Red, redder, madder : analysis and isolation of anthraquinones from madder roots (Rubia tinctorum)

The roots of Rubia tinctorum L. (madder) are the source of a natural dye. The dye components are anthraquinones with alizarin being the main dye component. Alizarin as such is present in madder root in only small quantities, most of the alizarin is present as its glycoside ruberythric acid. The sugar in this disaccharide is primeverose. Madder roots have been used to dye textiles in many parts of the world since ancient times. From 1600-1900 there was a heavy trade in madder throughout Europe. Madder root was an important export product for Holland. In 1868 Graebe and Liebermann discovered how to prepare alizarin synthetically. At the end of the 19 thcentury the madder culture rapidly declined due to the cheaper production of synthetic alizarin. Production of synthetic alizarin gives polluting side products. Nowadays the use and production of natural dyes becomes more popular due to the growing awareness for the environment and the need for alternative crops. An important element in the revitalisation of madder as an industrial crop is that the dye preparation from madder should be able to compete in quality and price with synthetic alizarin.Due to this renewed interest this research was initiated with this thesis as result.For the simultaneous identification of the anthraquinone glycosides and aglycones in extracts of madder root a high-pressure liquid chromatography method (HPLC) was developed. The anthraquinones were separated on an end-capped C 18 -RP column with a water-acetonitrile gradient as eluent and measured with ultra violet (UV) detection at 250 nm. For the identification of anthraquinones on-line a mass spectrometer (MS) and a diode-array detector were used.The main anthraquinones in an ethanol-water extract of madder root are the glycosides lucidin primeveroside and ruberythric acid and the anthraquinones pseudopurpurin and munjistin, which contain a carboxylic acid moiety. Beside these compounds also small amounts of the aglycones alizarin and purpurin could be detected and sometimes also lucidin was present.For the production of a commercially useful dye preparation from madder, the glycoside ruberythric acid should be hydrolysed to the aglycone alizarin, which is the main dye component. An intrinsic problem of the hydrolysis of ruberythric acid in madder root is the simultaneous conversion of lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin. The anthraquinone composition of the suspensions was analysed with HPLC-UV, HPLC-DAD and HPLC-MS.Stirring of dried madder root in water at room temperature for 90 min gave a suspension with pseudopurpurin, munjistin, alizarin and nordamnacanthal. Nordamnacanthal originates from lucidin primeveroside, which is hydrolysed to lucidin and subsequently oxidised to the corresponding aldehyde nordamnacanthal by an endogenous hydrolase and oxidase respectively. Nordamnacanthal is not mutagenic. During this conversion oxygen is obligatory and can be added by stirring the suspension. This stirring is an easy method for simultaneously hydrolysing ruberythric acid and to getting rid of the mutagenic lucidin.Different madder root cultivars were screened for their anthraquinone composition and amount of the main anthraquinones. The concentration of alizarin varied from 6.1 to11.8 mg/g root. If madder root was cultivated for three instead of two years the amount of alizarin increased from 6.7 mg/g to 8.7 mg/g.A number of different methods were compared for their capacity to isolate alizarin from the rest of the plant material. To make a first selection attention was mainly paid to the yield of alizarin. Three routes were selected as most promising for an industrial application. These three methods were tested at a larger scale. The first method consisted of the following steps: conversion of madder root (250 g) by endogenous enzymes, extraction of madder root with refluxing ethanol-water, hot filtration, evaporation to half of the original volume and precipitation at 4°C. An extract of 14.7 g was obtained which consisted for 35 % of anthraquinones. Of the total amount of alizarin available in the starting material 78% was extracted. The second method consisted of the following steps: extraction of madder root (250 g) with refluxing water, hot filtration, conversion of the glycosides in the filtrate by a madder root enzyme extract and precipitation at 4°C. An extract of 3.2 g was obtained of which 38 % were anthraquinones. Of the total amount of alizarin available in the starting material 19% was extracted. The third method consisted of the following steps: extraction of madder root with an aqueous surfactant solution, twice C 18 chromatography for extracting alizarin, elution of alizarin with methanol and evaporation. An extract of 17.1 g was obtained of which 11 % were anthraquinones. Of the total amount of alizarin available in the starting material 98% was extracted.For the development of an economically feasible route these three methods have to be further optimised. After optimisation the three routes have to be compared in terms of amount of extract obtained, alizarin content, dyeing capacity, costs and industrial applicability of the procedure.