ANTHRACENE DERIVATIVES IN TISSUE CULTURE OF SOME NIGERIAN CASSIA SPECIES
ANTHRACENE DERIVATIVES IN TISSUE CULTURE OF SOME NIGERIAN CASSIA SPECIES
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Date
1983-06
Authors
RAI, PREM PRAKASH
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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
Description
A thesis submitted to the Postgraduate School,
Ahrnadu Bello University, in partial fulfillment
of the requirements for the degree of
DOCTOR OF PHILOSOPHY Department of Pharmacognosy & Drug Development
Faculty of Pharmaceutical Sciences
Ahrnadu Bollo University, Zaria, Nigeria June, 1983
Keywords
ANTHRACENE,, DERIVATIVES,, TISSUE,, CULTURE,, NIGERIAN,, CASSIA,, SPECIES.