Biosynthesis of Furocoumarins: Further Studies on Ruta Graveolens
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BIOSYNTHESIS OF FUROCOUMARINS 813 Biosynthesis of Furocoumarins: Further Studies on Ruta graveolens F. DALL'ACQUA, A. CAPOZZI, S. MARCIANI, and G. CAPORALE Institute of Pharmaceutical Chemistry of the University, Centro di Studio per la Chimica del Farmaco e dei Prodotti biologicamente attivi del Consiglio Nazionale delle Ricerche, Padua (Italy) (Z. Naturforsch. 27 b, 813—817 [1972]; received March 20/April 24, 1972) The Authors have studied the biosynthesis of some furocoumarins contained in Ruta graveolens. Labeled 5,7- and 7,8-dihydroxycoumarins have been administered to the herb, however practically no incorporation of radioactivity was observed in the isolated furocoumarins. Moreover, with the aim to study in a more detailed way the role of rutaretin and marmesin, trapping experiments have been performed administering to the herb labeled umbelliferone together with unlabeled rutaretin and marmesin respectively. A very effective incorporation was observed into marmesin, in lesser extent into rutaretin. In trapping experiments carried out using labeled rutaretin together with unlabeled marmesin and "vice versa", the results obtained clearly show that while marmesin was converted into ruta- retin in a very effective way, rutaretin was transformed into marmesin only in a smaller extent. The Authors suggest the biogenetic origin of rutaretin and moreover formulate a new and more detailed aspect of the scheme for the biosynthetic pathway of furocoumarins in Ruta graveolens. The biogenesis of furocoumarins has been studied (III); from this compound other naturally occur- in various plants [Ficus carica 2, Pimpinella ring furocoumarins, for instance bergapten (IV) magna3'4, Ammi majus, Angelica archangelica, and xanthotoxin (V) can derive by means of hy- Heracleum lanatum, Ruta graveolens 5-9, Pastinaca droxylation and O-alkylation reactions. sativa10]. The general scheme of the biosynthetic More recently we have found that not only hy- pathway appears to be well established: it involves droxylation and O-alkylation reactions, but also de- in a first stage the formation of a 7-hydroxycouma- methoxylation reactions can take place in plants: in rin-derivative (the biogenesis of coumarins has fact we have observed that a reciprocal conversion already been largely studied); successively the furan between psoralen and xanthotoxin can occur6; ring is formed on this compound through an iso- moreover, 4',5' - dihydropsoralen, 4',5' - dihydro- prenylation reaction leading to a 4',5'-dihydrofuro- bergapten and 4/,5'-dihydroxanthotoxin proved to coumarin-intermediate; finally this last is trans- be precursors not only of the corresponding furo- formed into furocoumarin-derivative. coumarins (psoralen, bergapten and xanthotoxin, It has been found that 7-hydroxycoumarin (I) respectively) but also of the other two, although (umbelliferone) is the most effective general pre- only to a small extent5. cursor at the first stage. Recently BROWN 7- 8 sug- Concerning Ruta graveolens, some doubt could gested that the only natural intermediate at the stage arise about the preceding biogenetic scheme, be- of 4',5'-dihydrofurocoumarin-derivative is marme- cause marmesin till now has not been found present sin (II), which is then transformed into psoralen in this plant, although feeding experiments have OCH3 / ykAA, IV ovo v CH3 ^ II IH O'^V^O'^'O Fig. 1. General biosynthetic 0CH3 pathway proposed for psoralen V (III), bergapten(IV) and xantho- toxin (V). Requests for reprints should be sent to Dr. F. DALL'ACQUA, Universitä di Padova, Istituto di Chimica Farmaceutica, Via Marzolo 5, 35100 Padova (Italy). 814 F. DALL'ACQUA, A. CAPOZZI, S. MARCIANI, AND G. CAPORALE demonstrated that it may be a valid precursor for experiment was comprehended between the range furocoumarins (psoralen, bergapten and xantho- 10 —12 g. This was placed into 100 ml beakers con- taining a mixture formed by adding to the aqueous toxin) and especially for psoralen. On the other solution of the labeled compound to be administered, hand, another 4 ,5 -dihydrofurocoumarin-derivative an equal volume of an "aqueous nutritious solution" 17. has been isolated from Ruta graveolens, that is ruta- The system was illuminated with 500 W Osram HWL retin (VI), which is an 8-hydroxyderivative of mar- lamps; "metabolism time" lasted 7 days for feeding experiments with umbelliferone and its hydroxy- mesin n. derivatives, while for trapping experiments it lasted 54 hours. During the metabolic period "aqueous nutri- tious solution" was added to maintain constant the level of the liquid layer in the beakers. 3 OH VI Trapping experiments Fig. 2. Molecular structure of rutaretin (VI). Two different types of trapping experiments have been performed A) and B) : Very recently we have found that rutaretin (VI) A) 2.4 mg of tritiated umbelliferone was solubilized is well incorporated specifically into xanthotoxin in 40 ml of distilled water and successively 16 mg of unlabeled rutaretin was also dissolved in the same and also into psoralen and bergapten, even to a solution and this was administered to the herb as indi- 6 lesser extent . It may therefore be the natural cated in the section "feeding procedure". Analogous 4',5'-dihydrofurocoumarin-intermediate, at least for experiment was performed using unlabeled marmesin the biosynthesis of xanthotoxin. (15 mg) together with tritiated umbelliferone (2.6 mg); The studies reported in this paper have been per- B) 3 mg of tritiated rutaretin together with 10 mg of unlabeled marmesin were solubilized in 40 ml of formed to obtain further experimental information distilled water and this solution was then administered on the role of rutaretin, on its biogenetic origin and to Ruta graveolens as reported in the "feeding proce- on the possibility that marmesin also may be a phy- dure". Parallel experiment was carried out using 3 mg siological intermediate, perhaps present only in a of tritiated marmesin and 10 mg of unlabeled rutaretin, operating in the same experimental conditions. very small amount. The results obtained feeding Ruta graveolens with Isolation and purification of furocoumarins and of 5,7-dihydroxycoumarin (VII) and with 7,8-dihy- rutaretin and marmesin droxycoumarin (VIII) (daphnetin), as well as per- Isolation and purification of psoralen (III), bergap- forming some trapping experiments, in which marme- ten (IV) and xanthotoxin (V) were performed fol- lowing a procedure described elsewhere 6. sin and rutaretin were involved, allowed us to for- Rutaretin (VI) and marmesin (II) were isolated mulate a more detailed scheme for the biosynthetic and purified in a way similar to that described for pathway of psoralen and xanthotoxin in Ruta gra- furocoumarins with little change in the procedure for veolens. preparing the "coumarinic extract"1,2'18 as follows: the etheral coumarinic extract was washed three times with an equal volume of saturated sodium bicarbonate Materials and Methods aqueous solution. After this treatment, while marmesin and furocoumarins remained in the etheral solution 7-hydroxycoumarin or umbelliferone (I) 12, 5,7-di- (A), rutaretin passed into the aqueous bicarbonate hydroxycoumarin (VII) 13 and 7,8-dihydroxycoumarin phase; this last phase was acidified with 2n HCl and (VIII) 14 have been prepared in this Institute. then extracted with ether (B). From the two etheral Rutaretin (VI) 11 (extracted from Ruta graveo- 15 extracts (A) and (B) the solvent was distilled and the lens) and marmesin (II) (extracted from Aegle mar- residues were chromatographed as follows: melos, Correa) have kindly been provided by Prof. Dr. a) thin layer silica gel preparative chromatography G. SCHNEIDER and Prof. Dr. A. CHATTERJEE respecti- vely. 3H labeling procedure of all these substances has (Merck cat. 5717) using as solvent of development ethyl acetate : cyclohexane (3 : 1, v/v) ; been carried out according to the WILZBACH method 16. Chemical and radiochemical purification of labeled b) thin layer silica gel analytical chromatography compounds has been performed following a procedure (Merck cat. 5715) : solvent of development ethyl ace- described elsewhere 2. The specific radioactivity of these tate : cyclohexane (2 : 1, v/v) : at this stage chemical compounds is reported in Tables I, II and III. and radiochemical purity of the substances were tested. Spectrophotometrical measurements and radiochemical Feeding procedure determinations Cut ends of Ruta graveolens herb were employed; The different ethanolic solutions containing the generally the dry weight of this material for every various isolated compounds were examined spectro- BIOSYNTHESIS OF FUROCOUMARINS 815 photometrically both recording their u.v. spectra using It is well known that the biogenetic route starting a 124 double beam model Perkin-Elmer instrument and from umbelliferone and leading to furocoumarins successively determining their absorbance at X max involves 4',5'-dihydrointermediates; in the case of by means of a CF 4 Optica single beam spectro- photometer. Radiochemical measurements were per- Ruta graveolens recently it has been shown 6- 8 that, formed by a liquid scintillator spectrophotometer at this stage, marmesin (II) and rutaretin (VI) are model 3375 Packard using a dioxane base scintillator: involved. However while rutaretin is present in a naphthalene g 120, 2,5-diphenyl-oxazole g 4, 2,2'-p- valuable extent in this herb11 marmesin has not phenylen bis (5-phenyl-oxazole) g 0.075 in dioxane been till now identified. up to 1000 ml of solution. The apparatus efficiency in experimental