OPTIMISING ALKALOID YIELD IN CATHARANTHUS ROSEUS

Weld trials on Catharnnthua roseus were carried out to determine the best time to harvcst plants for production of alkaloids. Foliar application of plant nutrients containing the major and minor essential elements and plant hormones produced a significant increase in dry matter and alkaloid content in the leaves and roots of the plant. It was obsemd that the treatment with micro elements together with either cattle manure or NPK gave the highest increases in dry matter and alkaloid content.


INTRODUCTION
Many developing countries, including Sri .Lanka, grow plants for the production of pharmaceuticals.Catltorantltus roseus belongs to family Apocynaceae.There are three formas in C. roseus.The most common one, the forma rosea, was selected for our studies.Over seventy five alkaloids have been reported from this plant.'The two alkaloids vincristine and vinblastine, used as anticancer agents, are present in the leaves of C. roseus.Ajmalicine present in the roots has anti-fibriliar properties.Studies on optimising alkaloid .yieldsare required since there is a demand for the roots and leaves of C. roseus in the international market.Chatterjee and co-workers have observed that foliar spray of iron and magnesium had pronounced effects on the formation of total alkaloids?
The work presented here reports on the establishment of an optimum harvest time for C. roseus based on yields of total alkaloids and total dry matter and the effects of fertilizer, foliar application of plant nutrients, manures and stress conditions on the dry matter and alkaloid content.

METHODS AND MATERIALS
Setting up of the experimental plot: Fresh seeds were broadcast in plot of 4 m x 4.5 m area of sandy soil at CISIR, and irrigated twice a week.Thinning out of the plants were done from two weeks of the broadcasting of seeds to produce a spacing of 30 cm x 30 cm in between the plants.
Estimation of dry matter yield : Eight plants were randomly selected and uprooted every month for the estimation of dry matter and alkaloids in leaves, stem and roots.
Sampling was done from 4th month up to 12'~ mohth of maturity.Plant materials were dried in the shade and weights were taken at each maturity stage.
Estimation of total alkaloids yield : Powdered plant material was extracted with dilute acetic acid twice by percolation at room temperature.The resulting solutions .wereextracted with chloroform in acidic and basic media.The alkaloids were obtained by evaporating the chloroform extracts.This method was developed in our laboratory.
Estimation of sljmalicine content in the roots : A known quantity of the total alkaloid fraction was separated on silica gel GF tlc plates along with the authentic ajmalicine.The plates were developed in a toluene-ethyl acetate (1:l) solvent system.Ajmalicine content in the sample was estimated by tlc-densitometry at 280 nm in a Camag automatic scanning densitometer.
Studies on the effect of foliar application of plant nutrients on dry matter and alkaloid content: Field experiments were injtiated in August with foliar application of 'Maxi crop' solution (which was a mixture of major and minor elements and plant hormones) diluted 100 times with water.The field trial was conducted with four replicates in randomized complete block design at the CISIR experimental plots.The soil was clay loam with 50% clay, 18% silt, 30% sand.The pH of the soil was 5.8.
The seeds for the nursery were obtained from India.After the seedlings were maintained for 45 days in the nursery they were transplanted in the experimental plots with a spacing of 30 cm x 45 cm , and irrigated once a week during the dry period.There were two treatments: 1. Addition of nutrient solution 2. No addition These two treatments were replicated four, times to reduce experimental errors.First application of the foliar niltrient was done two months after transplahtation using a hand sprayer.Consequently nutrient solution was sprayed once a month, up to the.loth month.From the 4'h month upto the 12' b month after transplantation, 12 plants were harvested randomly from each plot at monthly intervals.After harvesting, each plant was separated into roots, leaves and stems.After drying, the weight of the dry matter was recorded and anaiysed for alkaloids aird ajmalicine content in the roots.3 Studies to determine the effect of fertilizer, manure and stress condition : The field experiments were initiated in October with treatments consisting of The experiments were wried out in a randomized complete block design with three replicates at the CISIR experimental plots.After the seedlings were maintained for 45 days in the nursery they were transplanted in the pperimental plots with the spacing of 30 cm. x 30 cm.-The fertilizer and manure treatments were given at the time of transplanting and the pruning was practised at the 3* month of maturity.Irrigation was done once a week during the dry period.
Sampling and determination of the dry matter yield : 10 -15 plants were harvested randomly from each plot at the 4th and loth months of maturity.After harvesting the roots and leaves of each plant were separated.They were then dried in the shade until constant moisture levels were reached.The dry matter yields were recorded before powdering and analysis.

RESULTS AND DISCUSSION
The changes in the dry matter during the growth of the plant is shown in the Figure 1.
This growth curve-is useful to study the growth pattern to cdculate the expected yield per unit area at dierent stages >bf mat&@.A rapid increase in the yields was observed after the sth month.Application of fertilizer after the .4'hmonth may therefore help to enhance the yields.
The loth month was selected to'harvest the plant for best economical yields as sufficient t i e is then available to prepare the land for re-planting.
Studies on the effects of the foliar application of plant nutrients indicated higher dry matter accumulation in treated plants (Figure 2) .Increase in the dry matter content is marked in treated plants upto 10 months of maturity.After the loth month, dry matter decreased in leaves possibly due to a higher rate of defoliation of leaves.
Figure 3 shows the change in alkaloid content in the whole plant with and without treatment.Regression analysis showed that the effect of treatment on the alkaloid content was significant (p c 0.05).The alkaloid content in whole plants was considered due to changes in roots, stems and leaves during transportation.These results are in agreement with those of Chatterjee et d? who a h dbser~ed that foliar application of iron and magnesium caused a sigd3cant increase in' alkaloid b e n t .
It is apparent that the application of the mixture of NPK and micro elements gave the highest yield of roots and alkaloids at maturity (Figures 4 and 5).It is also clear that the applihtion of cattle manure or NPK together with micro elements gave higher yields that when either was used alone.Higher increases in ajmalicine contents too were observed with these two treatments (Figure 6).The cattle manure when   From these studies it can be concluded that the best time to harvest the plant is at the loth month of maturity.The maximum utilization of the land is then possible as sufficient time is available to prepare the land for the next cycle.The foliar application of plant nutrients and application of NPKmicro element mixture or NPKcattle manure mixtures caused significant increases in the yields of dry matter and alkaloid contents.In addition these practices are economically feasible on a commercial scale.

3 IFigure 1 Flgure 4 :
Figure 1 The changes In the ykld of alkaloids In C roseus with f o k applbUoo ot plant nulrknb.

Figure 6 :
Figure 6: The eflects of dlflerent treatments on the Jmalklne content of rook.