Reacción #828724

Ecuación de reacción

2,4-dichlorophenoxyacetic acid

inositol

thiamine HCl

pyridoxine HCl

carborundum

nicotinic acid

→

diosgenin

Rendimiento 0.1%

Disolventes

HCl

Condiciones de reacción

Tratamiento posterior

1. 1
   workup.ADDITIONcontaining 0.1 mg
2. 2
   Otroon a rotary shaker (100 rpm, 28° C.)
3. 3
   OtroThe volume of growth medium per flask was
4. 4
   workup.ADDITIONwithout addition to the medium of elicitor in the form of oxidized BSA (20 μg./ml.)
5. 5
   workup.WAITAfter about 3 weeks
6. 6
   Filtraciónfiltered
7. 7
   Lavadowashed with water
8. 8
   Otrodried
9. 9
   ExtracciónThe cells were then extracted with chloroform at 60° C.
10. 10
    Filtraciónthe extract was filtered
11. 11
    Otroevaporated to dryness
12. 12
    Otrochromatographed
13. 13
    workup.DISSOLUTIONThe spots were then dissolved in chloroform

Procedimiento

Dioscorea deltoides (Mexican yam) cells (about 300-400 mg. fresh weight per flask) were grown in the dark in suspension in a growth medium, as described by Murashige and Skook (Physiol. Plantarum 1962 15: 473), containing 0.1 mg./l. 2,4-dichlorophenoxyacetic acid, 1 mg./l. nicotinic acid, 10 mg./l. thiamine-HCl, 1 mg./l. pyridoxine-HCl and 100 mg./l. inositol, in 125 ml. Erlenmeyer flasks on a rotary shaker (100 rpm, 28° C.). The volume of growth medium per flask was 30 ml., which contained about 10-20 mg. fine mesh carborundum and about 10 mg. sodium orthovanadate. An approximately equimolar amount of sodium metavanadate could be used instead of the orthovanadate. Comparative runs were carried out with and without addition to the medium of elicitor in the form of oxidized BSA (20 μg./ml.). After about 3 weeks, the cells were harvested, lyophilized, refluxed for 2 hours in 2N HCl, filtered, washed with water and dried. The cells were then extracted with chloroform at 60° C., the extract was filtered and evaporated to dryness, and the residue was taken up in chloroform and chromatographed. The spots were visualized with antimony chloride in chloroform. The spots were then dissolved in chloroform and the O.D. read in a spectrophotometer. Some determinations were made by GLC. In absence of elicitor there were obtained 0.1% (dry weight) diosgenin, compared with 3.8% (dry weight) in presence of elicitor, the latter representing a 38 fold enhancement of yield. The dry weight of cells at the end of the run was 1.6 g. per flask.

Fuente