Relationship between the hydroxylation capacity of Digitalis lanata plants and cell cultures

ZHIBI HU

Shanghai College of Traditional Chinese Medicine
Shanghai, People's Republic of China

ABSTRACT

Digitalis lanata plants are used for the isolation of digoxin. As by- products, they always contain high amounts of digitoxigenin glycosides. Biotransformation of cardenolides by plant cell cultures indicate a new possibility of making better use of these by-products. In order to find the cell strains with high and selective 12 b-hydroxylation capacity, the relationship between the hydroxylation capacity of D. lanata plants and cell cultures was studied.

Establishment of cell cultures

The suspension cultures tested in our screening were derived from stem, leaf, anther and ovary callus of 36 D. lanata plants, containing different levels of digoxin. For purposes of plant selection a hydroxylation test with 3H-b-methyldigitoxin was carried out. A total of 83 cultures were investigated, with addition of b-methyldigitoxin 3 days after inoculation.

Results

The results showed that there was some relationship between the hydroxylation rates of leaves and cell strains. However, no direct correlation was observed between digoxin content of the leaves and the hydroxylation rates of the corresponding cell cultures. In spite of nearly identical digoxin content of the plant, the hydroxylating capacity of the corresponding cell strains varied from high 12 b-hydroxylation to total lack of any activity. The variation in yield of b-methyldigoxin even occurred in the cell cultures derived from one parent plant. It was therefore concluded that the biotransformation ability of the strains were indeed different.

Selection of variant strains

During intensive selection, it was found that there was a special cell strain, 10/56-S-50-Se, with hydroxylating capacity much far exceeding that of the original strain, 10/56-S-50. Up to about 900 mg l^-1 of product yields were achieved in a 20 liter airlift reactor, which was nearly 3 times higher than the original strain.

Environmental controls - influence of different culture conditions on the hydroxylation capacity of cell cultures.

In the studies conducted to determine the influence of macroelements, carbon sources, and light on hydroxylation capacity of the cell cultures, it was found that there was no absolute correlation between the hydroxylation capacity of the plants and that of the cell cultures. From these results, it was concluded that in order to get the cell strains with high and selective biotransformation capacity, we should simultaneously adopt the following strategy:

(a) the screening of the cell from differentiated plants, with high digoxin contents and high hydroxylating activity;

(b) selection of variant cell strains, and

(c) environmental controls.