Isolation and structure elucidation of 5,7-disubstituted simple coumarins in the fruits of Heracleum mantegazzianum

In addition to bergapten (1), xanthotoxin (2), isopimpinellin (3), imperatorin (4), and pimpinellin (5), for the first time 5,7-disubstituted simple coumarins, limettin (5,7-dimethoxycoumarin) (6) and a new compound 5-methoxy-7-(3,3-dimethylalliloxy)-coumarin (7), have been isolated from the fruits of Heracleum mantegazzianum Somm. et Lev. The new compound 7 is a derivative of anisocoumarin B. The structures of 6 and 7 were determined on the basis of 1D and 2D NMR spectra.

Keywords: Limettin, 5,7-disubstituted-simple-coumarins, furanocoumarins, Heracleum mantegazzianum Somm. et Lev., Umbelliferae.

Heracleum mantegazzianum Somm. et Lev. (Umbelliferae) is an endemic plant naturally occurring at Caucasus (Heywood, 1971; Sascyperowa & Komissarenko, 1978). The plant is genetically related to Heracleum sibiricum L., but usually the aerial parts are more distinguished, reaching up to 160 cm (Sascyperowa & Komissarenko, 1978). This plant has been naturalised in Europe and North America as an ornamental (Heywood, 1971) or as feed for animals (Sascyperowa & Komissarenko, 1978). Reports on the biological activities of the plant constituents have suggested use for dermatitis (Heywood, 1971; Pira et al., 1989) and application in PUVA therapy (Beyrich, 1966). The essential oil, at the concentration up to 8% (Tkaczenko, 1987; Jain, 1969), possesses antimicrobial and antifungal properties. In addition to the essential oil and flavonoids (Siwon & Karlsen, 1976), coumarins have been isolated either from stem or fruits in abundance (Heywood, 1971; Sascyperowa & Komissarenko, 1978; Murray et al., 1982). The highest concentrations were detected in fruits (Pira et al., 1989), and all have been established as furanocoumarins (Heywood, 1971; Murray et al., 1982) with typical photosensitizing properties (Beyrich, 1966). Simple derivatives benzo-α-pirone have not been found in the fruits so far, and only small amounts of osthol and umbelliferone in the stems were described (Murray et al., 1982).

For the first time, our investigations report the isolation and structure determination of 5,7-disubstituted simple coumarins 6, 7 in the fruits of Heracleum mantegazzianum.

The mature fruits of Heracleum mantegazzianum (Umbelliferae) were collected at the district Slawinek, Lublin (Poland) from the Botanical Garden of the University of Maria Curie-Sklodowska in August 1994. A voucher specimen (No. 1981), identified by M.Sc. M. Byc, is deposited in the herbarium of Pharmacognosy Department, Medical University, Lublin, Poland.

The mature fruits of H. mantegazzianum (200 g) were pulverised and extracted with petroleum ether (POCH Gliwice, Poland, 60-70 Celsius) in a Soxhlet extractor for 48 h. The obtained extract concentrated in vacuum was kept in a refrigerator and the sediment, containing coumarins (TLC preliminary analysis on a silica gel, CH2Cl2-MeCN 98:2), dissolved in CH2Cl2 (E Merck, Darmstadt, Germany) was chromatographed on a Florisil (Fluka, 60-100 Mesh) column and eluted with a gradient of CH2Cl2-EtOAc (0-20%) to give 16 fractions. The first 4 fractions were collected together and further separated on a second Florisil (60-100 mesh) column eluted with a CH2Cl2-EtOAc (0-20%) gradient to yield 16 fractions. The combined fractions 1 and 2 were chromatographed on a silica gel 60 (230-400 mesh) column, eluted with a gradient CH2Cl2-EtOAc (0-10%) to give 5 fractions which were further separated by TLC on silica gel (E Merck, Darmstadt, Germany), using benzene-EtOAc 9:1, n-heptane-CH2Cl2-EtOAc 4:4:2, or CH2Cl2-MeCN 97.5:2.5. In this way, compounds 1-7 were isolated (Fig. 1).

The compounds 1-5 were identified on the basis of VR determined in micro-HPLC (Selko, Poland) analysis on Separon C18 250 x 1 mm column (65% aq. MeOH, flow rate 50 µl/min, UV detection at 254 nm) and Rf from TLC analysis (silica gel), n-heptane-CH2Cl2EtOAc 4:4:2 (UV detection at 254 nm). The VR and Rf values for 1-5 were in accordance with the values obtained for the corresponding standards and were as follows: 1 (VR 596 µl, Rf 0.77), 2 (VR 448 µl, Rf 0.71), 3 (VR 464 µl, Rf 0.87), 4 (VR 1076 µl, Rf 0.82), and 5 (VR 543 µl, Rf 0.70). For a mixture of 6 and 7, VR1 and VR2 of 623 and 665 µl and one spot, Rf 0.79, were determined. The fractions 3-5, after the second CC on the Florisil column, were combined and further separated on a silica gel 60 (230-400 Mesh) column eluted with a) a gradient (0-100%) of CH2Cl2-pet. ether, and b) CH2Cl2-EtOAc (0-20%), to obtain additional amounts of 1-5 after preparative TLC (silica gel), using benzene-EtOAc 9:1.

¹H-,13C-NMR, ¹H-¹H-COSY, ¹H-13C-HMQC and NOESY spectra for 6 and 7 were recorded on a Bruker A-300 NMR instrument.

The petroleum ether (60-70 Celsius) extract from the fruits of Heracleum mantegazzianum was found to be abundant in UV absorbing coumarins (preliminarily TLC analysis, UV detection).

Extensive chromatography of coumarin fractions by combination of the following two column chromatographic separations on florisil and the third separation on silica gel in gradient mode in each case followed by preparative TLC and RP-micro-HPLC analysis of the yielded five furanocoumarins 1-5 (Fig. 2) together with three coumarin fractions with Rf values and colour of fluorescence in UV light at 254 nm (1.0- violet, 0.63-blue, and 0.79- yellow-brown). The first two compounds were only in trace amounts and detailed NMR experiments were not performed. The last fraction (yellow-brown spot in UV light, 254 nm) was obtained in the amount about 5 mg (yellow-white flakes from methanol) and HPLC analysis revealed the mixture of two closely eluting coumarins (6-7). This mixture was further analysed by 1D and 2D NMR techniques.

Assignment of particular NMR signals to protons in the analysed coumarins mixture was possible especially by the application of ¹H-¹H-COSY, ¹H-13C-HMQC and NOESY techniques. The double bond in position C3-C4 in coumarins 6, 7 is proved by signals of olefinic protons in the region with δ = 6-9 ppm. These are doublets with coupling constants J3,4 = 9.8 Hz, typical for these structural fragments in coumarin skeleton (Tables 1 and 2). Substitution of aryl ring in compounds 6 and 7 was evidenced by two doublets at &delta ≈ 7 (for H-6) and &delta ≈ 7.6 (for H-8), with small value coupling constant J6,8 = 2.3-2.6 Hz (Tables 1 and 2), typical for meta-substituted protons in an aromatic ring. These assignments for H-3, H-4 and H-6, H-8 for 6 and 7 were clearly confirmed in ¹H-¹H-COSY and ¹H-13C-HMQC spectra (Tables 1 and 2). Two singlets in the ¹ H-NMR spectrum at &delta ≈ 4 (for H-5') and &delta ≈ 4.15 (for H-7') are typical for methoxyl groups. Identification and position of the methoxyl groups (H-5' and H-7') in 6 were additionally determined by ¹H-13C-HMQC and NOESY techniques. The NOESY spectrum revealed the close proximity of H-5' to H-4 and H-6, and H-7' to H-6 and H-8 (Tables 1 and 2). For compound 7, only one singlet at &delta ≈ 4.17 (for H-5') suggested for one methoxyl group at C5, as confirmed in ¹H-13C-HMQC and NOESY experiments. The presence of the isoprenoid chain in compound 7 at C7 was evidenced by two broad singlets at δ = 1.7 and 1.74 ppm (two methyl groups on double bond), doublet for methylene protons (CH2-7α) at δ = 4.85 ppm, which are coupled with vinyl proton (H-7β) for triplet at δ = 5.61 ppm (J = 6.5 Hz). A relatively high value of the chemical shift of CH2-7α protons proves that these protons are deshielded by an oxygen atom from one side, and a double bond from the other. The mutual relationship between these protons was clearly determined in ¹H-¹H-COSY and ¹H-13C-HMQC spectra.

Compound 6 (5,7-dimethoxycoumarin) is known in the literature (Gray, 1983) as limettin (citropten) and was isolated from some Citrus spp. (Rutaceae). This is the first report of the isolation of limettin from Heracleum mantegazzianum fruits and Umbelliferae. Compound 7 [(5-methoxy-7-(3,3-dimethylalliloxy)coumarin)] is a new compound not described yet in the scientific literature, although from the stem fraction of Clausena anisata (Rutaceae) its demethyl derivative [(5-hydroxy-7-(3,3-dimethylalliloxy)-coumarin)] has been isolated and named anisocoumarin B (Ngadju et al., 1989). Therefore, compound 7 is considered as 5-OMe-anisocoumarin B. The isolation of 7 from the fruits of Heracleum mantegazzianum suggests that similar biogenetic processes leading to 5,7-dioxygenated-7-O-prenylcoumarins in the Rutaceae should be also considered in the Umbelliferae.

Address correspondence to: Prof. Dr. K. Glowniak, Department of Pharmacognosy, Medical University, 12 Peowiaków St., 20-007 Lublin, Poland. Fax: + 48-81-53-289-03. E-mail: glowniak@hipokrates.am.lublin.pl.

DIAGRAM: Fig. 1. Isolation of coumarins from the fruits of Heracleum mantegazzianum.

DIAGRAMS: Fig. 2. The chemical structures of the identified coumarins.

Accepted: April 25, 2000