JJC

Jordan Journal of Chemistry Vol. 4 No.4, 2009, pp. 377-385

Preparation and HPLC Analysis of the Natural Pigments Obtained from Buckthorn (Rhamnus petiolaris Boiss) Dye Plants
Ozan Deveoglu*a, Recep Karadagb and Turkan Yurdunc
a b c Department of Chemistry, Faculty of Science and Letters, Marmara University, 34722, Goztepe, Kadkoy, Istanbul, Turkey Laboratory of Natural Dyes, Faculty of Fine Arts, Marmara University, 34718, Acbadem, Kadkoy, Istanbul, Turkey Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Marmara University, 34668, Haydarpasa, Istanbul, Turkey

Received on Sept. 27, 2009

Accepted on Dec. 2, 2009

Abstract
In this work, the buckthorn natural pigments, namely aluminium, tin and iron-buckthorn were obtained by the reaction of KAl(SO4)2.12H2O, SnCl2.2H2O and FeSO4.7H2O solutions with buckthorn (Rhamnus petiolaris Boiss) berries. A reversed-phase high performance liquid chromatography (HPLC) with diode-array detection (DAD) method was utilized for the identification of the buckthorn natural pigments. The extraction of dyestuffs from the natural pigments was carried out with hydrochloric acid / methanol / water (2:1:1; v/v/v) solution. According to the results of HPLC analysis of the natural pigments, it was observed that both rhamnetin and emodin that present in the pigments were precipitated by Al(III) whereas only emodin was precipitated by Sn(II) and Fe(II). CIELAB values of the natural pigments were measured.

Keywords: Rhamnus petiolaris; Dyestuff; Natural pigment; Rhamnetin; Emodin;

HPLC.

Introduction
Flavonoid, anthraquinone, and indigotin compounds that present in plants (Rhamnus petiolaris Boiss, Rubia tinctorum L., Reseda luteola L., Isatis tinctoria L. etc) and insects (Dactylopius coccus Costa, Kermes vermilio Planchon, Kerria lacca Kerr, etc.) and used in several fields as dye and natural pigment are known as natural dyestuffs[1-5]. Buckthorn dye plant has a very important place in natural dyestuff sources[6,7]. Buckthorn berries are an old Turkish dyestuff source[8]. The natural dyestuffs (such as flavonoids) are effective metal ion chelators[9,10]. Metal - flavonoid or metal - anthraquinone complexes are generated by the reaction of metals like aluminium(III) [KAl(SO4)2.12H2O], tin(II) [SnCl2.2H2O], and iron(II) [Fe(SO4).7H2O] with these dyestuffs[11]. In alkaline solutions, natural pigments are precipitated as insoluble metal-dyestuff complexes[12]. The composition of natural pigments depends not only on the species and origin of dye plant, but also on the procedures used for the extraction from dye plants and on the method used for natural pigment preparation. Moreover, their composition is influenced by ageing processes[10]. From the 14th to 19th centuries, natural pigments were primary constituents of the artists palette. Rhamnus lake was
* Corresponding author: Tel.: +902163384474, ext. 17; Fax: +902163367468 E-mail: ozan.deveoglu@marmara.edu.tr (O.Deveoglu)

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used for artistic techniques such as miniature, tempera and oil paint. The main dyestuff present in the aqueous extract from Rhamnus berries is reported to be a glycoside derivative of rhamnetin (3,5,3,4-tetrahydroxy-7-methoxyflavone)[13]. The identification of natural pigment and dyestuff analysis is one of the most important targets aimed in the scientific examination of paintings, textiles, illuminated manuscripts and other historic and archaeological materials[14]. Thus, several analytical techniques have been used in the identification of natural pigments for example gas chromatography/mass spectrometry[15], UV-visible spectrophotometry, thin layer chromatography[16-17], microspectrofluorimetry[18], high performance liquid chromatography[19], reversed phase liquid chromatography[20] and capillary electrophoresis with electrospray mass spectrometric detection[21], FT-IR spectroscopy[14] and raman spectroscopy[22]. Of these techniques, high performance liquid chromatography (HPLC) using a diode-array detection (DAD) is ideally suited for the identification of dyestuffs and natural pigments sampled from museum collections[23]. The colour of any natural pigment is the result of three combined factors: the spectrum of the light source, the spectral reflectivity of the natural pigment, and the spectral sensitivitiy of the eye. The CIELAB (1976) - system was introduced to describe colour as a result of these three factors. This system is a three - dimensional space, with coordinate axes L*, a* and b*. L* symbolizing the lightness of the colour (L*= 0: black, L*= 100: white), a* represents the green-red axis (a* negative: green, a* positive: red), and b* represents the blue-yellow axis (b* negative: blue, b* positive: yellow). Each natural pigment colour can be described as a series of values for L* a* and b*, and so as a point in this colour space[24-26].

Experimental
Plant, standard natural dyes and chemicals Buckthorn (Rhamnus petiolaris Boiss) was obtained from Laboratory for Natural Dyes, Faculty of Fine Arts, Marmara University. Standard natural dyes: rhamnetin (3,5,3',4'tetrahydroxy7-methoxyflavone), kaempferol (3,5,7,4'-tetrahydroxyflavone), emodin (1,3,8-trihydroxy-6-methylanthraquinone), isorhamnetin (3,5,7,4'-tetrahydroxy3'-methoxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) were obtained from Carl Roth (Karlsruhe, Germany). HCl, CH3OH, SnCl2.2H2O, FeSO4.7H2O and K2CO3 were obtained from Merck (Darmstadt, Germany), KAl(SO4)2.12H2O was obtained from Pancreac (Madrid, Barcelona). The HPLC mobile phase was prepared using Milli-Q water (Millipore, Bedford, MA, USA). Apparatus Hanna Instruments HI 8314 Membrane pH-meter, Heraeus D-6450 Hanau Oven, WiseStir MSH-20A Daihan Scientific Co. Stirrer, Shimadzu AEX-200G, Gesellschaft fr Labortechnik (GFL), GretagMacbeth SpectroEye Spectralphotometer were used.

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HPLC equipment Chromatographic experiments were carried out using an Agilent 1100 series system (Agilent Technologies, Hewlett-Packard, Germany) including a G1311A gradient delivery pump with a 50 l loop, a Rheodyne valve (7725i sample injector), a G1315A diode - array detector (Chromatograms were obtained by scanning the sample from 191 to 799 nm with a resolution of 2 nm and chromatographic peaks were monitored at 255 nm), a G1322A vacuum degasser and a G1316A thermostatted column compartment; the data were analyzed using an Agilent Chemstation. A NovaPak C18 analytical column (3,9 x 150 mm, 4 m, Part No WAT 086344, Waters) protected by a guard column filled with the same material was used. Analytical and guard columns were maintained at 30C. The HPLC gradient elution was performed using the method of Halpine et al[23] and Karapanagiotis et al.[27]. Chromatographic separations of the hydrolysed samples were performed using a gradient elution program that utilizes two solvents; solvent A: H2O - 0.1 % TFA (trifluoroacetic acid) and solvent B: CH3CN (acetonitrile) - 0.1 % TFA. The flow rate was 0.5 ml/min. and the applied elution program is described in table 1. Table 1: Parameters of gradient elution program Time (min) 0.0 1.0 20.0 25.0 28.0 33.0 35.0 45.0 Flow rate (ml/min) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 H2O-0.1 % CH3CN-0.1 % TFA (v/v) TFA (v/v) 95.0 95.0 70.0 40.0 40.0 5.0 5.0 95.0 5.0 5.0 30.0 60.0 60.0 95.0 95.0 5.0

Extraction of dyestuffs from Buckthorn 2 g of dried and ground buckthorn berries were transferred into a 400 ml beaker. 150 ml distilled water was then added. The mixture of buckthorn berries was heated to 100 C for 1 hour. Finally, the mixture was filtered to obtain the buckthorn extract (nonhydrolysed). Preparation of natural pigments 15% KAl(SO4)2.12H2O (alum) solution and 150 ml buckthorn extract were heated separately to 90 C and 60 C respectively. 10 ml of alum solution at 90 C was added to buckthorn extract at 60C. K2CO3 solution (0.1 M) was added to neutralize the mixture[10]. The mixture was cooled to room temperature to precipitate the natural pigment. After settlingdown, the mixture was filtered and the precipitate was washed with distilled water and dried on a filter paper at 101 C for 30 minutes. The dried aluminium natural pigment precipitate was powdered. The same process was repeated by using 20, 30, 40 and, 50 ml of alum solution to each part of 150 ml of buckthorn

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extract. These experiments were repeated to precipitate the natural pigments by using 5 % FeSO4.7H2O and 3 % SnCl2.2H2O solutions[28-30]. Extractions for HPLC analysis Dyestuff extraction was done by using the previously described method[31-33]. The samples were prepared as follows: 1 ) Pigment samples (each one approximately 3 mg) were hydrolysed by using H2O: MeOH: 37 HCl (1:1:2; v/v/v; 400 l) in conical glass tubes for precisely 8 min in a water-bath at 100 C to extract organic dyes. After rapid cooling under running cold water, the solution was evaporated just to dryness in a water-bath at 50-65 C under a gentle stream of nitrogen. The dry pigment residues were dissolved in 200 l of the mixture of MeOH: H2O (2:1; v/v). 2) One ml of the buckthorn extract was evaporated in a porcelain croze on a waterbath at 100C. The dry residue was hydrolysed according to above procedure. The hydrolysates of dry pigment residues and hydrolysates of dry buckthorn extract residues were dissolved in 200 l of the mixture of MeOH: H2O (2:1; v/v) and was centrifuged at 2500 rpm for 10 min. Then 5 l and/or 50 l of the supernatant were injected into the HPLC apparatus. Figures 1-5 show the HPLC chromatograms of the pigments, hydrolysates of buckthorn extract and without hydrolysates of buckthorn extract.

Figure 1: HPLC chromatogram of hydrolysed aluminium - buckthorn natural pigment Rhamnetin (27.4 min) and emodin (31.4 min) are identified.

Figure 2: HPLC chromatogram of hydrolysed iron-buckthorn natural pigment. Emodin (31.5 min) is identified.

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Figure 3: HPLC chromatogram of hydrolysed tin-buckthorn natural pigment. Emodin (31.5 min) is identified.

Figure 4: HPLC chromatogram of hydrolysed buckthorn extract. Rhamnetin (27.5 min) and Emodin (31.4 min) are identified.

Figure 5: HPLC chromatogram of non-hydrolysed buckthorn extract. Quercetin (18.6 min), Rhamnetin (27.3 min) and Emodin (30.8 min) are identified. Colour measurements L*, a* and b* values of the natural pigments were measured with GretagMacbeth SpectroEye Spectralphotometer. CIELAB graphs of natural pigments were drawn using measured values. Figures 9-11 show CIELAB colour graphs of the natural pigments.

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Results and discussion

In the present study, the complexes of buckthorn (Rhamnus petiolaris Boiss) with aluminium(III), iron(II) and tin(II) were obtained and analyzed qualitatively by reversed phase high performance liquid chromatography (RP-HPLC). Dyestuff extractions for HPLC analysis were done by using the previously described method[3133]

. The composition was identified based on the absorption spectra acquired with

standard reference dyestuff compounds (figures 6-8). HPLC analysis shows that rhamnetin and emodin were identified in aluminium - buckthorn natural pigments, and in hydrolysed and non-hydrolysed buckthorn extracts. Quercetin dyestuff was determined in the non-hydrolysed buckthorn extract. Emodin was identified in tinbuckthorn, and iron-buckthorn natural pigments. Rhamnetin flavonoid and emodin anthraquinone are metal chelates. It was found that the dyestuffs are presented in tinbuckthorn, iron-buckthorn, and aluminium-buckthorn pigments. The other dyestuffs (quercetin, isorhamnetin and kaempferol) did not form complexes with the metals that have been used in the study. The brightness and colour values of aluminium, iron, and tin-buckthorn pigments were determined by CIELAB colour space system. The best values for aluminium, iron, and tin-buckthorn pigments were observed in samples that have been prepared with 30, 30 and, 50 ml of the solution of metal salts, respectively. When yellow and blue colour values for aluminium and iron - buckthorn pigments were examined, it was observed that the best yellow and blue colour were obtained from the pigments that formed by using 30 ml of alum solution. The best yellow and blue colour values for tin buckthorn pigment was obtained from the pigment that formed by using 50 ml of tin solution.

Figure 6: Photodiode array spectra of (- - -) quercetin standard compound and (___) peak at 18.6 min from chromatogram in figure 5.

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Figure 7: Photodiode array spectra of (- - -) rhamnetin standard compound and (___) peak at 27.3 min from chromatogram in figure 5.

Figure 8: Photodiode array spectra of (___) emodin standard compound and (- - -) peak at 30.8 min from chromatogram in figure 5.

Figure 9: CIE LAB graph of aluminium - buckthorn natural pigment.

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Figure 10: CIE LAB graph of iron-buckthorn natural pigment.

Figure 11: CIE LAB graph of tin-buckthorn natural pigment.

Conclusion
In this study, the reaction of the dyestuffs present in buckthorn with aluminium(III), iron(II) and tin(II) has been used to prepare natural pigments. Results from the HPLC analysis of aluminium - buckthorn pigment show that rhamnetin and emodin are presented in the hydrolysed buckthorn extract. Emodin in tin and ironbuckthorn pigments and rhamnetin and quercetin with emodin in the non-hydrolysed buckthorn extract were determined. The effect of different amounts of metals on the colouring scale of the pigments were investigated.

Acknowledgements
The support by the EU through the INCO CT 2005 015406 MED COLOUR TECH (www.medcolourtech.org) is gratefully acknowledged.

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