Biotransformation of hydroxyanthraquinone glycosides in Cassia species

S.R. MALELE

Department of Pharmaceutical Sciences
Muhimbili Medical Centre
P.O. Box 65013
Dar es Salaam, Tanzania.

ABSTRACT

The development and application of tissue cultures in the production, biosynthesis and biotransformation of secondary metabolites is presented. Specific consideration is given to 1, 8 - dihydroxyanthraquinone derivatives of Cassia senna and Cassia artemisiodes. Plant Tissue Cultures, both static (solid) and in suspension (liquid) were established from seeds of same. Conditions for culture growth were investigated and optimised and cultures were maintained by sub-culturing for up to 32 passages.

Qualitative and quantitative analysis of hydroxyanthraquinone derivatives was investigated with emphasis on the application of HPLC. Total content and variation of these compounds in the species was carried out. Five compounds were identified and assayed, namely aloe-emodin, chrysophanol, emodin, physcion and rhein.

Incorporation of radio-active precursors (U-¹⁴C-acetate and (2-¹⁴C- malonate) were studied in cultures of the species, and their conversion into hydroxyanthraquinone derivatives has been instigated. Cultures were harvested at regular intervals, extracted and the hydroxyanthraquinones separated by HPLC before measurement of incorporated radioactivity.

Fluctuation of the radioactivity in the anthraquinone constituents occurred throughout the passage suggesting that biosynthesis and biotransformation were occurring simultaneously.

Plants of the same species were injected with (2-¹⁴C)-malonate, anthraquinones extracted at regular intervals and separated by HPLC prior to measurement of radioactivity.

Introduction

Anthraquinones are the largest group of natural quinones and historically the most important which for a long time have been used as dyes. The derivatives have cathartic activity and are used as purgatives and are widely employed in geriatric and pediatric medicine (Rada et al., 1974). Plant families which are the richest sources of this class of compounds (including important genera) are Polygonaceae (Rheum, Rumex and Polygonum), Rhamnaceae (Rhamaus and Zizyphus), Leguminoceae (Cassia), Rubiaceae (Morinda, Rubia and Galium, and Liliaceae (Threase and Evans, 1983).

Species such as Rheum palmatum (rhubarb), Aloe ferox, Cassia senna, and Rhamnus alnus have long been used as laxative drugs. They contain the anthraquinone derivatives, mainly as glycosides, which on hydrolysis yield aglycones which are hydroxyanthraquinone derivatives. The common polyhydroxyanthraquinone derivatives present in laxative drugs are 1,8 - dihydroxyanthraquinones (1,8 - DHAQ) and typical structures are given in Figure 1.

Fig. 1: Typical polyhydroxyanthraquinones

Biosynthesis of anthraquinones

Leristner et al., (1969) and Fairbairn et al. (1972) established that naturally occurring anthraquinones are synthesized by two completely separate pathways. Thus those of the emodin type (with substituents in both terminal rings A and C) are usually derived through the acetatemalonate (polyketide) pathway in both higher and lower plants, while the alizarin (without substituents in ring A) type of anthraquinones are derived through the shikimic acid pathway.

Pharmacology and mode of action

Sennosides have the highest purgative activity, followed by rhein monoglcosides, whereas the anthraquinone glycosides are less active and the aglycones have the least activity (Fairbairn et al., 1949, 1965, 1970).

The mechanism of action of anthraquinone glycosides involves the systematic deposition of these compounds to the site of action in the intestine, enzymatic cleavage of the sugar groups and the slow oxidation of the resulting compounds, thus releasing the free anthraquinones which act on the intestines to produce peristalsis (Fairbairn, 1964).

Plant tissue culture

Over the centuries, plants have made a major contribution to the health of mankind, particularly through their use as spices, flavours, fragrances, vegetable oils, soaps, natural gums, resins, drugs, insecticides and other significant industrial, medicinal and agricultural raw materials. Scraag (1986) noted that despite substantial advances in microbial and chemical production methods, plants still remain the source of active ingredients of some 25% of prescribed medicines. The continued demand of these compounds has encouraged scientists to search for reliable alternative sources. One of the significant contributions to the manipulative powers of modern biologists has been the development of tissue culture techniques. Plant cells in culture have been expected to produce secondary metabolites which are characteristic of the whole plant (Rai, 1976). Several patents dealing with the production from cultures of metabolites such as allergens, dios-genin, L-dopa, ginsenosides, glycyrrhixin, etc have been registered (Staba, 1982; Bajaj, 1988).

In this paper the establishment of tissue cultures of Cassia species and the careful phytochemical investigation of the controlled production of the hydroxyanthracene derivatives is discussed. An attempt to devise a sensitive, rapid and efficient analytical technique of these very closely related hydroxyanthracene derivatives by the use of HPLC will also be presented.

Materials and methods

Cultures

Cultures were established from seeds of Cassia artemisioides on Murashige and Skoog's modified tobacco medium. Cultures were incubated in the dark at 25°-27°C and maintained for more than 30 passages, each of 38 days. Static cultures were chosen for subsequent analysis rather than suspension cultures because they proved to give better results in the production of secondary metabolites. Anthraquinone content variation during a single passage of the culture was done with a view to subsequent investigation of the biotransformation of the compounds produced.

Phytochemical investigations

The phytochemical investigations followed the scheme shown in Figure 2.

Sensitivity screening for sennosides showed negative results. Nonetheless purification was carried out by column chromatography and preparative TLC. Five compounds - chrysophanol, emodin, physcion, aloe-emodin and rhein -were isolated and identified spectroscopically (UV, IR and MS) and by comparison of the melting points with those reported for chrysophanol, emodin, physcion, aloe-emodin and rhein.

Radio-tracer studies

Feeding technique

The precursors used were (1-¹⁴C)-acetate and (2-¹⁴C)-malonate. 0.1 mCi in 5 ml of each of the tracers was separately added onto the callus once the culture showed visible signs of growth. Cells were harvested at regular intervals, extracted and the compounds were separated by high performance liquid chromatography (HPLC). Plants were fed with ¹⁴C-malonate and radio-active incorporation monitored at regular intervals by HPLC. The malonate was fed at the leaf-base where an axillary bud was evident. The HPLC instrument consisted of Rheodyne rotary valve which was equipped with a 100 ml loop, in order to collect sufficient eluate from the column for scintillation studies.

Anthraquinones were consistently eluted in the sequence, aloe-emodin, rhein, emodin, chrysophanol and physcion. Using the reverse phase system, this elution sequence is broadly in accordance with their polarities: aloe-emodin polar, and physcion, least polar is eluted last.

Results

The results of the study to investigate the influence of ¹⁴C-acetate and ¹⁴C-malonate, intermediates in the biosynthesis of polyketides, on the production of hydroxyanthracene derivatives are shown in Figure 3A and 3B and also in Figure 4A and 4B. The incorporation rates of the two radio-tracers and the radio-activity values are given in Table 1.

Figure 2 : Schematic diagram for the entraction of Hydroxyanthracene derivatives

Figure 3A : Influence of acetate and malenate in rhein production in static cultures of Cassia senna

Figure 3B : Influence and radioactivity incorporation of acetate and malonate in emodin in static cultures of Cassia senna

Figure 4A : Influence and radioactivity incorporation of acetate and malonate in chrysophanol in static cultures of Cassia senna

Figure 4B : Influence and radioactivity incorporation of acetate and malonate in aloe-emodin in static cultures of Cassia senna

Fig 5 : Suggested transformation of anthroquinones derivatives

Comments:

(a) Anthracene derivatives were able to absorb the radio- tracers.

(b) Malonate was incorporated into hydroxyanthracene compounds at a higher rate than for acetate. The incorporation varied, chrysophanol being highest and with rhein much lower.

The suggested transformation of anthraquinone derivatives is given in Figure 5.

Discussion and Conclusion

From the results above the interconversions shown in Scheme 1 were found to occur.

Scheme 1