Salicin, salicylic acid-O-p-D-glucopyra-noside, is a naturally occurring glycoside found in the bark of willow trees (Salix spp.) and used as a template for the synthesis of acetylsalisylic acid, known as aspirin. Ancient man used it as an analgesic and antipyretic. Salicin plays an important role in plant-herbivore interactions[1, 2].
Varthemia persica DC. (Asteraceae) is an aromatic plant growing wild in central parts of Iran . The essential oil of V. persica has been reported to have several mono- and sesqueterpenes, specially b-eudesmol and spathulenol . There is no report on cell cultures of V. persica.
Plant cell cultures are capable of glucosylation of exogenously added phenols and their analogues. Several reports on bio-transformation of salicylaldehyde, salicyl alcohol, and salicylic acid as precursors for the production of glycosides have been published in the literature. Mizukami et al. studied glucosylation of salicyl alcohol in several cultured plant cells . High level production of salicin from salicylic acid has been achieved by cell suspension cultures of Catharanthus roseus [7, 8]. It was reported that salicylaldehyde is the optimal precursor of salicin in cultured cells of Datura meteloides and Coronilla varia . Also salicin was enzymatically synthesized from salicylic acid and salicyl alcohol using glu-canyltransferase of Bacillus subtilis macerans and sucrose dextransucrase of Leuconostoc mesenteroides . So far, there is no report on using cultured cells of V. persica for sali-cylaldehyde biotransformation studies.
2. Materials and methods
2.1. Cell cultures
Seeds of V. persica were surface sterilized in 30% w/v hydrogen peroxide containing 1% tween 80 for 2 min., then germinated on wet filter paper in Petri dishes in the dark at 25 °C. The cotyledons were then transferred onto Murashige and Skoog (MS) media containing 5 ppm ascorbic acid, 2 ppm 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 ppm kinetin (kin) . Calli were maintained by subculturing every 4 weeks, and suspension cultures were formed by agitation of 5 g callus to liquid medium until a suspension of free cells was formed. The suspensions were then placed on a rotary shaker running at 100 rpm, and were maintained by subsequent subculturing, using a dilution of 1 to 2, into new fresh liquid media.
The callus and suspensions were maintained in a 16 h light / 8 h dark cycle at 27 °C and subcultured every 4-6 weeks.
Suspension cultures grown over more than twenty generations were used for substrate feeding and bioconversion studies.
2.2. Substrate feeding and product extraction
The salicylic acid, salicylaldehyde, benzaldehyde, anisaldehyde, citral, eugenol, and safrole obtained from Sigma. Chemical purity (greater than 98%). Substrates were dissolved in a water-miscible solvent (ethanol 70%), which resulted in good mixing of the substrate upon addition to the aqueous medium. The substartes was added to suspension cultures to make a final concentration of 100 ppm, cell volume (50% p.v.). Control readings were made without addition of substartes to cultures and with addition of substartes to cell-free medium. The cultures were incubated under the conditions mentioned above. After the incubation period, the flask was swirled to ensure good mixing and two samples were removed with a 10-ml pre-sterilized, glass-tipples pipette. A new pipette was used for each sample .
The substrates and products were extracted from the dried cells which had been filtered from suspension cultures. The extract was reduced to a final volume of 1 ml and subjected to thin layer chromatography (TLC).
Table 1. Precursor fed to Varthemia persica cells suspension.
TLC analysis were carried out as following: the concentrated methanolic extracts obtained were spotted on precoated silicagel plates (Merck) and chromatographed in a saturated chamber containing EtO-AC: MeOH: H2O (77:13:10) solvent mixture. Visualisation of the separated bands was carried out under U.V. light (365 nm) . The glycosides were detected as a brown and red individual bands on extract obtained from salicylaldehyde feeding. The dried plates were then sprayed with vanillin-glacial acetic acid reagent.
Gas chromatography (GC) analysis were carried out as following: After 24 h of substrates feeding both the cells and the media were extracted using dichloromethane. The extract was reduced to a volume of 100 ml under nitrogen, then 0.1 ml was analyzed by gas liquid chromatography (GLC). GC analysis was carried out on a Perkin-Elmer 8500 GC with FID detector and a BP-1 capillary column (39 m×0.25 mm; film thickness 0.25 mm). The carrier gas was He with a flow rate of 2 ml/min., the oven temperature for first 4 min. was kept at 60 °C and then increased at a rate of 4 °C /min. until reached to the temperature of 280 °C, injector and detector temperatures were set at 280 °C. Confirmation of peak identity was effected by co-chromatography with standards .
Table2. Salicin bioproduced using Varthemia persica cells suspension.
3. Results and discussion
Several substrates including; two aromatic aldehydes, an aliphatic aldehyde, an aromatic acids, a phenol and an ether phenol were fed to V. persica cell suspension cultures to study the possible enzymatic conversion. No metabolism was detected over 48 h of incubation when the cultures were extracted with dichloromethane and methanol and extracts subjected to GC and TLC analysis, except for salicylaldehyde in which case salicin were detected as biotransformation products (Table 1).
V. persica cell suspension cultures were able to produce salicin from salicyl aldehyde when non-quantitative TLC analysis was viewed. No biotransformation was detected over 24 h when salicylic acid was administrated to the cells. Zenk (1967) suggested that salicyl alcohol is not a direct precursor of salicin, these findings may suggest the non necessity of alcohol formation for salicin production in these cultured cells . Terao et al. (1984) described a novel enzyme catalyzing a position-specific glycosylation of salicyl alcohol to form salicin in Gardenia jasminoides cell cultures . No salicyl alcohol formation was observed in our study. It has been reported that cell suspensions of some plant species glucosylate salicyl alcohol to form the phenolic glycoside salicin whereas cultures of other species form the corresponding alcohol glycoside, isosalicin, when they are fed with salicyl alcohol [6, 17]. It appears that the glycosylation reaction may vary with culture strains as well as chemical structures of substrates. No conversion was achieved when we administered benzaldehyde, anisaldehyde, citral, eugenol, and safrole to cultured cells of V. persica. Similarly, Tabata et al. (1988) have shown that from cell suspension cultures from ten different plant species, only six were able to glucosylate salicyl alcohol . Dombrowski and Alfermann (1992) found that suspensions from 12 different plant species converted salicyl alcohol to salicin and isosalicin or only isosalicin [19, 20].
V. persica cell cultures were not able to glucosylate benzaldehyde, anisaldehyde, and citral. The position of glycosylation in the substrate molecules seems to be specific. In this connection it has been reported that Bupleurum falcatum failed to glucosylate m-hydroxybenzoic acid, whereas some other culture strains glucosylated m- and p-hydroxybenzoic acid .
Interestingly, the salicylaldehyde administered was converted to the salicin within 24 h (Table 2), whereas other culture strains derived from different plant species produced salicin only within four days . It seems the successful glycosylation of an exogenous substrate requires a strong gluco-syltranferase activity. The interesting point to note is that the reverse reaction, conversion of salicin to salicylaldehyde, can be seen in plant defensive situation .
In plant cells, glucosylation reactions are of special interest because they facilitate the conversion of water-insoluble compounds to water-soluble compounds. Plant systems typically retain secondary metabolites intra-cellularly, therefore their accumulation might interfere with basic physiological functions, especially if the compounds are toxic or precipitate due to low solubility. Moreover, the investigation of phenolic glycosylation is of great importance in view of the possible application of plant cell culture to an efficient production of useful compounds.
This research was supported by a grant from the research council of the Isfahan University of Medical Sciences.