ANTHRACENE DERIVATIVES IN TISSUE CULTURE OF SOME NIGERIAN CASSIA SPECIES

ANTHRACENE DERIVATIVES IN TISSUE CULTURE OF SOME NIGERIAN CASSIA SPECIES

Abstract:

This study was undertaken to determine the nature and quantity of anthraquinones produced in callus cultures of four Nigerian species of Cassia: C. nodosa Roxb., c. alata L., .C. occidentalis L. and C. podocarpa Guill. 4 Perr. A visual selection procedure for a high yielding anthraquinone cell line from callus cultures of C. podocarpa was attempted for the first time. The use of plant tissue culture for secondary product biosynthesis, particularly in plants of pharmaceutical importance, holds promise for the controlled production of plant constituents. In all of the Cassia cultures studied, only hydroxyanthraquinones, anthrones, dianthrones, and anthraquinone monoglycosides were produced and the most pharmacologically active dianthrone glycosides based on rhein were particularly absent. Callus cultures from seedling hypocotyls and cotyledons of Cassia nodosa, <C. alata, C. occidentalis and C. podocarpa were established. Cultures were grown at 28 C under constant illumination in an incubator, and these cultures maintained a satisfactory growth on Plurashige and Skoog (M/S) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.6 mg/l) and kinetin (0.35 mg/l). For pigment production and tissue growth, the optimal concentration of 2,4—D was 0.6 mg/l medium in the presence of kinetin (0.4 mg/l). The results indicated that the production of anthraquinones was usually correlated with the rate of tissue growth, and appeared to depend on the relative quantities of 2,4-D and kinetin in the culture media. An approach to visual selection of high yeilding cell lines in C. podocarpa callus resulted in en increasing accumulation of anthraquinones from 1.3 to 1.8% dry weight of the material. It is (iv) suggested that anthraquinone synthesis in Cassia callus cultures is associated with brown pigmentation of tissues. The composition of the mixture of anthracene derivatives found in callus cultures of Cassia spp. was investigated using a number of chromatographic separation techniques. Various methods of paper, thin-layer (including “Vario-Ks-Chamber” with controlled humidity), column, and high performance liquid chromatography for the separation and identification of anthraquinones were critically evaluated. Some new solvent mixtures were developed which provided superior resolution of hydroxyanthraquinones in paper and thin-layer chromatographic systems. An improved system of high performance liquid chromatography (HPLC) utilising a reversed phase ion pairing technique was developed, which enabled the separation of a five component mixture containing principal anthraquinones: chrysophanol, physcion, emodin, rhein and aloe emodin. The resolution of rhein, one of the acidic components of many naturally occurring anthraquinone mixtures, had not been possible by HPLC methods reported previously. The amount of free and combined anthraquinones, and the total anthracene derivatives content was estimated colorimetrically. The amount of some individual anthraquinone compounds was determined on thin-layer adsorbent material using a ‘Vitatron’ densitometer. A densitometric method reported earlier was modified to enable the estimation of individual anthraquinones as equivalent to 1,2-dihydroxyanthraquinone, used as an internal standard. This modified procedure was simpler and did not involve the use of large quantities of reference substances. (v) Whilst a number of anthraquinones, anthrones and dianthrones were produced in all the Cassia cultures in low concentrations; anthraquinone glycosides were present in “fair amount” in callus cultures of C. podocarpa only. The total yield of anthraquinone compounds in C. podocarpa cultures was nearly the same as obtained in the leaves of the whole plant. However, sennosides were not detected in any of the Cassia cultures. It wae evident from the visual selection studies on C. podocarpa that biosynthetic capabilities of callus tissues could be improved progressively by selective subcultures. Qualitative and quantitative results, and the absence of sennosides particularly in cultures of C.Podocarpa, emphasize the variation in the anthracene derived constituents of the derived callus and the parent plant

ANTHRACENE DERIVATIVES IN TISSUE CULTURE OF SOME NIGERIAN CASSIA SPECIES