The growth improvement of aCardamine pratensis cell suspension can be obtained by the addition of casein hydrolysate to the medium. This component can be replaced by the same concentration of glutamine. Moreover on two cell suspensions,C. pratensis andSilene alba, this glutamine effect appears only when the medium has a phosphorus deficiency. Consequently, casein hydrolysate effectiveness would be due to its high concentration of glutamine and its contribution would balance the shortage of this amino acid synthesis when the medium has a phosphorus deficiency.
Silene alba cell suspensions were supplied with papaverine hydrochloride. Seven biotransformation compounds were obtained of which four were alkaloidal. The two most important compounds are regioselective demethylation products: 6-monodemethylpapaverine and Inthe field of vegetal cell biotransformation of alkaloids, little work has been performed on isoquinoline molecules. The bioconversion of reticuline by calluses of Papaver somniferum L. was studied previously (1). In this work we study the papaverine biotransformation by Si/ene alba Miller E. H. L. Krause cell suspension. Because S. alba cells are non-alkaloid producers, the biotransformation products are easily isolated.
MaterialsCell cultures of S. alba were grown in a Murashige and Skoog (2) liquid medium with sucrose (30 g.lj, adenine (7.4 106 M), 2,4-dichlorophenoxyacetic acid (5.l0_6 M) and kinetin (5. 10_6 M), as described earlier (3).The papaverine hydrochloride Codex was used as the substrate for these studies. The stability of a 1 g.l substrate solution was checked under experimental conditions after autoclaving in the culture medium; no new structures were demonstrated by thin layer chromatography (TLC).
MethodsCell suspensions with papaverine hydrochloride were incubated at 23°C C on a rotary shaker at 180 rpm under illumination of 25 sE.m2.s' for 12 hours per day. After inoculation and seven days of growth, dry weight (DW) evaluations were performed. Cells samples were collected by reduced pressure sieving and dried at 60° C for 48 hours.The culture medium was first extracted. Residues of extractions are extracted again after hydrolysis with concentrated H2S04 to pH 0.9 for 36 hours alternating between room temperature and reflux conditions. Samples were periodically checked for products by TLC. Extractions were performed after alkalinization to pH 10 with concentrated NH4 OH using methylene chloride. The organic phase was shaken with HCI 0.1 N (pH 4.5). The aqueous phase was alkalized with concentrated NH4OH and extracted with methylene chloride, subsequently dried over anhydrous Na2SO4 and evaporated under reduced pressure. It had been previously determined that this method is capable of achieving a 96 % efficiency in removing papaverine hydrochloride. The cells were lyophilized and extracted with methylene chloride in a Soxhlet apparatus. Crude extract was cleaned and extracted as performed with the culture medium.Extracts were then separated by TLC and densitometry. TLC was performed on Merck silica gel 60 F 254 plates. Three solvent systems were used: chloroform/acetone (60/40, v/v); benzene/methanol (75/25, v/v); and acetone/methanol/ammonia 85/5/2, v/v/v). UV light (254 and 366 nm) and Dragendorff's reagent were used for the detection of papaverine and its metabolic alkaloid derivatives. Densitometry was performed using a Vernon type pH 16 photometer with a Mazda 1803 incandescent lamp. The Kiesel gel 60 F Merck 0.25 mm thick plates were developed in a chloroform/acetone (70/30, v/v) solvent system. Spots were made visible by immersing the...
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