Coumarins from Eucalyptus viminalis leaves

Abstract

The genus Eucalyptus L'Her numbers over 500 species [1]. According to the USSR State Pharmacopoeia, XIth Ed., leaves of E. viminalis Labill. are an official plant raw material [2].We have previously reported research on hydroxycinnamic acids, flavonoids, and tanning agents from leaves of E. viminalis [3]. In continuation of that work we have isolated and identified six coumarins from this raw material.Air-dried leaves of E. viminalis (5.0 kg) were extracted with purified water (15.0 L) at 95°C for 2 h and left to stand for 12 h. The extraction was carried out three times with new portions of purified water. The aqueous extracts were combined (40.6 L), evaporated at 85°C in a vacuum-circulator apparatus at 690 mm Hg to a volume of 1.0 L, and left to stand for 4 d. The supernatant liquid was separated. Polysaccharides were precipitated first from the aqueous solution by adding a three-fold amount of EtOH (96%). The precipitate was filtered off. The filtrate was evaporated to an aqueous residue that was fractionated using CHCl 3 , EtOAc, and BuOH. The completeness of the fraction separations was monitored using PC and TLC.Next the CHCl 3 and EtOAc fractions were studied. Paper chromatography showed that the CHCl 3 fraction contained coumarins, chlorophylls, phospholipids, and triterpene compounds; the EtOAc extract, coumarins, flavonoids, and hydroxycinnamic acids. The CHCl 3 fraction was treated successively with NH 4 OH and NaHCO 3 and NaOH solutions (1%), by which the coumarins were transferred to the aqueous phase as salts. The resulting aqueous solutions were acidified with HCl to pH 4-5 and re-extracted with CHCl 3 (fractions 1, 2, and 3, respectively). The CHCl 3 and EtOAc fractions were evaporated. The dry solid was dissolved in a small amount of EtOH (96%), spotted using a capillary on chromatographic paper impregnated beforehand with formamide-acetone (1:3), and chromatographed in descending mode using CHCl 3 and hexane. Chromatograms were visualized using a diazo reagent and alcoholic KOH (10%) to identify at least six coumarintype compounds that were separated over a column of silica gel (fraction:sorbent, 1:250, h = 80, d = 3 cm, elution by C 6 H 6 :CHCl 3 with increasing concentration of the latter after 1 L). The separation was monitored using UV light and PC.Fraction 1 afforded 1 (0.058 g) and 2 (0.043); fraction 2, 3 (0.033); fraction 3, 4 (0.034); EtOAc fraction, 5 (0.048) and 6 (0.065).Physicochemical and chemical methods (PC, TLC, UV and IR spectroscopy, specific rotation, melting point, acid and enzymatic hydrolysis) were used to identify the isolated compounds [4]. Products from acid hydrolysis of 5 and 6 contained D-glucose according to polarography and chromatography using n-BuOH:CH 3 CO 2 H:H 2 O (4:1:2) and (CH 3 )2CO:n-BuOH:H 2 O (7:2:1).Thus, the compounds isolated from leaves of E. viminalis were identified as: 1, C 9 H 6 O 3 , mp 228-230°C, λ max 231, 258, 327 nm; 7-hydroxycoumarin or umbelliferone; 2, C 10 H 8 O 4 , mp 202-204°C, λ max 230, 255, 296, 346 nm; 6-methoxy-7-hydroxycoumarin...