, Japan, was accomplished by heating approximately 0.1–1 mg of fibers in 200 L of a solution of pyridine/water/1.0 M oxalic acid in water (95:95:10) at 100 ◦ C for 15 min. The mixture was cooled to room temperature and the solution was transferred to a 1.7-mL microcentrifuge tube by pipet. The fibers were washed with 30 L of water and the washings were added to the extract. The extract and washings were then cooled to <0 ◦ C and evaporated to dryness at room temperature in a vacuum desiccator overnight using a mechanical pump. The resulting residue was dissolved in 50 L of methanol/water (1:1), the solution was centrifuged at 12,000 rpm and 30 L of the supernatant was transferred to an insert in an autoinjector vial for analysis; 20 L was injected onto the HPLC column. In the foregoing procedure, only about 40% of the extract used for LC–MS analysis. In the case of certain extremely small museum samples, it was necessary to analyze as much of the extract as possible. In this case, after evaporation of the pyridine solution, the sample residue was mixed with 100 L of methanol. This solution was centrifuged at 12,000 rpm for 8 min, and 80 L of the supernatant was transferred to a new microcentrifuge vial. To the residual 20 L containing any precipitate was added an additional 50 L of methanol; this mixture was centrifuged, and about 50 L was removed and added to the 80 L of original supernatant. The combined washes (∼130 L) were evaporated to dryness, the residue was dissolved in 25 L of methanol/water (1:1), and all of this solution was transferred to the insert of an autoinjector vial; 20 L was injected. In this way, about 75% of the total extract could be used for analysis. Although we used a methanol/water solvent for extraction in earlier studies [12], we have switched to the pyridine/water
430
416
430
338
448
432 V 432
A. hispidus HCl 416 286
412
330 338
430
0 576
432
M. tinctorius
432 432 O 448
416
416
412 V 432 448
330
432
2.3. Dyed silk specimens Some 37 specimens from fragments of Nara (8th C) and Heian (9–12th C) period textiles were provided by the Museum of Fine Arts, Boston. Grass-derived dyes were found in several of the yellow and green specimens. The Nara specimen analyzed here is from a ¯ u-ji, ¯ fragment of a textile in Hory the oldest temple extant in Japan. In addition, threads from silk dyed by Uemura and by Yoshioka [11], using M. tinctorius, were provided by M. Kataoka of the Museum of Fine Arts. Samples of silk dyed with A. hispidus were provided by the Kihachijo Textile Co-op Association, Tokyo.
330 412
432
O 448
A. hispidus
416
432
mAU at 350nm
2.2. Extraction of plant material
432 430
432 O O 448 448
432 V 432
430
M. tinctorius HCl 416 286
Retention time (min) Fig. 1. Representative HPLC profiles of extracts from the plants, A. hispidus and M. tinctorius. Acid-treated extracts are marked “HCl.” The profiles shown were monitored at 350 nm; the molecular masses of some components are given above the peaks. O and V refer to the C-glucosides, orientin/isoorientin and vitexin/isovitexin, respectively.
mixture described here because (1) it is a better solvent for dyes of all color, (2) it allows for a higher extraction temperature and (3) its higher boiling point leads to less “bumping” during evaporation under vacuum. Oxalate anion [13] is a superior chelator of metal ions than is formate. 2.5. LC–MS analysis Extracts of plant material or of dyed silk were analyzed by HPLC–DAD–MS using an Agilent 1100 high performance liquid chromatography (HPLC) system consisting of an automatic injector, a gradient pump, a HP series 1100 diode array detector (DAD), and an Agilent series 1100 VL on-line electrospray ionization mass spectrometer (MS), essentially as described earlier by Zhang and Laursen [12]; the electrospray chamber was run at 350 ◦ C with a gas flow of 12 L/min and a Vcap of 3000 V. Operation of the system and data analysis were done using Chemstation software, and detection was generally done in the negative ion [M−H]− mode. Separation of dye components was made on a Vydac C18 reversed phase column (2.1 mm dia. × 250 mm long; 5-m particle size), operated at a flow rate of 0.25 mL/min. Columns were eluted with acetonitrile–water gradients containing 0.1% formic acid. In the course of this work, several gradients were used, but because the types of stationary and mobile phases were unchanged, the retention times of peaks were always in the same order. 2.6. UPLC–ESI-MS/MS experiments MS spectra were acquired using data-dependent acquisition (DDA) on a UPLC–MS/MS system comprised of an Acquity UPLC (Waters Corp., Milford, MA) and a linear ion trap quadrupole (LTQ) mass spectrometer (Thermo Electron Corp., San Jose, CA). The UPLC was equipped with a 2.1 mm × 150 mm T3 HSS reversed phase
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OH H3C
OH
O
OH
O HO
OH O
8
OH
OH
O
OH
OH O
OH
HO
O
OH
6 OH
OH
O
Luteolin 8-C-rhamnoside, 1 (or isomer) [M = 432]
OH
O
Arthraxin [M = 412]
O
Luteolin [M = 286]
O H3C
OCH3
OH
OH
OH
O
OH O
HO
OH
HO
OH
O
OH
O
Luteolin 8-C-4-ketorhamnoside, 3 (or isomer) [M = 430]
OCH3
OH Glucose-O
O
O
OH
OH
O
Luteolin 7-glucoside [M = 448]
Tricin [M = 330]
OH H3C
OH OH
O
OH
OH O
HO
OH
HO
O
O
OH
Apigenin 8-C-rhamnoside, 2 (or isomer) [M = 416]
OH HO
O
O
OH
8-Prenylapigenin [M = 338]
O
OH
+ 2 -CH2-
Dimethylluteolin? [M = 314]
Fig. 2. Putative structures of flavonoids referred to in this article.
column for separation with a gradient of 20% to 40% B over 40 min at a flow rate of 0.1 mL/min. Mobile phase A was 0.03% trifluoroacetic acid in water, and mobile phase B was 0.024% trifluoroacetic acid in acetonitrile. The separation was monitored at 350 nm. MS/MS data were acquired by data-dependent acquisition using pulsed Q dissociation (PQD) detection. PQD parameters were set at isolation width 2 m/z, normalized collision energy 35%, activation Q 0.7, and activation time (T) 0.1 ms; the threshold for MS/MS acquisition was set to 1000 counts. 3. Results and discussion One of the first observations made was that HPLC profiles of extracts of both M. tinctorius and A. hispidus were little changed by acid hydrolysis (Fig. 1), as discussed in detail below, although one small peak later shown to be luteolin 7-O-glucoside was converted to luteolin. These findings are in agreement with the observations of Wouters [4] for M. tinctorius, who used strong acid to extract dyes from textile fibers and UV–Vis detection of peaks. Furthermore, we found that UV–Vis spectra of individual peaks in the profiles were characteristic of flavones (e.g., luteolin), and that the HPLC retention times and observed molecular masses were consistent with flavones conjugated to sugar residues. Similar results were obtained for extracts of dyed silk obtained from Hachijo Island, except for some low mass peaks (see at the end of Section 3.3.). From the foregoing information we concluded that most of the dye components in these plants were probably flavonoid C-glycosides. As will be discussed below, one of the difficulties in establishing representative HPLC profiles for these dyes is that the profiles
change, depending on growth location and conditions, time of harvest, dyeing techniques, etc. Most of the components are the same qualitatively—even comparing M. tinctorius and A. hispidus—but the quantitative differences are considerable. Nevertheless some of the major components provide useful markers, in particular those having molecular masses of 432, 416 and 330. Both A. hispidus and M. tinctorius are grasses and members of the Poaceae (Gramineae) family, in which the major flavonoids are C-glycosides and tricin (M = 330) [14], so in retrospect it is not surprising that these types of compounds are good markers for grass-dyed textiles. Another characteristic of HPLC profiles for these grass-derived dyes is their general shape, as seen in Figs. 1–5, which is a low, rounded “hump” surmounted by sharp peaks. This yet-unexplained feature is not seen for other natural dye profiles, where one generally sees a flat baseline (unpublished observations). 3.1. LC–MS profiles of plant extracts The flavonoid components of A. hispidus and M. tinctorius in extracted plant material and dyed silk were separated by reversed phase HPLC, and eluted peaks were monitored at 350 nm, which is near the maximum absorbance of most flavonoids in this study, and in tandem by mass spectrometery, usually in the negative ion mode. Fig. 1 shows representative HPLC profiles of extracts A. hispidus and M. tinctorius. The ratios of components in the profiles are quite variable, depending on the environment where the plants were grown and when they were harvested, but three of the compounds, designated 1, 2 and 3 in Figs. 2 and 4, with respective molecular masses of
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381
416 (2) 337
Relative abundance
369
430 (3)
417
399
313
283
413
431
329 299
432 432 (1)
397 353
200
250
300
433
415
329
299
350
400
450
m/z Fig. 3. MS/MS positive ion fragments of predominant flavonoid C-glycoside components of extracts from A. hispidus. [The same patterns (not shown) are also seen for M. tinctorius.] Only the ions referred to in Fig. 4 are labeled. Molecular masses of the compounds themselves are given in the upper, left-hand corner of the panels.
432, 416 and 430 Da, predominate. As will be discussed later, some of these compounds do not necessarily predominate in historical textiles. Kaneta and Sugiyama [3] (who did not have the benefit of HPLC for analysis) reported finding only luteolin, luteolin 7-glucoside, tricin (M = 330) and a new flavonoid, arthraxin (M = 412), in extracts of A. hispidus and M. tinctorius. As can be seen in Fig. 1, and as we have observed in many extracts of plants grown under a variety of conditions (data not shown), these components are present only in very minor amounts. In fact, we only see large peaks for luteolin (M = 286) in acid-treated extracts, where the O-glycoside, luteolin 7-glucoside (M = 448), which elutes at about 12 min, has
0,4X+
HO
O
OH
B
O
HO
A
C
OH
O
OH O
HO
R
0,1X+
R = OH Luteolin 8-C-rhamnoside (1) M = 432 R = H Apigenin 8-C-rhamnoside (2) M = 416 Main ions obtained by PQD-MS-MS (1) / (2) 433/417 [M+H]+ 415/399 [M+H-18]+ -H2O: E 1+ 397/381 [M+H-36]+ -2H2O: E 2+ 353/337 [M+H-80]+ 0,4X+-2H 2O 329/313 [M+H-104]+ 0,2X+ 299/283 [M+H-134]+ 0,1X+
A
C
OH
O
CH3
HO 0,2X+
O HO
0,4X+
O
CH2OH
HO 0,2X+
HO 0,1X+
0,4X+
HO
CH3
HO 0,2X+
been hydrolyzed to luteolin. Nevertheless, luteolin is generally a rather large peak in historical dyed textiles (see Section 3.3). Kaneta and Sugiyama [3] also reported finding some of these flavonoids in Miscanthus sinensis Andersson and the common reed, Phragmites communis Trinius [= Phragmites australis (Cav.) Trin. ex Steud]. However we did not find large amounts of any flavonoids in either of these species and conclude that they would not be useful dyestuffs. The well-known C-glycosides, vitexin/isovitexin (M = 432) and orientin (luteolin 8-C-glucoside)/isoorientin (luteolin 6-Cglucoside) (M = 448), labeled, respectively, “V” and “O” elute between 5 and 10 min in Fig. 1 and are unchanged, as expected, by acid hydrolysis.
O
B
Vitexin Main ions obtained by PQD-MS-MS Vitexin 433 [M+H]+ 415 [M+H-18]+ -H2O: E1+ 397 [M+H-36]+ -2H2O: E2+ 367 [M+H-66]+ 2,3X+-2H2O 337 [M+H-96]+ 0,4X+-2H2O 313 [M+H-120]+ 0,2X+ 283 [M+H-150]+ 0,1X+
OH
HO O
HO 0,1X+
OH
OH
O
Luteolin 8-C-4-ketorhamnoside (3) M = 430 Main ions obtained by PQD-MS-MS (3) 431 [M+H]+ 413 [M+H-18]+ -H2O: E1+ 369 [M+H-62]+ 0,4X+-H2O 329 [M+H-102]+ 0,2X+ 299 [M+H-132]+ 0,1X+
OH
H2+O O
HO
A
C
OH
O
B
0,2B+
329
0,2X+
OH
OH
MS3
B OH
C +O
137
0,2B+
Fig. 4. Analysis of MS/MS fragmentation data for compounds 1, 2, and 3. The analysis is also shown for vitexin, which has the same molecular mass (432) as 1 (see [7]). The bottom of the figure shows the proposed consequence of MS3 fragmentation of the 0,2 X+ ion of 1. The ions present in greatest abundance are shown in bold font. Cleavage products are designated according to the nomenclature of Doman and Costello [23].
C. Mouri, R. Laursen / J. Chromatogr. A 1218 (2011) 7325–7330
432
A. hispidus on silk (Kihachijo)
330 286 416
338
M. M. tinctorius on on silksilk tinctorius (Yoshioka)
432
432
416 330
338
mAU at 350nm
416416 314314
Nara period M. tinctorius on silk (Uemura)
432
314
432
338
286
Heian period
Nara period
286 416 432 432
330
416 338
314
Retention time (min) Fig. 5. HPLC profiles (top to bottom panels, respectively) of extracts of yellow dyed silk specimens, from Hachijo Island (21st C), from the sample book of Yoshioka [11] (20th C), dyed silk sample collection of Uemura (20th C), the Heian period (9–12th C), and the Nara period (8th C).
One significant difference in the profiles of A. hispidus and M. tinctorius is the presence, in the M. tinctorius profile (Fig. 1), of a large peak of M = 576, which is the same mass as that for maysin [2 -O-␣rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)luteolin] described by Elliger et al. [15]. Treatment of the M. tinctorius extract with acid results in loss of the 576 peak (due to loss of a 146-Da rhamnosyl unit?) and appearance of a new one with M = 430 (Fig. 1). This new peak could be an epimer of the M = 430 (3) compound we analyzed below (see Section 3.2), since the both ␣-keto hydrogens can epimerize under acidic conditions. Unfortunately, the M = 576 peak is not useful for distinguishing between A. hispidus and M. tinctorius because it does not appear in either historical dyed textiles or in textiles we have dyed in our own laboratory (by the methods described in [16]). Possibly it is not adsorbed efficiently by the textile fibers due to its relatively high molecular weight. This idea is not without precedent since it has been noted that yarns dyed sequentially in the same madder dye bath had different colors: the first batch of yarn has a typical red color due to selective adsorption of alizarin and purpurin, whereas the second batch has a yellower shade due to adsorption by the higher molecular weight glycosides [17]. 3.2. MS–MS and MS3 analyses Fig. 3 shows MS/MS fragmentation profiles for the positive ions at [M+H]+ = 433, 417 and 431, corresponding to compounds 1, 2 and 3, respectively; Fig. 4 outlines the analyses of these profiles. Initially
7329
we suspected that the M = 432 peak might be vitexin (or isovitexin), which also is a C-glycoside, has the same mass and is widely found in plants, but comparison of retention times with vitexin, which is known to occur in large amounts in P. incarnata and C. monogyna, showed that 1 could not be vitexin. Furthermore, as shown in Fig. 4, the fragmentation patterns for vitexin [7] and 1 are significantly different, vitexin being the C-glucoside of apigenin. Further analysis of the fragments of 1 indicate that it is a 6-deoxyhexoside derivative of luteolin. Whether the sugar moiety of 1 is rhamnose or not cannot be stated with certainty at this point, but rhamnose is the most common deoxyhexose associated with flavonoids [8]. Nor can it be ascertained which anomeric form is present. MS3 fragmentation of the 0,2 X+ ion of 1 at m/z = 329 gave the 0,2 B+ fragment confirming the presence of the luteolin aglycone, rather than apigenin. The absence of 1,3 A+ fragments is consistent with substitution at the 8-position, rather than at the 6-position [7]. Substitution at the 8-position is also analogous to the structure of arthraxin (see Fig. 2), which is reported to have a C-8 carbon–carbon bond [3]. It can also be seen in Fig. 1, that there are at least three peaks (in addition to vitexin) that have molecular masses of 432. Although the largest and most consistently appearing peak, which elutes at about 20 min in Fig. 1, gives the fragmentation pattern shown in Fig. 4; the other M = 432 peaks display similar fragmentation patterns (data not shown). From this we conclude that all of the M = 432 peaks have the same sugar carbon skeleton. Since structure 1 has five chiral centers, many isomers are theoretically possible. Positional isomers (e.g., 6- and 8-substitution) are also possible. Compound 2, which differs from 1 by an oxygen atom (16 Da), shows a fragmentation pattern similar to that of 1, except that all of the fragments are 16 Da lower (Figs. 3 and 4). By analogy, we propose that compound 2 is apigenin 8-C-rhamnoside (or an isomer). Compound 3 differs from 1 by only 2 Da. As can be seen in Figs. 3 and 4, 3 shows only an E1 + ion (loss of one water) and not the E2 + ion (loss of two waters) seen for 1 and 2, since loss of two water molecules for a 4-keto-6-deoxyhexose would be impossible. On the other hand, 3 has the same 0,2 X+ and 0,1 X+ fragments seen for 1. Compound 3 is isomeric with (if not identical to) derhamnosylmaysin described by Elliger et al. [14]. 3.3. Flavonoid C-glycosides as textile dyes Fig. 5 shows three profiles of silk extracts where the dyestuff is known, and two where it is unknown. The “Kihachijo” specimen was textile material obtained from Hachijo Island in Japan, where only A. hispidus is used as a dyestuff. The other two specimens, “M. tinctorius on silk” were from the sample book of Yoshioka [11] and from a collection of dyed textiles samples prepared by Uemura. The Nara and Heian profiles were from extracts of threads from textiles in the collection of the Museum of Fine Arts, Boston. Some of these specimens were green, having been dyed, as the current studies have shown, with a yellow dye and indigo. The HPLC profiles (in Fig. 5) indicate that they were dyed with a grass-type dyestuff, such as A. hispidus or M. tinctorius. [In some other specimens, the yellow was a protoberberine dye (data not shown).] Earlier studies of some Nara period textiles by Uemura and Takagi [18,19] predated HPLC and could not distinguish between various flavonoids. The only recent published analysis of textiles of this age is by Nakamura et al. [20], who used spectroscopic methods that also do not distinguish between the thousands of known flavonoids. There are differences between the profiles of the plant extracts (Fig. 1) and the textile extracts shown in Fig. 5 (and in many other samples we have analyzed). Although most of the dyed textile samples show the M = 432 peak discussed above as a predominant peak, many of them also show strong luteolin (M = 286) and tricin (M = 330) peaks, as well as variable amounts of we suspect to be
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prenylluteloin (M = 338) and one of the dimethylluteolins (M = 314) (see Fig. 2), based on their molecular masses and HPLC retention times. All of the latter are known flavonoids. Of the aforementioned compounds, luteolin is not useful as a marker because it is found in many plants used for dyeing; luteolin and its derivatives are the most common of the flavone dyes. We still do not have an explanation for its prevalence in the dyed objects examined here, because it is present in very small amounts in unhydrolyzed plant extracts. It does, however, appear in HCl-treated extracts (Fig. 1), presumably due to hydrolysis of luteolin 7-O-glucoside. Arthraxin (M = 412; see Fig. 2), reported by Kaneta and Sugiyama [3] to be a major component of A. hispidus and M. tinctorius, is actually a minor component of plant extracts (Fig. 1) and is hardly detectable in textile extracts (Fig. 5). Dyeing of textiles and textile fibers typically employs as many as 20 repeated dyeing cycles (cf. [21]) in order to get intense, saturated colors. We suspect that the reason for the increased amounts of luteolin, tricin and other alkylated luteolins in dyed textiles is that these compounds have lower molecular weights and are more hydrophobic (they elute late on reversed-phase HPLC columns) than the flavonoid glycosides, and therefore are preferentially adsorbed by the textile fibers. Confirming this idea is our observation that we do not see any of the large, hydrophilic M = 576 component (which contains two sugars) of M. tinctorius (see Fig. 1) in extracts of silk that we have dyed (data not shown). There may be other explanations, but we do not think it is due to hydrolysis of the glycosidic linkage during the dyeing process, since we have never seen hydrolysis when dyeing with other flavonoid glycosides. 4. Conclusions Although we are unable, so far, to distinguish between textile fibers dyed with extracts of A. hispidus and M. tinctorius, there are several characteristics that distinguish this class of dyes from others: 1. The grass-derived dyes (e.g., from A. hispidus and M. tinctorius) are primarily C-glycosides, which are stable to hydrolysis by strong acids. Therefore profiles of acid-treated samples are similar to those of untreated ones. 2. The HPLC profiles of the flavonoid components of A. hispidus and M. tinctorius are rather distinctive, being characterized by a series of sharp peaks atop a sort of rounded “hump” between the earliest and latest eluting peaks (see Figs. 1 and 5). 3. One of the early-eluting C-glycoside peaks, and often the largest in dyes derived from A. hispidus and M. tinctorius, has M = 432; it is frequently followed, roughly a minute later (depending on the elution gradient), by another C-glycoside with M = 416. 4. Most grasses contain tricin (M = 330), which is often a large peak in dyed textiles and is also a useful marker for A. hispidus and M. tinctorius. Flavonoid C-glycosides have been known for many years, and undoubtedly occur in other known plant dyestuffs (cf. [22]), but
compounds 1, 2 and 3 are the first to be so documented. Grasses seem to have been little (if at all) used outside of Asia for dyeing, and the finding that the majority of flavonoids in A. hispidus and M. tinctorius are C-glycosides is consistent with reports [14] that most members of the Poaceae family are rich in flavonoid C-glycosides. Although the structures of 1, 2 and 3 remain to be elucidated absolutely, the first two of these compounds represent unique markers for textile fibers dyed with A. hispidus or M. tinctorius. Acknowledgements We are grateful to Ms. Monika Vecchi and Dr. Dingyi Wen at BiogenIdec for MS2 and MS3 experiments and Masumi Kataoka and Meredith Montague, Museum of Fine Arts for specimens of Japanese dyed textiles. For generous gifts of plant material, we would like to thank Dr. Yasushi Ibaragi, Tokushima Prefectural Museum; Dr. Eisuke Hayasaka, Fukui Botanical Garden; Dr. Yohei Sasaki, Kanazawa University; and the Kihachijo Textile Co-op Association, Tokyo. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2011.08.048. References [1] A. Waley, J.R. Allen, The Book of Songs, Grove Press, New York, 1996, p. 215 (In this poem, ‘Green ( )’ refers to Jinso = Arthraxon hispidus). [2] S. Goto, R. Yamakawa, Senryo-syokubutsu-fu (About Dye Plants), Hakuhosya, Kyoto, 1972 (in Japanese). [3] M. Kaneta, N. Sugiyama, Bull. Chem. Soc. Jpn. 45 (1972) 528. [4] J. Wouters, Dyes Hist. Archaeol. 12 (1994) 12. [5] G.C. Kite, E.A. Porter, F.C. Denison, R.J. Grayer, N.C. Veitch, I. Butler, M.S.J. Simmonds, J. Chromatogr. A 1104 (2006) 123. [6] F. Ferreres, A. Gil-Izquierdo, P.B. Andrade, P. Valentão, F.A. Tomás-Barberán, J. Chromatogr. A 1161 (2007) 214. [7] P. Waridel, J.-L. Wolfender, K. Ndjoko, K.R. Hobby, H.J. Major, K. Hostettmann, J. Chromatogr. A 926 (2001) 29. [8] T. Fossen, Ø.M. Andersen, in: Ø.M. Andersen, K.R. Markham (Eds.), Flavonoids: Chemistry, Biochemistry and Applications, Taylor & Francis, New York, 2006, p. 37. [9] H. Schilcher, Z. Naturforsch. B 23 (1968) 1393. [10] A. Rehwald, B. Meier, O. Sticher, J. Chromatogr. A 677 (1994) 25. [11] T. Yoshioka, Nihon no Iro, Shokubutsu Senryo no Hanashi (Japanese Colors: The Story of Natural Dyes), Shikosha, Kyoto, 1983 (in Japanese). [12] X. Zhang, R.A. Laursen, Anal. Chem. 77 (2005) 2022. [13] R. Marques, M.M. Sousa, M.C. Oliveira, M.J. Melo, J. Chromatogr. A 1216 (2009) 1402. [14] J.B. Harborne, C.A. Williams, Biochem. Syst. Ecol. 4 (1976) 267. [15] C.A. Elliger, B.G. Chan, A.C. Waiss Jr., R.E. Lundin, W.F. Haddon, Phytochemistry 19 (1980) 293. [16] E.S.B. Ferreira, A. Quye, H. McNab, A. Hulme, Dyes Hist. Archaeol. 18 (2002) 63. [17] D. Cardon, Natural Dyes: Sources, Tradition, Technology and Science, Archetype, London, 2007, p. 136. [18] R. Uemura, Y. Takagi, Shoryobukiyo 19 (1967) 63. [19] Y. Takagi, Shoryobukiyo 21 (1969) 48. [20] R. Nakamura, Y. Tanaka, A. Ogata, M. Naruse, Anal. Chem. 81 (2009) 5691. [21] S. Yoshioka, Shizen no iro wo someru (Natural Color Dyeing), Shikosha, Kyoto, 1996, p. 30 (in Japanese). [22] W. Nowik, J. Sep. Sci. 28 (2005) 1595. [23] B. Domon, C.E. Costello, Glycoconj. J. 5 (1988) 123.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Identification and partial characterization of C-glycosylflavone markers in Asian plant dyes using liquid chromatography–tandem mass spectrometry Chika Mouri, Richard Laursen ∗ Department of Chemistry, Boston University, Boston, MA 02215, USA
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 10 August 2011 Accepted 15 August 2011 Available online 19 August 2011 Keywords: C-glycosylflavones Mass-spectrometry Miscanthus tinctorius Arthraxon hispidus Kihachijo yellow dye Nara and Heian periods
a b s t r a c t Flavonoids in the grasses (Poaceae family), Arthraxon hispidus (Thunb.) Makino and Miscanthus tinctorius (Steudel) Hackel have long histories of use for producing yellow dyes in Japan and China, but up to now there have been no analytical procedures for characterizing the dye components in textiles dyed with these materials. LC–MS analysis of plant material and of silk dyed with extracts of these plants shows the presence, primarily, of flavonoid C-glycosides, three of which have been tentatively identified as luteolin 8-C-rhamnoside, apigenin 8-C-rhamnoside and luteolin 8-C-(4-ketorhamnoside). Two of these compounds, luteolin 8-C-rhamnoside (M = 432), apigenin 8-C-rhamnoside (M = 416), along with the previously known tricin (M = 330) and several other flavonoids that appear in varying amounts, serve as unique markers for identifying A. hispidus and M. tinctorius as the source of yellow dyes in textiles. Using this information, we have been able to identify grass-derived dyes in Japanese textiles dated to the Nara and Heian periods. However, due to the high variability in the amounts of various flavonoid components, our goal of distinguishing between the two plant sources remains elusive. © 2011 Elsevier B.V. All rights reserved.
1. 1Introduction For centuries, in China and Japan, and continuing even to the present day, extracts from members of the Poaceae (= Gramineae) family have been used to dye silk yellow. Specifically, the common grass, Arthraxon hispidus (Thunb.) Makino, which is regarded as a weed in many places, was mentioned as a dyestuff as early as the Warring States Period (475–221 BC) in China [1], and today is the source of the distinctive kihachijo (yellow of Hachijo island) dye used to color silk (usually for kimonos) on Hachijo Island, Japan [2]. In addition, extracts of Miscanthus tinctorius (Steudel) Hackel (Chinese grass) have also been used for centuries as a source for a yellow dye. Although these two plants are quite distinct anatomically, there is some confusion in the ancient literature about which plant is being referred to. Despite the long use of A. hispidus and M. tinctorius, there has been little study of the yellow dye components. Kaneta and Sugiyama [3] reported, based on material isolated from extracts, finding the flavonoids, luteolin 7-glucoside, luteolin, tricin and arthraxin (see structures in Fig. 2 below), the latter being a new, previously unreported compound, in extracts of both A. hispidus and M. tinctorius. Furthermore, Wouters [4] observed a large number of acid-stable, flavonoid-like HPLC peaks, in addition to luteolin,
∗ Corresponding author. Tel.: +1 617 353 2491; fax: +1 617 353 6466. E-mail address: [email protected] (R. Laursen). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.08.048
in acid extracts of wool dyed with M. tinctorius. However, the nature of those compounds was not determined. HPLC in combination with various forms of mass spectrometry has become one of the most useful tools for characterization of flavonoids [5–8], although, as in the case previously unreported compounds, purification followed by NMR analysis may also be necessary for complete characterization. In the present study our aim was to characterize at least some of the flavonoids in A. hispidus and M. tinctorius with the goal of identifying marker compounds that could be used to identify these dyestuffs in extracts of textiles. Since the majority of flavonoids in these plants turned out to be flavone C-glycosides, MS–MS and MS3 methods proved to be particularly helpful. Using the information obtained from these studies we have been able to demonstrate that the yellow dyes in certain ancient Japanese textiles were derived from A. hispidus or M. tinctorius. 2. Experimental 2.1. Materials Specimens of M. tinctorius were collected throughout the 2010 growing season at Mt. Takao, Kanazawa, Ishikawa (native plant, in a sunny location); at Asahi, Echizen-cho, Nyu-gun, Fukui (native plant, in a somewhat shady location) in west, central Japan; and Mukoterayama, Hachimancho, Tokushima, on Shikoku Island (cultivated plant, sunny location) in southern Japan. This plant was also
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purchased from Tanaka-nao in Tokyo, where it is sold as a dyestuff. Specimens of A. hispidus were collected at various times during 2009 and 2010 in Kanazawa and were also obtained from Hachijo Island in 2009 and 2010. For reference to the C-glycoside, vitexin, we analyzed samples of Passiflora incarnata and Crataegus monogyna (purchased from Mountain Rose Herbs), both of which contain large quantities of vitexin (as well as orientin and isoorientin) [9,10].
Specimens of A. hispidus and M. tinctorius and other plants were extracted by heating about 10 mg of the ground, dry plant with 1.0 mL of methanol/water (1:1) at 65 ◦ C for 1 h. The supernatant liquid was removed by pipet and was centrifuged at 12,000 rpm for about 5 min, after which the clear supernatant was subjected to analysis by LC–MS. This solution was diluted with methanol/water (1:1) if necessary. In some cases, the plant extracts were heated in 2 M HCl at 95 ◦ C for 60 min to hydrolyze O-glycosides.
O 448
V 432
2.4. Extraction of dyed silk Extraction of either laboratory-dyed silk or fabric from Hachijo Island, Japan, was accomplished by heating approximately 0.1–1 mg of fibers in 200 L of a solution of pyridine/water/1.0 M oxalic acid in water (95:95:10) at 100 ◦ C for 15 min. The mixture was cooled to room temperature and the solution was transferred to a 1.7-mL microcentrifuge tube by pipet. The fibers were washed with 30 L of water and the washings were added to the extract. The extract and washings were then cooled to <0 ◦ C and evaporated to dryness at room temperature in a vacuum desiccator overnight using a mechanical pump. The resulting residue was dissolved in 50 L of methanol/water (1:1), the solution was centrifuged at 12,000 rpm and 30 L of the supernatant was transferred to an insert in an autoinjector vial for analysis; 20 L was injected onto the HPLC column. In the foregoing procedure, only about 40% of the extract used for LC–MS analysis. In the case of certain extremely small museum samples, it was necessary to analyze as much of the extract as possible. In this case, after evaporation of the pyridine solution, the sample residue was mixed with 100 L of methanol. This solution was centrifuged at 12,000 rpm for 8 min, and 80 L of the supernatant was transferred to a new microcentrifuge vial. To the residual 20 L containing any precipitate was added an additional 50 L of methanol; this mixture was centrifuged, and about 50 L was removed and added to the 80 L of original supernatant. The combined washes (∼130 L) were evaporated to dryness, the residue was dissolved in 25 L of methanol/water (1:1), and all of this solution was transferred to the insert of an autoinjector vial; 20 L was injected. In this way, about 75% of the total extract could be used for analysis. Although we used a methanol/water solvent for extraction in earlier studies [12], we have switched to the pyridine/water
430
416
430
338
448
432 V 432
A. hispidus HCl 416 286
412
330 338
430
0 576
432
M. tinctorius
432 432 O 448
416
416
412 V 432 448
330
432
2.3. Dyed silk specimens Some 37 specimens from fragments of Nara (8th C) and Heian (9–12th C) period textiles were provided by the Museum of Fine Arts, Boston. Grass-derived dyes were found in several of the yellow and green specimens. The Nara specimen analyzed here is from a ¯ u-ji, ¯ fragment of a textile in Hory the oldest temple extant in Japan. In addition, threads from silk dyed by Uemura and by Yoshioka [11], using M. tinctorius, were provided by M. Kataoka of the Museum of Fine Arts. Samples of silk dyed with A. hispidus were provided by the Kihachijo Textile Co-op Association, Tokyo.
330 412
432
O 448
A. hispidus
416
432
mAU at 350nm
2.2. Extraction of plant material
432 430
432 O O 448 448
432 V 432
430
M. tinctorius HCl 416 286
Retention time (min) Fig. 1. Representative HPLC profiles of extracts from the plants, A. hispidus and M. tinctorius. Acid-treated extracts are marked “HCl.” The profiles shown were monitored at 350 nm; the molecular masses of some components are given above the peaks. O and V refer to the C-glucosides, orientin/isoorientin and vitexin/isovitexin, respectively.
mixture described here because (1) it is a better solvent for dyes of all color, (2) it allows for a higher extraction temperature and (3) its higher boiling point leads to less “bumping” during evaporation under vacuum. Oxalate anion [13] is a superior chelator of metal ions than is formate. 2.5. LC–MS analysis Extracts of plant material or of dyed silk were analyzed by HPLC–DAD–MS using an Agilent 1100 high performance liquid chromatography (HPLC) system consisting of an automatic injector, a gradient pump, a HP series 1100 diode array detector (DAD), and an Agilent series 1100 VL on-line electrospray ionization mass spectrometer (MS), essentially as described earlier by Zhang and Laursen [12]; the electrospray chamber was run at 350 ◦ C with a gas flow of 12 L/min and a Vcap of 3000 V. Operation of the system and data analysis were done using Chemstation software, and detection was generally done in the negative ion [M−H]− mode. Separation of dye components was made on a Vydac C18 reversed phase column (2.1 mm dia. × 250 mm long; 5-m particle size), operated at a flow rate of 0.25 mL/min. Columns were eluted with acetonitrile–water gradients containing 0.1% formic acid. In the course of this work, several gradients were used, but because the types of stationary and mobile phases were unchanged, the retention times of peaks were always in the same order. 2.6. UPLC–ESI-MS/MS experiments MS spectra were acquired using data-dependent acquisition (DDA) on a UPLC–MS/MS system comprised of an Acquity UPLC (Waters Corp., Milford, MA) and a linear ion trap quadrupole (LTQ) mass spectrometer (Thermo Electron Corp., San Jose, CA). The UPLC was equipped with a 2.1 mm × 150 mm T3 HSS reversed phase
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OH H3C
OH
O
OH
O HO
OH O
8
OH
OH
O
OH
OH O
OH
HO
O
OH
6 OH
OH
O
Luteolin 8-C-rhamnoside, 1 (or isomer) [M = 432]
OH
O
Arthraxin [M = 412]
O
Luteolin [M = 286]
O H3C
OCH3
OH
OH
OH
O
OH O
HO
OH
HO
OH
O
OH
O
Luteolin 8-C-4-ketorhamnoside, 3 (or isomer) [M = 430]
OCH3
OH Glucose-O
O
O
OH
OH
O
Luteolin 7-glucoside [M = 448]
Tricin [M = 330]
OH H3C
OH OH
O
OH
OH O
HO
OH
HO
O
O
OH
Apigenin 8-C-rhamnoside, 2 (or isomer) [M = 416]
OH HO
O
O
OH
8-Prenylapigenin [M = 338]
O
OH
+ 2 -CH2-
Dimethylluteolin? [M = 314]
Fig. 2. Putative structures of flavonoids referred to in this article.
column for separation with a gradient of 20% to 40% B over 40 min at a flow rate of 0.1 mL/min. Mobile phase A was 0.03% trifluoroacetic acid in water, and mobile phase B was 0.024% trifluoroacetic acid in acetonitrile. The separation was monitored at 350 nm. MS/MS data were acquired by data-dependent acquisition using pulsed Q dissociation (PQD) detection. PQD parameters were set at isolation width 2 m/z, normalized collision energy 35%, activation Q 0.7, and activation time (T) 0.1 ms; the threshold for MS/MS acquisition was set to 1000 counts. 3. Results and discussion One of the first observations made was that HPLC profiles of extracts of both M. tinctorius and A. hispidus were little changed by acid hydrolysis (Fig. 1), as discussed in detail below, although one small peak later shown to be luteolin 7-O-glucoside was converted to luteolin. These findings are in agreement with the observations of Wouters [4] for M. tinctorius, who used strong acid to extract dyes from textile fibers and UV–Vis detection of peaks. Furthermore, we found that UV–Vis spectra of individual peaks in the profiles were characteristic of flavones (e.g., luteolin), and that the HPLC retention times and observed molecular masses were consistent with flavones conjugated to sugar residues. Similar results were obtained for extracts of dyed silk obtained from Hachijo Island, except for some low mass peaks (see at the end of Section 3.3.). From the foregoing information we concluded that most of the dye components in these plants were probably flavonoid C-glycosides. As will be discussed below, one of the difficulties in establishing representative HPLC profiles for these dyes is that the profiles
change, depending on growth location and conditions, time of harvest, dyeing techniques, etc. Most of the components are the same qualitatively—even comparing M. tinctorius and A. hispidus—but the quantitative differences are considerable. Nevertheless some of the major components provide useful markers, in particular those having molecular masses of 432, 416 and 330. Both A. hispidus and M. tinctorius are grasses and members of the Poaceae (Gramineae) family, in which the major flavonoids are C-glycosides and tricin (M = 330) [14], so in retrospect it is not surprising that these types of compounds are good markers for grass-dyed textiles. Another characteristic of HPLC profiles for these grass-derived dyes is their general shape, as seen in Figs. 1–5, which is a low, rounded “hump” surmounted by sharp peaks. This yet-unexplained feature is not seen for other natural dye profiles, where one generally sees a flat baseline (unpublished observations). 3.1. LC–MS profiles of plant extracts The flavonoid components of A. hispidus and M. tinctorius in extracted plant material and dyed silk were separated by reversed phase HPLC, and eluted peaks were monitored at 350 nm, which is near the maximum absorbance of most flavonoids in this study, and in tandem by mass spectrometery, usually in the negative ion mode. Fig. 1 shows representative HPLC profiles of extracts A. hispidus and M. tinctorius. The ratios of components in the profiles are quite variable, depending on the environment where the plants were grown and when they were harvested, but three of the compounds, designated 1, 2 and 3 in Figs. 2 and 4, with respective molecular masses of
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381
416 (2) 337
Relative abundance
369
430 (3)
417
399
313
283
413
431
329 299
432 432 (1)
397 353
200
250
300
433
415
329
299
350
400
450
m/z Fig. 3. MS/MS positive ion fragments of predominant flavonoid C-glycoside components of extracts from A. hispidus. [The same patterns (not shown) are also seen for M. tinctorius.] Only the ions referred to in Fig. 4 are labeled. Molecular masses of the compounds themselves are given in the upper, left-hand corner of the panels.
432, 416 and 430 Da, predominate. As will be discussed later, some of these compounds do not necessarily predominate in historical textiles. Kaneta and Sugiyama [3] (who did not have the benefit of HPLC for analysis) reported finding only luteolin, luteolin 7-glucoside, tricin (M = 330) and a new flavonoid, arthraxin (M = 412), in extracts of A. hispidus and M. tinctorius. As can be seen in Fig. 1, and as we have observed in many extracts of plants grown under a variety of conditions (data not shown), these components are present only in very minor amounts. In fact, we only see large peaks for luteolin (M = 286) in acid-treated extracts, where the O-glycoside, luteolin 7-glucoside (M = 448), which elutes at about 12 min, has
0,4X+
HO
O
OH
B
O
HO
A
C
OH
O
OH O
HO
R
0,1X+
R = OH Luteolin 8-C-rhamnoside (1) M = 432 R = H Apigenin 8-C-rhamnoside (2) M = 416 Main ions obtained by PQD-MS-MS (1) / (2) 433/417 [M+H]+ 415/399 [M+H-18]+ -H2O: E 1+ 397/381 [M+H-36]+ -2H2O: E 2+ 353/337 [M+H-80]+ 0,4X+-2H 2O 329/313 [M+H-104]+ 0,2X+ 299/283 [M+H-134]+ 0,1X+
A
C
OH
O
CH3
HO 0,2X+
O HO
0,4X+
O
CH2OH
HO 0,2X+
HO 0,1X+
0,4X+
HO
CH3
HO 0,2X+
been hydrolyzed to luteolin. Nevertheless, luteolin is generally a rather large peak in historical dyed textiles (see Section 3.3). Kaneta and Sugiyama [3] also reported finding some of these flavonoids in Miscanthus sinensis Andersson and the common reed, Phragmites communis Trinius [= Phragmites australis (Cav.) Trin. ex Steud]. However we did not find large amounts of any flavonoids in either of these species and conclude that they would not be useful dyestuffs. The well-known C-glycosides, vitexin/isovitexin (M = 432) and orientin (luteolin 8-C-glucoside)/isoorientin (luteolin 6-Cglucoside) (M = 448), labeled, respectively, “V” and “O” elute between 5 and 10 min in Fig. 1 and are unchanged, as expected, by acid hydrolysis.
O
B
Vitexin Main ions obtained by PQD-MS-MS Vitexin 433 [M+H]+ 415 [M+H-18]+ -H2O: E1+ 397 [M+H-36]+ -2H2O: E2+ 367 [M+H-66]+ 2,3X+-2H2O 337 [M+H-96]+ 0,4X+-2H2O 313 [M+H-120]+ 0,2X+ 283 [M+H-150]+ 0,1X+
OH
HO O
HO 0,1X+
OH
OH
O
Luteolin 8-C-4-ketorhamnoside (3) M = 430 Main ions obtained by PQD-MS-MS (3) 431 [M+H]+ 413 [M+H-18]+ -H2O: E1+ 369 [M+H-62]+ 0,4X+-H2O 329 [M+H-102]+ 0,2X+ 299 [M+H-132]+ 0,1X+
OH
H2+O O
HO
A
C
OH
O
B
0,2B+
329
0,2X+
OH
OH
MS3
B OH
C +O
137
0,2B+
Fig. 4. Analysis of MS/MS fragmentation data for compounds 1, 2, and 3. The analysis is also shown for vitexin, which has the same molecular mass (432) as 1 (see [7]). The bottom of the figure shows the proposed consequence of MS3 fragmentation of the 0,2 X+ ion of 1. The ions present in greatest abundance are shown in bold font. Cleavage products are designated according to the nomenclature of Doman and Costello [23].
C. Mouri, R. Laursen / J. Chromatogr. A 1218 (2011) 7325–7330
432
A. hispidus on silk (Kihachijo)
330 286 416
338
M. M. tinctorius on on silksilk tinctorius (Yoshioka)
432
432
416 330
338
mAU at 350nm
416416 314314
Nara period M. tinctorius on silk (Uemura)
432
314
432
338
286
Heian period
Nara period
286 416 432 432
330
416 338
314
Retention time (min) Fig. 5. HPLC profiles (top to bottom panels, respectively) of extracts of yellow dyed silk specimens, from Hachijo Island (21st C), from the sample book of Yoshioka [11] (20th C), dyed silk sample collection of Uemura (20th C), the Heian period (9–12th C), and the Nara period (8th C).
One significant difference in the profiles of A. hispidus and M. tinctorius is the presence, in the M. tinctorius profile (Fig. 1), of a large peak of M = 576, which is the same mass as that for maysin [2 -O-␣rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)luteolin] described by Elliger et al. [15]. Treatment of the M. tinctorius extract with acid results in loss of the 576 peak (due to loss of a 146-Da rhamnosyl unit?) and appearance of a new one with M = 430 (Fig. 1). This new peak could be an epimer of the M = 430 (3) compound we analyzed below (see Section 3.2), since the both ␣-keto hydrogens can epimerize under acidic conditions. Unfortunately, the M = 576 peak is not useful for distinguishing between A. hispidus and M. tinctorius because it does not appear in either historical dyed textiles or in textiles we have dyed in our own laboratory (by the methods described in [16]). Possibly it is not adsorbed efficiently by the textile fibers due to its relatively high molecular weight. This idea is not without precedent since it has been noted that yarns dyed sequentially in the same madder dye bath had different colors: the first batch of yarn has a typical red color due to selective adsorption of alizarin and purpurin, whereas the second batch has a yellower shade due to adsorption by the higher molecular weight glycosides [17]. 3.2. MS–MS and MS3 analyses Fig. 3 shows MS/MS fragmentation profiles for the positive ions at [M+H]+ = 433, 417 and 431, corresponding to compounds 1, 2 and 3, respectively; Fig. 4 outlines the analyses of these profiles. Initially
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we suspected that the M = 432 peak might be vitexin (or isovitexin), which also is a C-glycoside, has the same mass and is widely found in plants, but comparison of retention times with vitexin, which is known to occur in large amounts in P. incarnata and C. monogyna, showed that 1 could not be vitexin. Furthermore, as shown in Fig. 4, the fragmentation patterns for vitexin [7] and 1 are significantly different, vitexin being the C-glucoside of apigenin. Further analysis of the fragments of 1 indicate that it is a 6-deoxyhexoside derivative of luteolin. Whether the sugar moiety of 1 is rhamnose or not cannot be stated with certainty at this point, but rhamnose is the most common deoxyhexose associated with flavonoids [8]. Nor can it be ascertained which anomeric form is present. MS3 fragmentation of the 0,2 X+ ion of 1 at m/z = 329 gave the 0,2 B+ fragment confirming the presence of the luteolin aglycone, rather than apigenin. The absence of 1,3 A+ fragments is consistent with substitution at the 8-position, rather than at the 6-position [7]. Substitution at the 8-position is also analogous to the structure of arthraxin (see Fig. 2), which is reported to have a C-8 carbon–carbon bond [3]. It can also be seen in Fig. 1, that there are at least three peaks (in addition to vitexin) that have molecular masses of 432. Although the largest and most consistently appearing peak, which elutes at about 20 min in Fig. 1, gives the fragmentation pattern shown in Fig. 4; the other M = 432 peaks display similar fragmentation patterns (data not shown). From this we conclude that all of the M = 432 peaks have the same sugar carbon skeleton. Since structure 1 has five chiral centers, many isomers are theoretically possible. Positional isomers (e.g., 6- and 8-substitution) are also possible. Compound 2, which differs from 1 by an oxygen atom (16 Da), shows a fragmentation pattern similar to that of 1, except that all of the fragments are 16 Da lower (Figs. 3 and 4). By analogy, we propose that compound 2 is apigenin 8-C-rhamnoside (or an isomer). Compound 3 differs from 1 by only 2 Da. As can be seen in Figs. 3 and 4, 3 shows only an E1 + ion (loss of one water) and not the E2 + ion (loss of two waters) seen for 1 and 2, since loss of two water molecules for a 4-keto-6-deoxyhexose would be impossible. On the other hand, 3 has the same 0,2 X+ and 0,1 X+ fragments seen for 1. Compound 3 is isomeric with (if not identical to) derhamnosylmaysin described by Elliger et al. [14]. 3.3. Flavonoid C-glycosides as textile dyes Fig. 5 shows three profiles of silk extracts where the dyestuff is known, and two where it is unknown. The “Kihachijo” specimen was textile material obtained from Hachijo Island in Japan, where only A. hispidus is used as a dyestuff. The other two specimens, “M. tinctorius on silk” were from the sample book of Yoshioka [11] and from a collection of dyed textiles samples prepared by Uemura. The Nara and Heian profiles were from extracts of threads from textiles in the collection of the Museum of Fine Arts, Boston. Some of these specimens were green, having been dyed, as the current studies have shown, with a yellow dye and indigo. The HPLC profiles (in Fig. 5) indicate that they were dyed with a grass-type dyestuff, such as A. hispidus or M. tinctorius. [In some other specimens, the yellow was a protoberberine dye (data not shown).] Earlier studies of some Nara period textiles by Uemura and Takagi [18,19] predated HPLC and could not distinguish between various flavonoids. The only recent published analysis of textiles of this age is by Nakamura et al. [20], who used spectroscopic methods that also do not distinguish between the thousands of known flavonoids. There are differences between the profiles of the plant extracts (Fig. 1) and the textile extracts shown in Fig. 5 (and in many other samples we have analyzed). Although most of the dyed textile samples show the M = 432 peak discussed above as a predominant peak, many of them also show strong luteolin (M = 286) and tricin (M = 330) peaks, as well as variable amounts of we suspect to be
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prenylluteloin (M = 338) and one of the dimethylluteolins (M = 314) (see Fig. 2), based on their molecular masses and HPLC retention times. All of the latter are known flavonoids. Of the aforementioned compounds, luteolin is not useful as a marker because it is found in many plants used for dyeing; luteolin and its derivatives are the most common of the flavone dyes. We still do not have an explanation for its prevalence in the dyed objects examined here, because it is present in very small amounts in unhydrolyzed plant extracts. It does, however, appear in HCl-treated extracts (Fig. 1), presumably due to hydrolysis of luteolin 7-O-glucoside. Arthraxin (M = 412; see Fig. 2), reported by Kaneta and Sugiyama [3] to be a major component of A. hispidus and M. tinctorius, is actually a minor component of plant extracts (Fig. 1) and is hardly detectable in textile extracts (Fig. 5). Dyeing of textiles and textile fibers typically employs as many as 20 repeated dyeing cycles (cf. [21]) in order to get intense, saturated colors. We suspect that the reason for the increased amounts of luteolin, tricin and other alkylated luteolins in dyed textiles is that these compounds have lower molecular weights and are more hydrophobic (they elute late on reversed-phase HPLC columns) than the flavonoid glycosides, and therefore are preferentially adsorbed by the textile fibers. Confirming this idea is our observation that we do not see any of the large, hydrophilic M = 576 component (which contains two sugars) of M. tinctorius (see Fig. 1) in extracts of silk that we have dyed (data not shown). There may be other explanations, but we do not think it is due to hydrolysis of the glycosidic linkage during the dyeing process, since we have never seen hydrolysis when dyeing with other flavonoid glycosides. 4. Conclusions Although we are unable, so far, to distinguish between textile fibers dyed with extracts of A. hispidus and M. tinctorius, there are several characteristics that distinguish this class of dyes from others: 1. The grass-derived dyes (e.g., from A. hispidus and M. tinctorius) are primarily C-glycosides, which are stable to hydrolysis by strong acids. Therefore profiles of acid-treated samples are similar to those of untreated ones. 2. The HPLC profiles of the flavonoid components of A. hispidus and M. tinctorius are rather distinctive, being characterized by a series of sharp peaks atop a sort of rounded “hump” between the earliest and latest eluting peaks (see Figs. 1 and 5). 3. One of the early-eluting C-glycoside peaks, and often the largest in dyes derived from A. hispidus and M. tinctorius, has M = 432; it is frequently followed, roughly a minute later (depending on the elution gradient), by another C-glycoside with M = 416. 4. Most grasses contain tricin (M = 330), which is often a large peak in dyed textiles and is also a useful marker for A. hispidus and M. tinctorius. Flavonoid C-glycosides have been known for many years, and undoubtedly occur in other known plant dyestuffs (cf. [22]), but
compounds 1, 2 and 3 are the first to be so documented. Grasses seem to have been little (if at all) used outside of Asia for dyeing, and the finding that the majority of flavonoids in A. hispidus and M. tinctorius are C-glycosides is consistent with reports [14] that most members of the Poaceae family are rich in flavonoid C-glycosides. Although the structures of 1, 2 and 3 remain to be elucidated absolutely, the first two of these compounds represent unique markers for textile fibers dyed with A. hispidus or M. tinctorius. Acknowledgements We are grateful to Ms. Monika Vecchi and Dr. Dingyi Wen at BiogenIdec for MS2 and MS3 experiments and Masumi Kataoka and Meredith Montague, Museum of Fine Arts for specimens of Japanese dyed textiles. For generous gifts of plant material, we would like to thank Dr. Yasushi Ibaragi, Tokushima Prefectural Museum; Dr. Eisuke Hayasaka, Fukui Botanical Garden; Dr. Yohei Sasaki, Kanazawa University; and the Kihachijo Textile Co-op Association, Tokyo. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2011.08.048. References [1] A. Waley, J.R. Allen, The Book of Songs, Grove Press, New York, 1996, p. 215 (In this poem, ‘Green ( )’ refers to Jinso = Arthraxon hispidus). [2] S. Goto, R. Yamakawa, Senryo-syokubutsu-fu (About Dye Plants), Hakuhosya, Kyoto, 1972 (in Japanese). [3] M. Kaneta, N. Sugiyama, Bull. Chem. Soc. Jpn. 45 (1972) 528. [4] J. Wouters, Dyes Hist. Archaeol. 12 (1994) 12. [5] G.C. Kite, E.A. Porter, F.C. Denison, R.J. Grayer, N.C. Veitch, I. Butler, M.S.J. Simmonds, J. Chromatogr. A 1104 (2006) 123. [6] F. Ferreres, A. Gil-Izquierdo, P.B. Andrade, P. Valentão, F.A. Tomás-Barberán, J. Chromatogr. A 1161 (2007) 214. [7] P. Waridel, J.-L. Wolfender, K. Ndjoko, K.R. Hobby, H.J. Major, K. Hostettmann, J. Chromatogr. A 926 (2001) 29. [8] T. Fossen, Ø.M. Andersen, in: Ø.M. Andersen, K.R. Markham (Eds.), Flavonoids: Chemistry, Biochemistry and Applications, Taylor & Francis, New York, 2006, p. 37. [9] H. Schilcher, Z. Naturforsch. B 23 (1968) 1393. [10] A. Rehwald, B. Meier, O. Sticher, J. Chromatogr. A 677 (1994) 25. [11] T. Yoshioka, Nihon no Iro, Shokubutsu Senryo no Hanashi (Japanese Colors: The Story of Natural Dyes), Shikosha, Kyoto, 1983 (in Japanese). [12] X. Zhang, R.A. Laursen, Anal. Chem. 77 (2005) 2022. [13] R. Marques, M.M. Sousa, M.C. Oliveira, M.J. Melo, J. Chromatogr. A 1216 (2009) 1402. [14] J.B. Harborne, C.A. Williams, Biochem. Syst. Ecol. 4 (1976) 267. [15] C.A. Elliger, B.G. Chan, A.C. Waiss Jr., R.E. Lundin, W.F. Haddon, Phytochemistry 19 (1980) 293. [16] E.S.B. Ferreira, A. Quye, H. McNab, A. Hulme, Dyes Hist. Archaeol. 18 (2002) 63. [17] D. Cardon, Natural Dyes: Sources, Tradition, Technology and Science, Archetype, London, 2007, p. 136. [18] R. Uemura, Y. Takagi, Shoryobukiyo 19 (1967) 63. [19] Y. Takagi, Shoryobukiyo 21 (1969) 48. [20] R. Nakamura, Y. Tanaka, A. Ogata, M. Naruse, Anal. Chem. 81 (2009) 5691. [21] S. Yoshioka, Shizen no iro wo someru (Natural Color Dyeing), Shikosha, Kyoto, 1996, p. 30 (in Japanese). [22] W. Nowik, J. Sep. Sci. 28 (2005) 1595. [23] B. Domon, C.E. Costello, Glycoconj. J. 5 (1988) 123.