Production of alizarin extracts from Rubia tinctorum and assessment of their dyeing properties

Industrial Crops and Products 26 (2007) 151–162

Production of alizarin extracts from Rubia tinctorum and assessment of their dyeing properties D. De Santis, M. Moresi ∗ Dipartimento di Scienze e Tecnologie Agroalimentari, Universit`a della Tuscia, Via S. C. de Lellis, 01100 Viterbo, Italy Received 19 April 2006; accepted 7 February 2007

Abstract In this work several experiments were performed in 50 cm3 shaken-tubes, thus allowing methanol to be selected as the most appropriate leaching solvent for alizarin from roots of common madder (Rubia tinctorum). Methanol at 25 ◦ C was found to be able to extract not only free alizarin but also its glycosidic forms, thus resulting in an overall alizarin extraction yield of 2.9 ± 0.1 g kg−1 of dried material when leaching madder root particles with 100 dm3 of methanol kg−1 . Further extraction tests using a liquid–solid ratio of 40 dm3 kg−1 in a 1-dm3 stirred extractor allowed the production of a methanolic extract, which was then dried under vacuum. The solid residues were re-dissolved in ethanol so as to avoid methanol vapours exhaling from dyeing baths. Dyed standard specimens of raw cotton and wool exhibited almost the same reddish-yellow hue, even if those coloured with the ethanolic extract had a lighter colour intensity and a more pinkish shade than those dyed with Rubia root particles. Whatever the dyeing procedure used, the colour intensity or hue of cotton specimens was found to be brighter or more pinkish than the wool ones. These characteristics were also more evident for the cotton specimens dyed with the ethanolic extract. The fastness properties of dyed cotton and wool specimens were evaluated and it was found that all the dyed specimens were not or just slightly affected by manual washing at 40 ◦ C, acid or basic perspiration tests, and it was also found that the resistance to fading of dyed wool specimens was generally greater than that of cotton ones. © 2007 Elsevier B.V. All rights reserved. Keywords: Colour fastness tests; Dyeing tests; Extraction yield; Alizarin; Rubia tinctorum; Solvent selectivity

1. Introduction The world-wide demand for fibres and colorants of natural origin is increasing probably as a result of the greater awareness of the general consumers in the USA, Europe and Japan towards the highly pollutant procedures affecting not only fibre cultivation, but also their

∗ Corresponding author. Tel.: +39 0761 357494; fax: +39 0761 357498. E-mail address: [email protected] (M. Moresi).

0926-6690/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2007.02.002

transformation into coloured textiles, as well as the noxious effects of some synthetic dyes on human health as sources of skin cancer, disorders and allergic contact dermatitis (Borland, 2000; Francalanci et al., 2001; Glover, 1995). Many research projects have so far been scheduled to evaluate the techno-economic feasibility of alternative dye crops (Marotti, 1997; Vetter, 1997), such as amaranth (Amaranthus tricolor L., A. cruentus L.) (Angelini et al., 1997a; Huang and Elbe, 1986), common madder (Rubia tinctorum L.) (Angelini et al., 1997c,d; Vetter, 1997), woad (Isatis tinctoria L.) (Kokubun et al., 1998;

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Maier et al., 1990; Vetter, 1997), weld (Reseda luteola L.) (Angelini et al., 1997b; Cerrato et al., 2002; Vetter, 1997), and several plant species indigenous to Northeastern India (Bhuyan and Saikia, 2005). More specifically, R. tinctorum L. is a perennial plant naturally growing in Southern Europe, including Southern Britain and Mediterranean countries, which produces a red dye (alizarin) from its roots (Angelini et al., 1997c,d; Vetter, 1997). These roots are covered with a blackish rind, beneath which they are reddish, with a pale yellow pith. In France, after drying, the outer layer is threshed off and powdered and packed separately as an inferior product called mall. The stripped roots are generally dried and then powdered. Root harvesting is carried out in September and October when the plant is at least 15-month-old. However, the maximum harvesting yields of the order of 3–4 metric tonnes (MT) of dry roots per hectare are associated with about 3-year-old plants (Angelini et al., 1997c,d). Madder has been cultivated as a source of dyestuff since antiquity in central Asia and Egypt, where it was grown as early as 1500 bc. Cloth dyed with madder root pigment was found in the tomb of the Pharaoh Tutankhamun, in the ruins of Pompeii, and ancient Corinth. In the Middle Ages, Charlemagne encouraged madder cultivation (Ball, 2002). In 1826, the French chemist Pierre-Jean Robiquet found that there were two colorants in madder root, i.e., the red alizarin and the more rapidly fading purpurin. The alizarin component became the first natural dye to be synthetically reproduced in 1868 when the German chemists Carl Graebe and Carl Liebermann, working for BASF, developed a method for preparing it commercially from anthracene. About the same time, the English dye chemist William Perkin independently discovered the same synthetic route, although the BASF group filed its patent before Perkin by only 1 day. Since the synthetic alizarin could be produced at less than half the cost of the natural product, the cultivation of madder rapidly collapsed (Ball, 2002). Further information on the historical development, cultivation, harvesting and dyeing techniques of madder, as well as on the purification and structures of isolated compounds, was reported by Derksen and van Beek (2002a). Besides sugars and tannins, the madder root contains several dye pigments and the most important one is alizarin, its pigment code is Pigment Red 83 and its colour index number, CI, is 75,330 (Piccaglia, 1997). From chemical point of view, alizarin is a hydroxyl derivate of anthraquinone (i.e., 1,2dihydroxyanthraquinone), as shown in Fig. 1.

Fig. 1. Main molecular structures of the anthraquinones present in Rubia tinctorum roots (Bosakova et al., 2000; Derksen et al., 1998, 2002b,c, 2004; Schweppe, 1993).

So far, some 36 anthraquinones have been detected in R. tinctorum by various workers (Bosakova et al., 2000; Derksen et al., 1998, 2002b, 2004; Schweppe, 1993). Table 1 just lists the essential ones, such as purpurin, xanthopurpurin, rubiadin, munjistin, lucidin, pseudo-purpurin, etc. Though pseudo-purpurin or xanthopurpurin yields the orange or yellow dye, respectively, alizarin is the most interesting of the colouring substances. It occurs as orange-red crystals, almost insoluble in water, but readily soluble in alcohol, ether, the fixed oils, and alkaline solutions (Derksen and van Beek, 2002a). Actually, natural alizarin varies from scarlet to pink to red with a bluish tint depending on the strengths of alkali or acid used. A strong alkali will create a violet-blue colour, a diluted alkali a violet red, while a strong acid will give rise to a yellowish red. The alcoholic and aqueous solutions are rose-coloured and the ethereal ones golden-yellow. The colour of the madder lakes is influenced by the metallic salts used: iron free alum yields the best red, the alumina lake is rose red or bluish red with calcium, the tin lake is red-violet, the iron lake is black-violet, and the chrome lake is brown-violet or red-brown The aluminium mordant of alizarin is the well known dye named as Turkey Red, used to dye cotton and wool with excellent fastness (Derksen and van Beek, 2002a). Actually, the old dyeing techniques and recipes as picked up from practical books (Lonardoni, 1995; Lundborg, 1983) are to be customized to modern industrial-scale dyeing processes. For instance, because of their toxicity traditional heavy metal mordants (i.e., tin, copper and chrome) are to be substituted with less problematic mordants, such alum and iron sulphate (Lonardoni, 1995; Lundborg, 1983; Vetter, 1997). Also, appropriate mordant combination are to be established and standardised to improve fastness and broaden the colour shades of naturally dyed textiles. It is thus worth citing the high fastness colours obtained using common madder roots to dye wool, feathered-leather and cotton

Table 1 Common and chemical abstract index names and corresponding raw formulas and molecular masses of the main anthraquinone glycosides and aglycones present in Rubia tinctorum roots as referred to Fig. 1 Common name

Raw formula

MM (Da)

1 2 3 4 5 6 7

Alizarin Purpuroxanthin Quinizarin Dantron Anthraflavin Rubiadin Munjistin

C14 H8 O4 C14 H8 O4 C14 H8 O4 C14 H8 O4 C14 H8 O4 C15 H10 O4 C15 H8 O6

240 240 240 240 240 240 284

8

Nordamnacanthal

C15 H8 O5

268

9

Lucidin

C15 H10 O5

270

10 11 12 13 14

Anthragallol Purpurin 5-hydroxyalizarin Flavopurpurin Pseudo-purpurin

C14 H8 O5 C14 H8 O5 C14 H8 O5 C14 H8 O5 C15 H8 O7

256 256 256 256 300

15

Ruberythric acid

C25 H26 O13

534

16

Lucidin primeveroside

C26 H28 O14

564

Chemical abstract index name

R1

R2

R3

R4

R5

R6

R7

R8

1,2-Dihydroxyanthraquinone 1,3-Dihydroxyanthraquinone 1,4-Dihydroxyanthraquinone 1,8-Dihydroxyanthraquinone 2,6-Dihydroxyanthraquinone 1,3-Dihydroxy-2-methylanthraquinone 1,3-Dihydroxy-2-carbonic acid anthraquinone 1,3-Dihydroxy-2methoxyanthraquinone 1,3-Dihydroxy-2-(hydroxymethyl) anthraquinone 1,2,3-Trihydroxyanthraquinone 1,2,4-Trihydroxyanthraquinone 1,2,5-Trihydroxyanthraquinone 1,2,6-Trihydroxyanthraquinone 1,3,4-Trihydroxy-2-carbonic acid anthraquinone 1-Hydroxy-2-[(6-O-␤-d-xylopyranosyl-␤-dglucopyranosyl)-oxy]-anthraquinone 1-Hydroxy-2-(hydroxymethyl)-3-[(6-O-␤-dxylopyranosyl-␤-d-glucopyranosyl)-oxy]anthraquinone

–OH –OH –OH –OH –H –OH –OH

–OH –H –H –H –OH –CH3 –COOH

–H –OH –H –H –H –OH –OH

–H –H –OH –H –H –H –H

–H –H –H –H –H –H –H

–H –H –H –H –OH –H –H

–H –H –H –H –H –H –H

–H –H –H –OH –H –H –H

–OH

–CHO

–OH

–H

–H

–H

–H

–H

–OH

–CH2 OH

–OH

–H

–H

–H

–H

–H

–OH –OH –OH –OH –OH

–OH –OH –OH –OH –COOH

–OH –H –H –H –OH

–H –OH –H –H –OH

–H –H –OH –H –H

–H –H –H –OH –H

–H –H –H –H –H

–H –H –H –H –H

–OH

–O-primeverose

–H

–H

–H

–H

–H

–H

–OH

–CH2 OH

–O-primeverose

–H

–H

–OH

–H

–H

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premordanted with an optimal mixture of urea, ammonia, and calcium oxalate (Oenal et al., 2004). Another open question is how to assure a good contact between natural fibres and dye plant materials, as well as to guarantee an easy separation of dyed fibres from exhausted solid residues. The use of dyeing bags containing the plant material of concern appears to be feasible on small-dyeing scales only (Vetter, 1997), such a method requiring long dyeing times and bringing low fibre brightness (Hatamipour and Shafikhani, 1999). In principle, it would be much easier for the industrial dyers willing to revive the natural dyeing techniques to replace madder root particles with concentrated extracts of its pigments, provided that the solvent chosen guarantees a series of properties as follows: (a) its extraction capacity is extremely high for practically all the natural pigments present in the raw materials of interest, but almost nil for waxes, colloidal and albuminous materials; (b) its boiling temperature and latent heat of vaporisation is quite low to allow its separation at low temperatures with minimum energy consumption; (c) its reactivity with colours and pigments is insignificant to avoid any loss in the colour quality. Leaching of madder pigments from the Rubiaceae family has been carried out under solvent refluxing, as well as ultrasonic-assisted (Weng and Sheu, 2000) or microwave-assisted (Dabiri et al., 2005) extraction, using aqueous solutions of ethanol (Derksen et al., 1998, 2002b; Zolt´an et al., 1993) or methanol (Dabiri et al., 2005; Weng and Sheu, 2000) at different volumetric fractions, ether (Angelini et al., 1997d), chloroform (Lodhi et al., 1994), or tetrahydrofuran (Derksen et al., 2004). A few chemicals (such as benzene, hexane, ether, ethyl acetate, chloroform and tetrahydrofuran) are regarded as appropriate to dissolve free aglycones (Angelini et al., 1997d; Derksen et al., 2004; Lodhi et al., 1994), while other ones (e.g., methanol, ethanol, and water) are more specific for dissolving the glycosides (Angelini et al., 1997d; Dabiri et al., 2005; Derksen et al., 1998). From an industrial point of view it would be easier to resort to solid extracts despite there is at present no definite answer to this prospective solution. The simplest extract would be a watery one although not all the dye pigments are water-soluble. Use of organic solvents as those given above might give rise to extracts incompletely water-soluble. A better way to obtain water-soluble powder extracts seems to resort to an alkaline solvent (Vetter, 1997) or to water at temperatures

below 65 ◦ C under oxygenation of the solid–liquid mixture to provide alizarin free of lucidin (Derksen et al., 2002c). The present investigation, therefore, was aimed at identifying the most appropriate leaching solvent for alizarin-based pigments from pre-ground and sieved madder roots, and at producing a concentrated extract of their natural pigments. Further work was also carried out to assess the dyeing capacity of such an easy-touse concentrated extract of alizarin as compared to that of madder root powder on raw cotton or wool specimens, and to determine the fastness properties of the dyed specimens by adopting standard test methods. 2. Materials and methods The vegetable matrix used in this work consisted of about 4-year-old roots of R. tinctorum L. plant, that were cultivated, collected, and naturally dried. The samples were kindly provided by the Cooperative farm “La Campana” (Montefiore dell’Aso, Ascoli Piceno, Italy). For all dyeing tests both premordanted raw cotton (CIELAB co-ordinates: L∗o = 65.1 ± 0.1, ao∗ = 0.1 ± 0.4; bo∗ = −0.8 ± 0.1; density 0.02 g cm−2 ) and wool (L∗o = 73.3 ± 0.4, ao∗ = 2.5 ± 0.5, bo∗ = −8.4 ± 0.2; −2 density 0.0225 g cm ) fabrics were used. 2.1. Pre-treatments of the vegetal matrix The roots were ground using a laboratory-scale miller IKA (mod. MF109) and sieved through screens with 0.5-mm circular openings. Its humidity, after drying at 105 ◦ C for 24 h, was 9.3 ± 0.2% (w/w). 2.2. Pigment leaching and estimation of extraction yields To select the best solvent for alizarin, distilled water (W) and a few other solvents, such as acetone (Ac), ethanol (EtOH), ether (Et2 O) and methanol (MeOH), all of analytical grade, were tested by using particles sieved through 0.5-mm openings (ds ). By using screwcapped FEP (Fluorinated ethylene propylene) 50-cm3 tubes, 0.5 g of pre-ground, madder root powder was suspended in 20 cm3 of solvent. The tubes were put in a thermostatic bath at 25 ◦ C. After 12 h of extraction, any tube was quickly cooled by immersion in iced water and centrifuged at 10,000 rev min−1 (13,776 × g) for 10 min at room temperature. The residue was washed twice with 20 cm3 of fresh solvent. Once all supernatants had been recovered in a 100-cm3 graduated cylinder, other fresh solvent was added up to a constant volume of 60 cm3 . The

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extract was appropriately diluted and filtered by using a micro-syringe equipped with 0.45 ␮m PTFE Millex Filter unit (Millipore Co., Bedford, USA) before injecting 20 mm3 in the HPLC (High Pressure Liquid Cromatograph) system. Once the alizarin concentration (in g m−3 ) had been determined, it was multiplied by the final volume of supernatant (6 × 10−5 m3 ), thus yielding the overall amount of pigment (g) extracted. This was referred to the initial vegetal dry matter (dm) used, thus allowing the extraction yield (YES ) in g kg−1 dm to be estimated. Alternatively, the supernatants were filtered through a pre-weighed Whatman GF/C disc (47 mm) and further diluted with the appropriate solvent up to a final volumetric dilution factor of 40, before assessing their absorption spectrum in the range of 200–750 nm using a UV–vis Spectrometer Lambda25 (Perkin-Elmer Instruments, Norwalk, CT, USA). 2.3. HPLC analysis Alizarin in any supernatant was determined by using a Waters Associates (Milford, Mass., USA) high-performance liquid chromatograph mod. 600. The apparatus was equipped with a high-pressure volumetric pump, a UV–vis Perkin-Elmers LC-95 detector, a Waters Guard pre-column Nova-Pak C18 , a reverse phase Waters Nova-Pak C18 column (3.9 mm × 300 mm), and a Spectra Physics Data Jet integrator. Pre-filtered samples containing alizarin were injected in the column kept at room temperature, using a mobile phase differently composed of pure methanol (A) and 10% (v/v) aqueous acetic acid (B). The elution programme involved two different isocratic conditions using a constant overall flow rate of 0.6 cm3 min−1 . The initial mobile phase consisted of 60% A and 40% B for 30 min, while the second one of 100% A for the subsequent 10 min to regenerate the column. Alizarin detection (retention time of about 12.5 ± 0.5 min) was performed at 255 nm (Angelini et al., 1997d). Standard solutions containing 0.25–10 g of alizarin per m3 of methanol were prepared and analysed in triplicate by HPLC. The peak area of the chromatograms was linearly correlated to the alizarin concentration in the injected sample. 2.4. Total alizarin content of the vegetal matrix Two analytical methods were used to determine the amount of free and bound alizarin in the pre-ground and sieved Rubia roots used in this work. The method 1 was derived from Angelini et al. (1997d), which was different from that described by

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Lodhi et al. (1994). The above dried powder (250 mg) was extracted with 25 cm3 of ether (Et2 O) for 12 h and filtered through Whatman No. 1 paper (Whatman International Ltd., Maidstone, UK). The plant material residue was washed with Et2 O (4 × 12.5 cm3 ) until the filtrate became colourless. All ethereal extracts were combined and evaporated to dryness under vac¨ ¨ uum in a Rotavapor BUCHI mod. R110 (BUCHI Labortechnik AG, Flawil, Switzerland). The resulting solid residue (containing free antraquinones) was re-dissolved in methanol (25 cm3 ), thus allowing its content of free alizarin to be evaluated by HPLC. The ether-exhausted plant material was dried, extracted with methanol (25 cm3 ) for 8 h, and filtered. Further washings, at least for four times, of such residue with methanol (4 × 12.5 cm3 ) were performed until the filtrate became colourless. All the resulting filtrates were collected together and brought to a final volume of 75 cm3 with methanol before determining the corresponding alizarin concentration by HPLC. According to Lodhi et al. (1994), an aliquot (15 cm3 ) of the methanolic filtrate was evaporated to dryness and the residue was dissolved in 10 cm3 of 5% (v/v) HCl and hydrolysed at 65 ◦ C for 24 h. The aglycones released were extracted by adding 50 cm3 of ethyl acetate. Once the organic phase had been washed with water (4 × 20 cm3 ) to get rid of sugars and salts, it was fully evaporated under vacuum. Then, the solid residue was newly dissolved in methanol (25 cm3 ), filtered through a 0.45 ␮m PTFE Millex Filter unit (Millipore Co., Bedford, USA), and finally analysed by HPLC. According to the method 2, 250 mg of the dried powder was extracted with 25 cm3 of methanol for 12 h and filtered (Whatman No. 1 paper). The plant material residue was washed with methanol (4 × 12.5 cm3 ) until the filtrate became colourless. All the resulting filtrates were collected together and brought to a final volume of 75 cm3 by adding methanol. By using the same procedure given above, it was possible to hydrolyse the antraquinone glycosides extracted and then assess the overall alizarin content of the methanolic extract. 2.5. Scaling-up of the extraction process The extraction process was scaled-up in a 1-dm3 jacketed Pyrex laboratory extractor, equipped with a portable, 40 mm marine-type propeller mixer IKA (mod. EUROSTAR) mounted vertically on centre with baffles at the wall. Once the extractor had been filled with 0.8 dm3 of HPLC-grade methanol (Sigma–Aldrich Srl, Milan, I), the liquid temperature was set to 25.0 ± 0.1 ◦ C by re-circulating a thermostatic liquid within the extrac-

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tor jacket and setting the stirrer speed to 700 rev min−1 . Through one of the openings of the extractor cover, 20 g of dm (ds ≤ 0.5 mm) were then added, thus obtaining a liquid–solid ratio of 40 dm3 kg−1 . After 12 h, the exhausted particles were settled to the bottom of the liquid, thus allowing a clear solution containing 26 ± 2 g m−3 of alizarin equivalent to be recovered and evaporated at 40 ◦ C under vacuum ¨ in a Rotavapor BUCHI mod. R110. The resulting solid residue was fully re-dissolved in 150 cm3 of ethanol, thus yielding a concentrated extract containing 119 ± 8 g m−3 of alizarin equivalent. This avoided toxic methanol vapours exhaling from the dyeing baths. 2.6. Preparation of fabric specimens Raw cotton and wool fabrics were subdivided into standard specimens (10 cm × 4 cm) in accordance with the Italian Association of Textile Colourists (1997). 2.7. Mordanting procedure For mordanting 10 g of raw wool specimens, the procedure adopted (Lundborg, 1983) consisted of first dissolving 2.5 g of alum [KAl(SO4 )2 ·12H2 O] in 400 cm3 of demineralised warm water. The previously wet specimens were then dipped in the mordanting solution and the system was heated up to 90 ◦ C for 1 h. After 1 h, the suspension was cooled to room temperature. So also for mordanting 10 g of raw cotton specimens, the following procedure (Lundborg, 1983) was used in which 2.5 g of alum [KAl(SO4 )2 ·12H2 O] and 1.0 g of cream of tartar (C4 H5 KO6 ) were first dissolved in 250 cm3 of demineralised warm water and then the previously wet specimens were dipped in the mordanting solution. The suspension was heated up to the boiling point for 2 h. The suspension was then cooled to room temperature. 2.8. Dye-bath preparation and dyeing In accordance with Lonardoni (1995) and Lundborg (1983), 10 g of fabrics were dyed by leaching 10 g of madder root powder (ds ≤ 0.5 mm) in 500 cm3 of deionised water at room temperature overnight (12 h). After recovering the supernatant by centrifugation, the solid residue was washed twice with de-ionised water. All supernatants were collected and some de-ionised water was added to re-constitute the initial dye-bath volume (500 cm3 ), thus yielding a final concentration of alizarin equivalent of circa 14 g m−3 .

When using the alizarin concentrated extract, deionised water was added so as to obtain the above concentration of alizarin equivalent. Premordanted raw wool or cotton specimens (10 g) were dipped in the dye-bath (500 cm3 ) mentioned above. The suspension was heated up to 90 ◦ C for wool and to boiling for cotton for 2 h, stirring regularly. After allowing the specimens to cool off in the pot, they were rinsed in tap water repeatedly until the rinsed water was clear and then dried in the shade. 2.9. Colour fastness standard tests All dyed specimens were submitted to a few colour fastness standard test methods, such as manual washing at 40 ◦ C, and acid (pH 5.5) and basic (pH 8) perspiration (Associazione Nobilitazione Tessile, 1997). 2.10. Colour measurements The colour of specimens, before and after dyeing or after any colour fastness test, was measured by using a portable colour-measuring instrument (mod. D25-PC2; Hunterlab, Restow, Virginia, USA) with a diffuse (0/45◦ ) illuminating viewing geometry and a 50-mm diameter of specimen aperture. After calibrating the instrument, any specimen was mounted between two glass plates and its colour was assayed in three different (left, central and right) positions by recording the resulting CIELAB co-ordinates (L*, a*, and b*). 3. Results and discussion 3.1. Leaching solvent selection To select the leaching solvent capable of maximising the extraction yield of alizarin, several trials were carried out by using ether, acetone, ethanol, methanol or distilled water at 25 ◦ C for 12 h. Once the yield of alizarin (YES ) in any of the above solvents had been determined (Table 2), it was possible to estimate its corresponding selectivity coefficient (K) with respect to water as follows: K=

YES YEW

(1)

In accordance with Angelini et al. (1997d), ether was able to dissolve only the free aglycones, thus providing quite a low extraction yield coefficient of 0.18 ± 0.03 g kg−1 , practically coincident with that pertaining to acetone. The yield coefficients for ethanol and water were definitively greater (i.e., about 0.34 and 1.06 g kg−1 ,

D. De Santis, M. Moresi / Industrial Crops and Products 26 (2007) 151–162 Table 2 Extraction yields (YES ) and selectivity coefficients (KS ), as well as concentrations (cAE ) of alizarin equivalent in the extracts, when 0.5mm sieved Rubia root particles were leached with different solvents at 25 ◦ C using a liquid/solid ratio of 40 dm3 per kg dry matter for 12 h Solvent

YES (g kg−1 )

Ether Acetone Ethanol Methanol Water

0.18 0.18 0.34 1.5 1.06

± ± ± ± ±

0.03 0.04 0.02 0.2 0.08

cAE (g m−3 ) 4.5 4 8.5 37.5 26

± ± ± ± ±

0.3 1 0.6 0.8 2

KS 0.17 0.32 1.42 1.00

respectively) than the former, but smaller than that for methanol (1.5 ± 0.2 g kg−1 ). Thus, methanol was found to be the most selective solvent for extraction of alizarin, followed by water, ethanol, acetone and ether. The concentrations (cAE ) of alizarin equivalent in the corresponding extracts were about 38, 26, 8, 4 and 5 g m−3 , respectively (Table 2). Fig. 2 shows the absorption spectra of the resulting Rubia root extracts diluted with the corresponding solvent by an overall volumetric factor of 40 as compared to those of the synthetic alizarin in methanol and methanolic extract of the ether-exhausted root residues. It can be noted that the methanolic extracts of the madder roots as such or previously leached with ether presented absorption maxima at 240 nm, while the synthetic alizarin in methanol exhibited its absorption peak at 260 nm. Such peaks were absent in all the other extracts examined, thus

Fig. 2. Absorption spectra of a methanolic solution containing 10 g m−3 of synthetic alizarin (䊉), several Rubia root extracts using methanol (), water (), ethanol (*) or acetone (), and a methanolic extract of the ether-exhausted root residues (♦), all diluted with the corresponding solvent by an overall volumetric factor of 40: optical density (OD) against wavelength (λ).

157

confirming the greater selectivity of methanol for Rubia anthraquinones. 3.2. Total alizarin content of the vegetal matrix The amounts of free and bound alizarin in the Rubia roots used in this work were determined by means of two different analytical methods and the results obtained were recorded in Table 3. In the first method, ether was used to recover the free alizarin and in the second one methanol was used to recuperate the bound alizarin and then acid hydrolysis was carried out to free the aglycones. It was possible to assess that the Rubia roots used contained about 0.21 ± 0.03 g of free alizarin per kg of dry matter, while the glycoside form was about 2.6 ± 0.4 g kg−1 , thus resulting in an overall alizarin content of 2.8 ± 0.3 g kg−1 . By submitting the madder roots to a unique extraction process using methanol, it should have been possible to recover both free and bound forms of alizarin. In fact, the subsequent acid hydrolytic treatment yielded an overall alizarin content of 2.9 ± 0.1 g kg−1 , practically coincident with that assessed via the analytical method 1 above. By referring to Table 3, it was possible to get proof of the accuracy of both these two analytical procedures by comparing the HPLC-detected alizarin concentrations in the methanolic extracts of the madder roots as such (1.5 ± 0.2 g kg−1 ) or previously leached with ether (1.3 ± 0.2 g kg−1 ), their difference being coincident with the free aglycones selectively removed by ether (0.21 ± 0.03 g kg−1 ). These data also showed that the alizarin content assessed by HPLC in the case of methanolic solutions containing bound forms of alizarin was greatly underestimated with respect to the case in which all the alizarin forms had been hydrolysed to free the aglycones (Table 3). Thus, the extraction yields shown in Table 2 were to be used as a rough guide to screen the effectiveness of extraction of alizarin with different solvents. The overall amount of alizarin present in the Rubia roots used here was about three times more than that (0.94 ± 0.02 g kg−1 ) determined by Lodhi et al. (1994) in R. tinctorum roots harvested from greenhouse grown plant samples supplied by the King’s College of London (UK), but from 33 to 42% of that assessed by Angelini et al. (1997d) in 5-, 15- or 30-month-old Rubia roots, this being equal to 8.2, 6.8 or 7.1 g kg−1 , respectively. A great deal of variation (15.6–39.4 g kg−1 ) in the overall anthraquinones present in root-stock and root samples of plants originating from 11 different habitats after their acidic hydrolysis was detected by Boldizsar et al.

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Table 3 Free (aglycones) and bound (glycosides) alizarin present in 0.5 mm sieved R. tinctorum roots, as determined by selective extraction steps using one or two solvents in sequence Analytical methods

1 2

Solvent 1

Ether –

YES1 (g kg−1 )

0.21 ± 0.03 –

Solvent 2

Methanol Methanol

Overall alizarin content (g kg−1 )

Acid hydrolysis Before, YES2 (g kg−1 )

After, YES2 (g kg−1 )

1.3 ± 0.2 1.5 ± 0.2

2.6 ± 0.4 2.9 ± 0.1

(2004) by using HPLC, UV–vis spectrophotometric and mass spectrometric methods with alizarin, purpurin, and lucidin varying in the ranges of 9.6–21.8, 3.7–12.3, and 1.8–5.7 g kg−1 , respectively. Thus, it is possible to confirm the large variation in the overall alizarin contents as due to differences in Rubia genotype and species.

2.8 ± 0.3 2.9 ± 0.1

points (0, 0), (a*, b*), and (a*, 0), and tangent of hue angle (tanh*), which is expressed by the ratio b*/a*. It can be noted from Fig. 3a that both the chroma (25.8) and lightness coefficient (26.8) of the point R (representative of all wool specimens dyed with madder root) were significantly smaller than those (30.5 and 38.2, respectively) of the point E (representative of all wool

3.3. Dyeing tests Premordanted standard specimens of raw cotton or wool were submitted to a few preliminary dyeing tests using Rubia root particles (ds ≤ 0.5 mm) and the alizarin concentrated extracts. The colour of any generic jth dyed specimen was measured by recording its CIELAB co-ordinates (L∗j , aj∗ , and bj∗ ) in three different (left, central and right) positions, which were averaged as shown in Table 4. For any dyed specimen, the differences between any measured CIELAB co-ordinate in any of the three positions tested and its corresponding average value listed in Table 4 was found to be statistically insignificant at the 95% confidence level, thus involving no colour change, not only within the same jth dyed specimen, but also within each set of dyed cotton or wool specimens examined. Owing to such limited variations in the CIELAB coordinates, the colour of both sets of dyed specimens was represented by the average values of the CIELAB co-ordinates shown on the last row of Table 4, thus allowing their hue sequence and hue-angle orientation to be plotted in the (a* and b*) plane (Fig. 3). When using Rubia root particles (R) or the alizarin concentrated extract (E), both the average values of a* and b* were positive. Therefore, their corresponding characteristic colour points fell in the first quadrant (Fig. 3) and were labelled as reddish-yellow, the shades of red being generally predominant (a* > b*). To quantify properly the colour of dyed specimens, two other colour √ parameters were estimated, that is chroma C∗ (= a∗2 + b∗2 ), which represents the hypotenuse of a right angled triangle created by joining

Fig. 3. Colour representation of (a) wool and (b) cotton specimens dyed using 0.5-mm sieved Rubia root particles (R: ) or methanolic extracts (E: ) in the CIELAB (a* and b*).

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159

Table 4 Colour characteristics of wool or cotton standard specimens dyed using 0.5 mm sieved Rubia root particles or alizarin concentrated extracts: average values and standard deviations of the CIELAB co-ordinates (L*, a*, b*) measured in three different (left, central and right) positions Dyed specimen no.

Rubia root powder L*

Wool specimens 1 2 3 4 5 6 7 8 9 10 Average Cotton specimens 1 2 3 4 5 6 7 8 9 10 Average

25.9 26.1 26.9 27.3 26.7 27.1 26.7 27.2 27.0 26.9

a* ± ± ± ± ± ± ± ± ± ±

0.3 0.4 0.6 0.2 0.2 0.2 0.2 0.4 0.8 0.2

26.8 ± 0.6

39.2 39.0 38.7 41.2 40.4 40.2 39.1 40.7 39.3 38.6

Alizarin extract

± ± ± ± ± ± ± ± ± ±

0.3 0.6 0.5 0.4 0.4 0.4 0.2 0.2 0.6 0.9

39.6 ± 1.0

20.4 20.4 20.3 20.6 21.8 21.3 21.3 21.0 20.7 21.3

b* ± ± ± ± ± ± ± ± ± ±

0.3 0.4 0.2 0.9 1.2 1.3 0.3 0.8 0.8 0.5

20.9 ± 0.8

19.8 20.2 20.0 19.1 19.7 19.6 19.3 19.1 19.6 20.4

± ± ± ± ± ± ± ± ± ±

0.8 0.7 0.3 1.0 0.8 0.4 0.4 1.1 0.3 0.6

19.7 ± 0.7

specimens dyed with the ethanolic extract of alizarin). On the contrary, the hue angle of the former (27.8◦ ) was a little smaller than that (35.8◦ ) of the latter. Thus, dyed wool specimens with Rubia root particles presented a greater colour intensity with a darker shade in the red hue, while those dyed with the ethanolic extract had a lighter colour intensity with a more pinkish shade in the orange-red hue. It can be derived from Fig. 3b that the chroma (24.3) of the point R was just a little greater than that (22.1) of the point E. On the contrary, both the hue angle (22.2◦ ) and lightness coefficient (39.6) of the former were significantly smaller than those (36◦ and 53.3, respectively) of the latter. So, dyed cotton specimens with the extracted dye presented a lighter colour intensity with a more pinkish shade of red hue, thus appearing more rose-coloured than those tinted with Rubia roots. To quantify the differences in metric lightness and chromaticity for the two sets of dyed specimens under study, the colour difference (Ej ) between any generic jth dyed cotton specimen and the original raw one (the

15.3 15.4 15.4 15.3 15.0 14.6 14.8 15.6 14.8 15.0

L* ± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.4 0.1 0.7 0.5 0.3 0.2 0.4 0.4

15.1 ± 0.4

14.1 14.6 14.6 13.7 13.8 14.1 14.0 14.1 15.1 15.1

± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.2 0.1 0.2 0.3 0.2 0.1 0.3 0.5

14.4 ± 0.5

37.2 39.0 35.6 38.9 39.0 39.4 39.4 37.0 39.0 37.5

a* ± ± ± ± ± ± ± ± ± ±

0.3 0.6 0.3 0.8 0.1 0.3 0.5 0.7 0.3 0.5

38.2 ± 1.3

55.5 52.9 53.4 52.5 52.6 52.7 52.8 53.2 53.3 54.1

± ± ± ± ± ± ± ± ± ±

0.4 0.3 0.5 0.1 0.4 0.8 0.3 0.7 0.6 0.3

53.3 ± 1.0

28.1 25.6 27.1 25.8 24.9 27.7 27.6 29.1 26.4 27.2

b* ± ± ± ± ± ± ± ± ± ±

0.9 0.6 0.5 0.6 0.5 0.4 1.6 1.2 0.4 1.2

27.0 ± 1.4

18.3 21.1 20.2 20.7 21.2 20.4 22.0 21.0 20.4 19.8

± ± ± ± ± ± ± ± ± ±

0.6 0.7 0.3 0.1 1.0 0.3 0.5 0.9 0.2 0.6

20.5 ± 1.1

14.1 14.8 14.7 14.5 14.6 13.7 13.9 13.7 14.0 14.1

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.2 0.4 0.3 0.2 0.5 0.1 0.1

14.2 ± 0.4

7.6 8.4 8.4 8.5 8.3 8.4 8.3 8.2 8.5 8.3

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.4

8.4 ± 0.3

CIELAB co-ordinates of which being reported in Section 2) was calculated as the square root of the squares of the respective differences of L*, a* and b*:  Ej = (L∗j − L∗o )2 + (aj∗ − ao∗ )2 + (bj∗ − bo∗ )2 (2) As a consequence of the different dyeing procedures used, Ej ranged from 54.9 to 56.0 with an average value of 55.3 ± 0.5 when using madder root particles and from 47.0 to 50.6 with an average value of 48 ± 1 when using the ethanolic extract. As plotted in Fig. 4a, both dyeing procedures gave rise to a uniform difference of colour between dyed and raw wool specimens. Fig. 4b gave an overall idea of the uniformity of colour achieved when dyeing premordanted cotton specimens with madder root particles or the alizarin extract, their colour differences E between any of the 10 dyed cotton specimens and the premordanted raw cotton ones (the CIELAB co-ordinates of which being reported in Section 2) presented average values of 36 ± 1 or 25 ± 1, respectively.

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Fig. 5. Average values of the colour difference (E) between any dyed and raw (a) wool or (b) cotton specimens as estimated in a set of 10 premordanted specimens dyed with 0.5-mm sieved Rubia root particles (R) and methanolic extracts (E) after dyeing (T) or different colour fastness tests (manual washing at 40 ◦ C, MW; acid, AP, and basic, BP, perspiration).

Fig. 4. Colour difference (E) between any dyed and raw (a) wool or (b) cotton specimens as estimated in a set of 10 premordanted specimens dyed with 0.5-mm sieved Rubia root particles (R: ) or methanolic extracts (E: ).

When using the alizarin extract, the colour intensity of wool and cotton specimens had a more definite pink hue than those tinted with madder root particles. 3.4. Colour fastness tests The above two sets of dyed specimens were submitted to the most significant colour fastness standard tests for underwear, viz. manual washing at 40 ◦ C, and acid (pH 5.5) and basic (pH 8) perspiration tests (Associazione Nobilitazione Tessile, 1997). As shown in Fig. 5a, for the wool specimens dyed with madder root particles or with the alizarin extract, their

initial values of E (55.3 ± 0.5 and 48 ± 1, respectively) were practically unaffected (59 ± 1 and 48 ± 1, respectively) by manual washing at 40 ◦ C, as well as acid or basic perspiration tests. From Fig. 5b it was evident that for the cotton specimens dyed with Rubia root particles their initial value of E (36 ± 1) was not affected by the above colour fastness standard tests, while the initial colour (25 ± 1) of cotton specimens dyed with the alizarin extract tended to fade slightly, thus attenuating the colour difference (E) between dyed specimens and premordanted raw cotton ones to 24 ± 1, 23 ± 1 or 22 ± 2 by basic perspiration, home laundering or acid perspiration tests, respectively. As shown in Fig. 5, the resistance to fading of wool specimens dyed using either madder root particles or alizarin extracts was generally greater than that of cotton dyed with the extract. This paralleled the greater resistance to light of madder root dyed wool specimens with respect to cotton ones, as previously assessed via accelerated xenotests by Angelini et al. (1997d).

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4. Conclusions A series of experiments performed in 50 cm3 shakentubes have shown methanol to be the best solvent for extracting either free or bound forms of alizarin. Further tests in a 1 dm3 stirred extractor allowed the production of a concentrated extract at 119 ± 8 g m−3 of alizarin equivalent, the dyeing properties of which were further assessed on cotton and wool specimens. A series of preliminary dyeing tests on premordanted standard specimens of raw cotton and wool gave rise to dyed specimens with the same reddish-yellow hue, even if those coloured with the ethanolic extract had a lighter colour intensity with a more pinkish shade than those dyed with Rubia root particles. Whatever the dyeing procedure used, the colour intensity and hue of cotton specimens were lighter and more pinkish than the wool ones, and both these characteristics were more evident for the cotton specimens dyed with the ethanolic extract. The significant colour fastening standard tests for underclothing that were carried out in the dyed samples exhibited quite constant colour differences (E). In both cases, the resistance to fading of dyed wool specimens was generally greater than that of cotton ones. From the above study, it might be concluded that the colour components extracted by methanol from the roots of R. tinctorum would help to standardise the natural-dyeing procedures within statistically insignificant differences in colour intensity. At the same time, the textile manufacturer would not be burden with the disposal problems of wet madder root residues. Thus, the dye extracted might be an alternative to synthetic dye for dyeing cotton and wool. Acknowledgements The authors would like to thank the skilful help of Mr. Paolo Caporro and Mr. Alfonso Cerrato during the experimental work. References Angelini, L., Belloni, P., Bertacchi, A., 1997a. Amaranto. In: Marotti, M. (Ed.), Le piante coloranti. Edagricole, Bologna, pp. 49–53. Angelini, L., Belloni, P., Bertacchi, A., 1997b. Reseda. In: Marotti, M. (Ed.), Le piante coloranti. Edagricole, Bologna, pp. 108–111. Angelini, L., Belloni, P., Bertacchi, A., 1997c. Robbia. In: Marotti, M. (Ed.), Le piante coloranti. Edagricole, Bologna, pp. 112–115. Angelini, L.G., Pistelli, L., Belloni, P., Bertoli, A., Panconesi, S., 1997d. Rubia tinctorum a source of natural dyes: agronomic evaluation, quantitative analysis of alizarin and industrial assays. Ind. Crops Prods. 6, 303–311. Associazione Nobilitazione Tessile, 1997. Progetto standard di qualit`a, 3rd ed. PRO.NO.TEX Srl, Milano, pp. 23, 34 and 37.

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