Two validated HPLC methods for the quantification of

PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 15, 397–406 (2004) DETERMINATION OF ANTHRAQUINONES IN RUBIA TINCTORUM Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.800

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Two Validated HPLC Methods for the Quantification of Alizarin and other Anthraquinones in Rubia tinctorum Cultivars Goverdina C. H. Derksen, Gerrit P. Lelyveld, Teris A. van Beek,* Anthony Capelle and Æde de Groot Laboratory of Organic Chemistry, Natural Products Chemistry Group, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands

Direct and indirect HPLC-UV methods for the quantitative determination of anthraquinones in dried madder root have been developed, validated and compared. In the direct method, madder root was extracted twice with refluxing ethanol–water. This method allowed the determination of the two major native anthraquinone glycosides lucidin primeveroside and ruberythric acid. In the indirect extraction method, the anthraquinone glycosides were first converted into aglycones by endogenous enzymes and the aglycones were subsequently extracted with tetrahydrofuran–water and then analysed. In this case the anthraquinones alizarin, purpurin and nordamnacanthal may be determined. The content of nordamnacanthal is proportional to the amount of lucidin primeveroside originally present. The indirect extraction method is easier to apply. Different madder cultivars were screened for their anthraquinone content. Copyright © 2004 John Wiley & Sons, Ltd. Keywords: HPLC; quantitative analysis; alizarin; ruberythric acid; madder; Rubia tinctorum; cultivars.

INTRODUCTION The roots of Rubia tinctorum (madder) are the source of a natural dye and have been used to dye textiles in many parts of the world since ancient times. The dye components are anthraquinones with alizarin, the hydrolysis product of ruberythric acid, being the main dye constituent. At the end of the nineteenth century the cultivation of madder rapidly declined owing to the introduction of a cheap synthetic process for the production of alizarin (Thomson, 1971; Schweppe, 1993). As a result of the polluting side products that are formed during the synthesis of alizarin, and the growing popular awareness of the environment, increasing efforts are being made to revive the cultivation of madder as a dye plant. One important element in the revitalisation of natural alizarin as a commercial commodity is that farmers should be able to supply plentiful amounts of cheap, high-quality, plant material. This could be achieved by the selection of a cultivar of R. tinctorum with advantageous agronomic characteristics such as plant density, root density, root thickness, growing rate, resistance to diseases and content of ruberythric acid and alizarin. For the quantification of anthraquinones in madder roots a simple and reliable analytical method is necessary. For the rapid quantification of anthraquinones in cell suspension cultures most researchers have used the method developed by Zenk et al. (1975) (e.g. Leistner, 1975; Schulte et al., 1984; Khouri et al., 1986; Strobel et al., 1990; van der Heijden et al., 1994; Kuzovkina et al., 1996; Ramos-Valdivia et al., 1998; van der Plas et al., 1998). In this method, the absorption of an 80% ethanol ∗ Correspondence to: T. A. van Beek, Laboratory of Organic Chemistry, Natural Products Chemistry Group, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands. Email: [email protected]

Copyright © 2004 John Wiley & Sons, Ltd. Copyright © 2004 John Wiley & Sons, Ltd.

extract of a cell suspension culture is measured at 434 nm, the absorption maximum of alizarin. A modification of the method, consisting of adding one drop of 5% potassium hydroxide and measuring at 520 nm, has also been used for quantification purposes (Suzuki et al., 1984; Wijnsma et al., 1985; van der Heijden et al., 1994). A disadvantage is that no distinction can be made between the different anthraquinones present in the extracts. In order to obtain information about individual anthraquinones HPLC-UV is necessary, but most HPLC methods described in the literature can be used to quantify only one or two anthraquinones (Tóth et al., 1993; Lodhi et al., 1994; Krizsán et al., 1996; Angelini et al., 1997; Novotná et al., 1999; Bosáková et al., 2000). In this paper the development of two new methods for the quantification of the most abundant anthraquinones in R. tinctorum, and their application in the screening of several cultivars of this species, are described.

EXPERIMENTAL Chemicals. Acetonitrile (HPLC grade), chloroform and tetrahydrofuran were obtained from Lab-Scan Analytical Sciences (Dublin, Ireland). Ammonium formate, formic acid and ethanol were obtained from Acros (Geel, Belgium). Ultrapure water was obtained from a combined Seradest LFM 20 Serapur Pro 90 C apparatus (Seral, Beun de Ronde, Abcoude, Netherlands). All HPLC solvents were degassed prior to use by vacuum filtration over a 0.45 µm membrane filter (type RC; Schleicher and Schuell, Einbeck Germany). Unless mentioned otherwise, filtration implies that the madder suspension was filtered through a filter paper (Schleicher and Schuell) under reduced pressure in a Büchner funnel. All solvent ratios of eluents or solvent mixtures Received 5 January 2004 Phytochem. Anal. 15: 397–406 (2004) Revised 15 May 2004 Accepted 15 May 2004

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are volume ratios. Dimethylsulphoxide-d6 (Acros) was used as the solvent for NMR spectroscopy. Reference compounds. Alizarin (1,2dihydroxyanthraquinone) and purpurin (1,2,4trihydroxyanthraquinone) were purchased from Acros (Geel, Belgium), and crude ruberythric acid was from Carl Roth (Karlsruhe, Germany). Lucidin primeveroside and ruberythric acid were separated and purified by droplet counter-current chromatography as described earlier (Derksen et al., 1998). Xanthopurpurin and the mutagenic lucidin (Brown and Brown, 1976; Brown and Dietrich, 1979; Brown, 1980; Tikkanen et al., 1983; Yasui and Takeda, 1983; Poginsky et al., 1987, 1991; Westendorf et al., 1988, 1990; Wölfe et al., 1990; Ino et al., 1995) were synthesised in two steps according to the method of Murti et al. (1970) and purified by chromatography on silica (Derksen et al., 1998). Pseudopurpurin, munjistin and nordamnacanthal were isolated from R. tinctorum: dried madder root (50 g) was stirred in ultrapure water (2 L) at room temperature for 2 h and the aqueous layer was extracted twice with ethyl acetate (500 mL). The two immiscible layers were separated with a separatory funnel, the ethyl acetate layers were combined and both the ethyl acetate layer, containing nordamnacanthal, and the aqueous layer, containing the acidic anthraquinones pseudopurpurin and munjistin, were further processed as follows. (i) The ethyl acetate layer was dried under reduced pressure (1.2 g), redissolved in tetrahydrofuran– water (1:1) and transferred to a column containing 15 g of C18 Bondelut (40 µm; Baker, Deventer, Netherlands). The column was eluted with tetrahydrofuran–water (1:1) (before use the eluent was flushed through with nitrogen) and 18 coloured fractions were collected. The last coloured fraction (no. 18) was obtained by eluting the column with 100% tetrahydrofuran. The fractions were analysed by HPLC, and those which contained nordamnacanthal were stored in a refrigerator overnight, after which time most of the precipitated nordamnacanthal was obtained by filtration. The remaining solutions and filtrates containing nordamnacanthal were pooled and t-butylmethyl ether (10%) was added, which caused phase separation. The organic layer was dried and pooled with the precipitated nordamnacanthal. The resulting product (57 mg) was stored in a freezer under nitrogen. (ii) The original aqueous layer was filtered and the filtrate (1800 mL) was added directly to a Sephadex LH20 column and eluted with water. Brown, red and yellow fractions were obtained and analysed by HPLC. Pure fractions were pooled and freeze-dried with a Christ Alpha 1–2 freeze dryer (Salm en Kipp, Breukelen, The Netherlands) to yield pseudopurpurin (87 mg; red fraction) and munjistin (32 mg; yellow fraction). Purity reference compounds. The purity of all reference compounds was checked by HPLC–UV/vis (photodiode array detection; Derksen et al., 2002), MS (HPLC-ESI/ NI; Derksen et al., 2002), qualitative 400 MHz 1H-NMR, 100 MHz 13C-NMR, and quantitative 400 MHz 1H-NMR spectroscopy. For the latter technique, maleic acid was used as internal standard and dimethylsulphoxide-d6 as solvent (van Beek et al., 1993). Plant material. Roots from several cultivars of R. tinctorum and one of R. cordifolia were collected on 12 Copyright © 2004 John Wiley & Sons, Ltd.

November 1999 in Zuidbroek, Groningen, The Netherlands. Two- and three-year-old roots from the same cultivar were collected in Borkel en Schaft, Brabant, The Netherlands. Herbarium accession numbers are available on request from the Department of Taxonomy of Wageningen University, The Netherlands. The fresh roots were dried at 45°C for 1 week in an oven with forced ventilation. The weights of the roots before and after drying were compared using an analytical balance. The dried roots were powdered in a Retsch Grindomix model GM200 (Emergo, Landsmeer, The Netherlands) for 1 min at 7000 rpm, for 1 min at 8500 rpm and for 1 min at 10,000 rpm. Quantification method based on the glycosides present in madder root (direct extraction method). Dried and powdered root material (2.5 g) was refluxed with 100 mL water–ethanol (1:1) for 2 h and the suspension was filtered. The total volume (mL) of the filtrate was determined by decanting into a measuring cylinder. A sample (100 µL) of the filtrate was diluted with 900 µL of water–methanol–formic acid (1:1:0.005). The residue from filtration was refluxed with 50 mL water–ethanol (1:1) and after 2 h the suspension was filtered. The volume of the filtrate was determined (mL). A sample (500 µL) of the filtrate was diluted with 500 µL of water–methanol–formic acid (1:1:0.005). All experiments were performed in triplicate. Quantification method based on aglycones formed by hydrolysis of the corresponding glycosides (indirect extraction method). Dried and powdered madder root (0.25 g) was stirred in 10 mL of ultra pure water for 1 h at 45°C (or for 2 h at 25°C). After stirring, 40 mL tetrahydrofuran–water–formic acid (1:1:0.005) were added and the mixture stirred for another 30 min at room temperature. A sample (1000 µL) was diluted with 1000 µL of tetrahydrofuran–water–formic acid (1:1:0.005). All experiments were performed in triplicate. Direct extraction method—extraction efficiency. Dried and powdered root material (2.5 g) was refluxed with 100 mL water–ethanol (1:1). After 2 h the suspension was filtered, the volume of the filtrate was determined (mL) and a sample (100 µL) diluted with 900 µL water– methanol–formic acid (1:1:0.005). The residue was refluxed with 100 mL water–ethanol (1:1). After 2 h the suspension was filtered and a sample (500 µL) was taken. Extraction and filtration of the residue was repeated twice and samples (500 µL) were again taken. Finally the residue was extracted and filtered for a fourth time but with 100 mL of tetrahydrofuran instead of water– ethanol. All 500 µL samples were diluted with 500 µL water–methanol–formic acid (1:1:0.005). The volumes (mL) of the filtrates were determined. Indirect extraction method—influence of extraction volume and time. Five samples of 2.5 g of dried and powdered madder root were stirred in 100 mL water at 45°C. After 1 h, volumes of 25, 50, 100, 200 or 400 mL of tetrahydrofuran–water–formic acid (1:1:0.005) were added to samples 1–5 respectively and each solution was stirred. After 1 h and after 64 h of stirring, samples of 1000 µL were taken. The experiment was performed in duplicate. Phytochem. Anal. 15: 397–406 (2004)

DETERMINATION OF ANTHRAQUINONES IN RUBIA TINCTORUM

Indirect extraction method—influence of sample size on standard deviation. Two series of four different suspensions of dried and powdered madder root were stirred in water (2.5 g in 100 mL, 1.25 g in 50 mL, 0.63 g in 25 mL and 0.25 g in 10 mL) for 1 h at 45°C. After 1 h, volumes of 400, 200, 100 and 40 mL of tetrahydrofuran–water– formic acid (1:1:0.005) were added, respectively, to the different suspensions. After 1 h stirring, samples of 1000 µL were taken. Indirect extraction method—influence of temperature. Six portions of 0.25 g of dried and powdered madder root were stirred in 10 mL of water of 45°C. After 1 h, 40 mL of tetrahydrofuran–water–formic acid (1:1:0.005) was added to each solution. Two were refluxed for 1 h, two were stirred at 45°C and two were stirred at room temperature. After 1 h, samples of 1000 µL were taken. All samples were diluted with 1000 µL of tetrahydrofuran– water–formic acid (1:1:0.05). Recovery—direct extraction method. Dried and powdered madder root (7.5 g) was refluxed in 100 mL ethanol–water (1:1). After 2 h the suspension was filtered, the volume of filtrate adjusted to 300 mL with ethanol–water (1:1), the filtrate divided into three equal portions of 100 mL and transferred to three round bottomed flasks. From each flask a sample of 100 µL was taken, diluted with 900 µL methanol–water–formic acid (1:1:0.005) and analysed (to give the 100% value). The three filtrates were refluxed and filtered and the residues were refluxed and filtered again as described for the direct extraction method. Samples were taken from the filtrates and analysed, and the recovery thus determined. Recovery—indirect extraction method. Dried and powdered madder root (1 g) was stirred in 40 mL water at 45°C for 1 h after which 160 mL of water was added. The solution was heated (90°C) and filtered, the filtrate cooled and three portions of 50.00 mL taken and transferred to three round bottomed flasks. From each solution a sample (500 µL) was taken, diluted with 500 µL tetrahydrofuran–water–formic acid (1:1:0.005) and analysed by HPLC (to give the 100% value). The three madder root water filtrates were lyophilised, resuspended in 10 mL of water and treated as described for the indirect extraction method. Samples were taken, analysed by HPLC and the recovery thus determined. Liquefaction. Dried and powdered madder root (0.25 g) was suspended in 10 mL of water or 10 mL of 0.02 M sodium acetate buffer (pH 4.5) and 200 µL aliquots of commercial enzyme preparations Viscozyme L (a pectinase with some cellulolytic and hemicellulolytic activity; Novo Nordisk) and Celluclast 1.5 L (mainly cellulase activity; Novo Nordisk, Bagsvaerd, Denmark) were added (triplicate experiments). One half of the samples were stirred for 2 h while the other half were stirred for 80 h at 45°C. After stirring, 40 mL of tetrahydrofuran–water–formic acid (1:1:0.005) were added. The diluted samples were stirred for 30 min at 45°C and a 1000 µL sample was diluted with 1000 µL of tetrahydrofuran–water–formic acid (1:1:0.005). HPLC analysis. All of the obtained (diluted) samples were filtered through a 0.45 µm, 25 mm diameter Copyright © 2004 John Wiley & Sons, Ltd.

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membrane filter (type RC; Schleicher and Schuell) and analysed by HPLC. Analyses were carried out at room temperature on an Alltima (Alltech, Breda, Netherlands) end-capped C18 column (250 × 4.6 mm i.d.; 100 Å pore size, 5 µm particle size) equipped with a pre-column. Prior to use, solvents were filtered through a 0.45 µm, 50 mm diameter membrane filter (type RC; Schleicher and Schuell) and sonicated for 15 min in a Retsch Transsonic 570 bath (Emergo, Landsmeer, The Netherlands). Chromatography was carried out using a mobile phase consisting of ammonium formate–formic acid buffer (0.2 m, pH 3) containing EDTA (30 mg/L) (solvent A) and acetonitrile (solvent B). A linear gradient programme was applied as follows: 0–6 min isocratic with 27% B; 6–20 min linear increase to 60% B; 20– 23 min isocratic with 60% B; 23–25 min linear increase to 70% B; 25–35 min isocratic with 70% B; 35–40 min linear decrease to 27% B. The flow-rate during the analysis was 1.0 mL/min and peaks were detected at 254 nm. In Table 1, the correlation coefficient of the calibration curves, the molar extinction coefficient, and the detection range for the different anthraquinones are reported. Although detection at 434 nm is more selective, detection at 254 nm was more sensitive and did not pose a problem in terms of co-elution with impurities. The HPLC-apparatus was a Gilson (Goffin-Meyvis, Bergen op Zoom, The Netherlands) system comprising a 305 piston pump, a 306 piston pump, an 811 C dynamic mixer and an 805 manometric module connected to a 234 autoinjector and a Shimadzu (‘s-Hertogenbosch, the Netherlands) Chromatopac C-R3A integrator. Detection was performed using a Gilson 116 UV detector. HPLC-PAD and HPLC-MS experiments were carried out as described earlier (Derksen et al., 2002).

RESULTS AND DISCUSSION For the large-scale screening of madder roots a simple and selective quantitative extraction of the main anthraquinones is necessary. The main bottleneck is not the separation and detection, which can be easily performed by HPLC-UV, but the instability and poor solubility of several anthraquinones. The main anthraquinone glycosides are easily converted into their aglycones by endogenous enzymes (Derksen et al., 2003) and the anthraquinone carboxylic acids are readily decarboxylated. Because of this, two different methods for the quantitative determination of anthraquinones in madder were developed and compared. Pre-treatment of plant material The water content of fresh roots varied from 62 to 69%. Fresh roots were air-dried for one week and ground in a mixer at three different increasing speeds. With this grinding procedure the endogenous enzymes were not denatured. A qualitative comparison of the chromatograms obtained from fresh roots and from the same roots after drying did not show any degradation or changes as a result of the drying or grinding. The roots could be stored for at least 4 years at room temperature without significant deterioration of the anthraquinones or of the endogenous enzymes. Phytochem. Anal. 15: 397–406 (2004)

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Table 1. Purity, range of anthraquinone concentrations used for the calibration curve, calibration curve equation, correlation coefficient (r 2) and molar extinction coefficients (ε) at 254 nm and at 434 nm Compound Lucidin Ruberythric acid Pseudopurpurin Munjistin Lucidin Alizarin Purpurin Nordamnacanthal

Purity (%)

Minimum– maximum (mg/L)

Calibration curve (x = mg/L; y = area)

r2

ε 254 nm (L/mol cm)

ε 434 nm (L/mol cm)

92 86 54 52 74 96 53 53

1.8–46.2 1.8–43.9 2.1–53.8 2.1–51.9 2.6–65.5 5.1–127.1 2.2–54.2 2.1–53.1

y = 5835x + 539 y = 7694x + 727 y = 9814x − 7343 y = 20820x − 915 y = 13028x − 2681.7 y = 15094x + 19666 y = 14427x + 1261.2 y = 9369x − 12734

1.0000 0.9999 0.9999 1.0000 1.0000 1.0000 0.9997 0.9976

2.19 × 104 3.33 × 104 2.89 × 104 3.51 × 104 0.80 × 104 2.82 × 104 4.64 × 104 3.00 × 104

4.52 × 103 5.78 × 103 3.87 × 103 6.59 × 103 4.73 × 103 6.03 × 103 7.65 × 103 5.60 × 103

Figure 1. Structures of anthraquinones.

Figure 2. HPLC profile of a water–ethanol (1:1) extract of madder root (direct extraction method). Key to peak identity: LP, lucidin primeveroside; RA, ruberythric acid; PP, pseudopurpurin; MU, munjistin; AL, alizarin; and PU, purpurin. (For chromatographic protocol see Experimental section.)

Quantification method based on the glycosides in madder root (direct extraction method) The main anthraquinones in madder roots are the glycosides ruberythric acid and lucidin primeveroside, and the anthraquinone carboxylic acids munjistin and pseudopurpurin (Fig. 1). Several solvents such as ethanol, methanol, water and a 0.5 m sodium hydroxide solution were tested for their ability to dissolve these compounds: ethanol–water (1:1) was found to be the most efficient. Extraction with ethanol–water was performed at different temperatures, namely, reflux, 80°C, 45°C and room temperature. Refluxing was found to be the best because at high temperatures filtration of the extract was much faster and a higher yield of anthraquinones was obtained. At lower temperatures the filter paper became clogged almost immediately, leading to poor recoveries. Increasing the particle size Copyright © 2004 John Wiley & Sons, Ltd.

did not solve this problem and increased the standard deviation. The extraction efficiency for lucidin primeveroside and ruberythric acid was determined by repeating the extraction of madder root four times. Of the total amount of anthraquinones present, >95% was extracted during the first extraction and ca. 99% was extracted after the second. The amount of sample (2.5 g) and solvent necessary for the above described extraction procedure was quite large. However, halving the sample size and the amount of solvent to 1.25 g and 50 mL, respectively, significantly decreased the reproducibility (RSD >15%). The final procedure of the direct extraction method consisted of extracting 2.5 g of madder root with 100 and 50 mL of ethanol–water (1:1). A typical chromatogram is depicted in Fig. 2. As part of the validation, the precision of the extraction method and the injection/integration procedure was Phytochem. Anal. 15: 397–406 (2004)

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Table 2. Concentrations of anthraquinones as determined by the direct ethanol–water extraction and the indirect extraction method. For every compound two relative standard deviations (RSD) are given: ‘RSD overall’ was obtained by injecting five extracts once, ‘RSD 1 extract’ was obtained by injecting one extract five times Compound Lucidin Ruberythric acid Pseudopurpurin Munjistin Alizarin Purpurin Nordamnacanthal

Direct method (mg/g root)

RSD overall

RSD 1 extract

Indirect method (mg/g root)

RSD overall

RSD 1 extract

24.58 10.11 3.72 2.30 0.54 2.08 0.65

3.5 3.3 5.6 6.1 7.6 12.5 20.0

1.2 1.2 1.7 1.0 0.7 3.9 —

0 0 8.64 3.69 6.33 1.06 12.53

— — 4.4 4.5 2.6 8.6 5.9

— — 1.3 2.3 1.9 8.2 1.8

Figure 3. Influence of extraction volume in the indirect extraction method showing the yield of anthraquinones (and standard deviation) after extracting a sample of 2.5 g madder root, which had been stirred for 1 h at 45°C in 100 mL of water, with different amounts of tetrahydrofuran– water–formic acid (1:1:0.005).

determined. Five samples (2.5 g each) from the same batch were taken, extracted and analysed as described for the direct extraction method. The RSD for the individual anthraquinones varied between 3.5 and 20.0% (Table 2). When the same HPLC sample extract was injected five times, the RSD varied between 0.7 and 3.9%. Based on these results it was decided to take three samples from every batch and inject every obtained HPLC sample once. Finally recovery experiments were performed. The recoveries were 93.8, 95.4, 94.8, 95.4, 96.4 and 107.8% for lucidin primeveroside, ruberythric acid, pseudopurpurin, munjistin, alizarin and purpurin, respectively. The high recovery of purpurin could be caused by the fact that pseudopurpurin is easily decarboxylated, but this is not confirmed by a lower recovery of pseudopurpurin. The most likely explanation is a high RSD for purpurin. Purpurin is the most troublesome compound of all of the anthraquinones dealt with in this study in terms of giving precipitations. The recovery values for all of the other analytes were around 95%. An explanation for these values could be precipitation during the filtration step. Quantification method based on aglycones (indirect extraction method) The fact that during stirring of madder root in water the glycosides in madder root can be easily converted into alizarin and nordamnacanthal by endogenous enzymes Copyright © 2004 John Wiley & Sons, Ltd.

(Derksen et al., 2003) was used to develop a quantification method. Because alizarin and nordamnacanthal are insoluble in water, they need to be redissolved before sampling. The solubility of the most important aglycone, alizarin was determined in various solvents such as chloroform, ethyl acetate, acetone, ethanol, tetrahydrofuran and 0.5 m sodium hydroxide. As tetrahydrofuran was the superior solvent, a mixture of water and tetrahydrofuran was chosen. In the initial procedure madder root (2.5 g) was stirred in water (100 mL) for 1 h at 45°C, following which 400 mL of tetrahydrofuran–water–formic acid (1:1:0.005) were added and the mixture stirred at room temperature for 1 h to dissolve the precipitated anthraquinone aglycones. Higher temperatures (45°C and reflux) were tried but did not lead to a higher yield. Next, a reduction in the sample size and the amount of solvent was tried. The necessary volume of solvent could be reduced from 400 to 200 mL tetrahydrofuran–water– formic acid when 2.5 g of madder root was used without any effect on recovery and reproducibility (Fig. 3). A further decrease in volume affected the results negatively. With regard to the duration of stirring, 1 h was found to be sufficient; after 24 h of stirring the same results were obtained. In an attempt further to reduce the amount of solvent, the sample size was lowered whilst keeping the ratio between the weight of madder root and the volume of water and added tetrahydrofuran–water–formic acid constant (1:40:160). The results are depicted in Fig. 4 and show that the sample size could be lowered to 0.25 g Phytochem. Anal. 15: 397–406 (2004)

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Figure 4. Influence of the sample size on the yield of the extracted anthraquinone aglycones (and standard deviation) for the indirect extraction method. The ratio between the weight of madder root and the volume of water and added tetrahydrofuran–water–formic acid was kept constant at (1:40:160).

Figure 5. HPLC profile of a madder root extract obtained after hydrolysis with endogenous enzymes (indirect extraction method). Key to peak identity: PP, pseudopurpurin; MU, munjistin; AL, alizarin; PU, purpurin; and ND, nordamnacanthal. (For chromatographic protocol see Experimental section.)

of ground madder root. Experiments with 0.125 g were also performed but were practically more difficult to perform without negatively affecting the yield or standard deviation. Thus for the indirect extraction method a 10 times lower amount of solvent was needed relative to the direct extraction method. Also for the indirect extraction method, the precision of the method and the injection/integration procedure was determined (Table 2). A typical HPLC profile is depicted in Fig. 5. In the method described above an acidified tetrahydrofuran–water solution was added to the madder root suspension, which led to decarboxylation of pseudopurpurin (Derksen et al., 2002) to purpurin. Probably all of the purpurin detected is formed from pseudopurpurin because, when no acid was present, no purpurin could be detected. However, when no acid is present, only half of the amount of the formed nordamnacanthal will dissolve in tetrahydrofuran–water and this is unacceptable for a quantitative determination of lucidin primeveroside. The amount of pseudopurpurin originally present in madder root can be calculated by multiplying the amount of purpurin by the factor 300/256 (the molecular weight of pseudopurpurin divided by the molecular weight of purpurin). It remains unclear whether both purpurin and pseudopurpurin are present in the fresh roots. In all of the analyses of madder root extracts performed in the course of this study purpurin was found. Copyright © 2004 John Wiley & Sons, Ltd.

The recovery experiments were performed with an aqueous madder root extract with known amounts of the different anthraquinones. The recoveries were 104, 115, 108, 106 and 110% for pseudopurpurin, munjistin, alizarin, purpurin and nordamnacanthal, respectively. A reason for the high recovery could be the evaporation of some tetrahydrofuran during the stirring of the solution at 45°C. As it is possible that not all anthraquinones are released by these extraction methods, madder root was treated with two commercial liquefaction enzymes, namely Viscozyme L (a pectinase with some hemicellulolutic and cellulolytic activity) and Celluclast 1.5 L (a cellulase). The amounts of anthraquinones determined in these samples were the same as those determined with organic solvents, indicating that no anthraquinones are strongly bound to polysaccharides or cell walls. The direct extraction method with ethanol–water did not give the same results for pseudopurpurin and munjistin as the indirect tetrahydrofuran–water method (Table 2). To check if this was caused by the extraction solvent, madder root was also extracted with tetrahydrofuran–water without prior enzymatic hydrolysis of the glycosides. In this case, more pseudopurpurin and munjistin were detected but still not as much as after conversion. This could point to the presence of a pseudopurpurin glycoside and a munjistin glycoside but no proof for this could be found with either HPLC-PAD or HPLC-MS. Phytochem. Anal. 15: 397–406 (2004)

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Figure 6. The concentration of lucidin primeveroside and ruberythric acid in mg/g root as determined by the direct ethanol–water extraction method and the concentration of nordamnacanthal and alizarin in mg/g as determined by the indirect tetrahydrofuran–water extraction method for different cultivars of Rubia tinctorum.

Method application Different cultivars of R. tinctorum were screened for their content of lucidin primeveroside and ruberythric acid with the direct extraction method (ethanol–water extraction) and for their content of pseudopurpurin, munjistin, alizarin, purpurin and nordamnacanthal with the indirect extraction method (conversion with endogenous enzymes). The results are depicted in Figs 6 Copyright © 2004 John Wiley & Sons, Ltd.

and 7. According to the supplier, all of the roots tested were of cultivars of R. tinctorum with the exception of batch 18, which was possibly R. cordifolia. However, according to the literature R. cordifolia should contain alizarin (Wijnsma et al., 1985; Schweppe, 1993). Thus the identity of batch 18 remains unclear. The original concentration of lucidin primeveroside and ruberythric acid can be calculated from the concentration of nordamnacanthal and alizarin as determined Phytochem. Anal. 15: 397–406 (2004)

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Figure 7. The concentrations of pseudopurpurin, munjistin and purpurin found in different cultivars of Rubia tinctorum after conversion of the glycosides and extraction with tetrahydrofuran–water (indirect method).

by the indirect extraction method. On average the concentration of ruberythric acid that was determined by recalculation from the alizarin content in the indirect method was 18% higher than the concentration of ruberythric acid as determined with the direct ethanol– water extraction. For lucidin primeveroside, the indirect method based on a recalculation of nordamnacanthal gave a 7% higher result than the direct ethanol–water method. For cultivar 16, the difference is relatively large. An explanation can be found in the recovery experiments. The recovery results (see above) show that the direct glycoside determination gave in general 5% lower values than anticipated while the indirect aglycone determination gave values that were 5–10% too high. When this is taken into account, the results found for the two different methods in the screening experiments are not significantly different. The concentrations that were found for lucidin primeveroside, ruberythric acid, alizarin and nordamnacanthal are depicted in Fig. 6. The highest concentrations of lucidin primeveroside were found in cultivars 11, 8 and 16, the lowest in cultivar 5. With the direct and indirect extraction method the highest concentrations of ruberythric acid and alizarin were found in cultivars 8, 19, 6 and 14. Cultivar 5 had a much higher concentration of alizarin (10.0 mg/g) as determined by the indirect extraction method than expected from the 9.4 mg/g ruberythric acid (~4.2 mg/g alizarin) measured by the direct extraction method. However in Fig. 6 it is not taken into account that during the direct ethanol–water extraction, together with 9.4 mg/g ruberythric acid, 3.2 mg/g alizarin was also extracted. A similar phenomenon can be observed for lucidin primeveroside and nordamnacanthal in this cultivar. So, before the enzymatic reaction was applied to these madder roots, the concentration of Copyright © 2004 John Wiley & Sons, Ltd.

alizarin and nordamnacanthal was already relatively high, possibly due to storage under humid conditions. This explains why cultivar 5 had the lowest concentration of ruberythric acid and lucidin primeveroside but not the lowest concentration of alizarin and nordamnacanthal. The concentration of pseudopurpurin, munjistin and purpurin found with the indirect tetrahydrofuran–water extraction method is depicted in Fig. 7. The highest amounts of pseudopurpurin and purpurin in madder root were found in cultivars 18, 16 and 8. For munjistin the highest levels were found in samples 18, 17 and 19. Because pseudopurpurin is easily converted to purpurin during the analysis, the amount of pseudopurpurin and purpurin is depicted in one graph. In earlier days madder roots were harvested in the autumn of the third year (Zuurdeeg, 1995). For a more economically feasible production it would be attractive if the harvesting could take place after only 2 years. In this case, the costs of one more year of cultivation must be offset against the difference in alizarin content and biomass after 2 and 3 years. The concentration of anthraquinones was determined with the indirect extraction method and the results for 2- and 3-year-old madder roots are presented in Table 3. If madder root is cultivated for 3 years instead of 2, the concentration of alizarin in the roots is 30% higher while the biomass increases 10–15%. Two methods for the quantitative analysis of anthraquinones in madder root were developed and compared. The indirect extraction method requires less material, is simpler to perform and shows a slightly better precision than the direct ethanol–water extraction method for the quantitative determination of the glycosides. In the direct method the suspension must be refluxed, filtered and the remaining residue has to be Phytochem. Anal. 15: 397–406 (2004)

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405

Table 3. Concentrations of anthraquinones (mg/g) in 2- and 3-year-old madder roots, and the percentage increase in the third year relative to the second year Compound Pseudopurpurin Munjistin Alizarin Purpurin Nordamnacanthal

Two-year-old roots

Three-year-old roots

Percentage increase second to third year

7.7 5.8 6.7 3.7 11.3

7.4 6.2 8.7 3.5 13.4

−4 +7 +30 −5 +19

refluxed again, which is labour-intensive. In the indirect method, the temperature is much lower and the suspension simply needs to be stirred. The method was performed at 45°C but, if more convenient, it can also be performed at room temperature. In this case the suspension should be stirred for 2 h instead of 1 h. However, it must be emphasised that the compounds determined by the indirect extraction method are not originally present in madder root. In the case of the indirect extraction method, the HPLC analysis time can be reduced. With the present HPLC method, the first aglycone (pseudopurpurin)

elutes only at 15 min. Thus, the gradient system could start at a higher percentage of acetonitrile because no glycosides are present. The concentration of alizarin varied between 6.1 and 11.8 mg/g root for the different cultivars screened in this research. This difference has to be taken into account when a cultivar is selected for the commercial production of alizarin. Beside the content of alizarin in the root, agronomic characteristics such as plant density, root density, root thickness, resistance against abiotic and biotic influences and cost–benefit ratio for 2- or 3-year-old roots are also important.

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