Fitoterapia 72 Ž2001. 376᎐381
Furanocoumarins from the aerial parts of Dorstenia contrajer¨a A. Caceres a , L. Rastrelli b, F. De Simoneb, G. De Martino b, C. Saturnino b, P. Saturnino b, R. Aquino b,U a
Departamento de Citohistologıa, ´ Escuela de Quimıca ´ Biologica, Uni¨ ersidad de San Carlos de Guatemala, Zona 12, 01002 Guatemala b Dipartimento di Scienze Farmaceutiche, Uni¨ ersita ` di Salerno, In¨ ariante 11 r C, 84084 Fisciano, Salerno, Italy
Received 2 October 2000; accepted in revised form 21 November 2000
Abstract A new glycosylated furanocoumarin, ␣-L-rhamnopyranosyl-Ž1 ª 6.--D-glucopyranosylbergaptol (1), has been isolated from Dorstenia contrajer¨ a together with three known furanocoumarins, catechin and epicatechin. Their structures were established using high field 2D NMR techniques. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Dorstenia contrajer¨ a; Furanocoumarins; Flavonoids
1. Introduction Dorstenia is a herbaceous genus of the family Moraceae found throughout the Central and South American countries and particularly rich in furanocoumarins. Dorstenia contrajer¨ a L. is a shrub native in moist thickets and forests from South Mexico to Venezuela and Peru, and diffused in the lesser Antilles north of Puerto Rico, in Guatemala, Panama and Costa Rica w1,2x. The entire plant, including rhizome and roots, is used in local traditional medicines such as febrifuge, U
Corresponding author. Tel.: q39-08-9964356; fax: q39-08-9962828. E-mail address:
[email protected] ŽR. Aquino.. 0367-326Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 0 . 0 0 3 2 8 - 2
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emmenagogue, remedy for cold and diarrhea, and for the treatment of snake-bites. In previous investigations it has been proved that the dichloromethane extract of the whole plant possesses a slight larvicidal activity and antimicrobial properties but the active compounds were not identified w3x. The present paper is concerned with the isolation of a new glycosylated furanocoumarin 1 from the methanol extract of the aerial parts of this plant, in addition to bergapten ( 2 ) and 4- ww3- Ž4,5-dihydro-5,5-dimethyl-4-oxo-2furanyl.butylxoxyx-7H-furow3,2-gxw1xbenzopyran-7-one (3), both previously isolated w3x, bergaptol (4), catechin and epicatechin.
2. Experimental 2.1. Plant material D. contrajer¨ a aerial part was collected at Chimaltenango, Guatemala, in January 1996 and identified by J. Castillo. An authenticated voucher specimen was deposited at the Herbarium of the Facultad de Agronomia, Universidad de San Carlos de Guatemala.
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2.2. Extraction and isolation The powdered, dried aerial parts Ž300 g. were defatted with petrol Ž4.5 g. and CHCl 3 Ž14.1 g. in a Soxhlet apparatus and extracted successively at room temperature with MeOH Ž15.6 g.. Part of the MeOH extract Ž8 g. was partitioned between n-BuOH and H 2 O to afford a n-BuOH-soluble portion Ž3.5 g. which was chromatographed on a Sephadex LH-20 column using MeOH as eluent. Fractions Ž9 ml. were collected and checked by TLC ŽSi-gel, n-BuOHrAcOHrH 2 O 60:15:25.. Fractions 35᎐45 Ž450 mg. containing the crude coumarin mixture, were submitted to RP-HPLC on a C-18 -Bondapack column Ž30 cm = 7.8 mm, flow rate 2.5 ml miny1 . using MeOHrH 2 O ᎏ 1:1 as the eluent to yield compounds 1 Ž20 mg; t R , 11.0 min., 2 Ž57 mg; t R , 25 min., 3 Ž24 mg; t R , 30 min. and 4 Ž15 mg; t R , 22 min.. Fractions 60᎐65 Ž324 mg. were separated in the same conditions using MeOHrH 2 O 7:3 giving catechin Ž8 mg; t R , 15 min. and epicatechin Ž4 mg; t R , 19 min.. 2.3. Methods FAB MS in negative ion mode was recorded on a VG ZAB instrument ŽXE atoms of energy 2᎐6 kV.; NMR: CD3 OD, Bruker DRX-600 spectrometer operating at 599.19 MHz for 1 H and 150.858 for 13 C; DEPT, DFQ-COSY w7x, HSQC w8x and HMBC w8x experiments were performed using the UXNMR software package; the selective excitation spectra, 1D TOCSY w9x were acquired using waveform generator-based GAUSS shaped pulses, mixing times ranging from 100 to 120 ms, and MLEV-17 spin-lock field of 10 kHz preceded by a 2.5-ms trim pulse; chemical shifts are expressed in ␦ Žppm. referring to solvent peaks: ␦ H 3.34 and ␦ C 40.0 for CD3 OD. 2.4. Spectral data Ž . Compound 1. Mp 240᎐242⬚C; w ␣ x 25 D : q150 c 0.5, MeOH ; FABMS mrz: 509 wM-Hxy, 363 wŽM-H.-146xy, 201 wŽM-H.-Ž146 q 162.xy; IR bands ŽKBr.: 1730᎐1710, 1610, 1550, 1450, 890 cmy1 ; UV max ŽMeOH.: 222, 251, 258, 266, 308 nm; 1 H and 13 C-NMR Žsee Table 1..
3. Results and discussion Separation of the methanol extract of D. contrajer¨ a aerial parts by Sephadex LH-20 column and RP-HPLC yielded six major compounds, five of which were identified as bergapten (2 ) and 4-ww3-Ž4,5-dihydro-5,5-dimethyl-4-oxo-2furanyl.butylxoxyx-7H-furow3,2-gxw1xbenzopyran-7-one (3) w3x; bergaptol (4), the major furocoumarin found in D. brasiliensis w4x; catechin and epicatechin w5x, never reported previously in the genus. Compound 1 had a molecular formula C 23 H 26 O 13 as determined by a combination of 13 C, DEPT 13 C-NMR and FAB MS analyses. The FAB MS spectrum in
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Table 1 1 H and 13 C-NMR Data of compound 1 Ž600 MHz. a Position
␦C
2 3 4 5 6 7 8 4a 8a 2⬘ 3⬘ 1⬘⬘ 2⬘⬘ 3⬘⬘ 4⬘⬘ 5⬘⬘ 6⬘⬘
161.9 113.8 146.8 152.1 116.4 158.0 96.1 108.8 153.0 148.0 105.1 105.5 75.1 77.7 71.1 77.1 68.2
1⬘⬘⬘ 2⬘⬘⬘ 3⬘⬘⬘ 4⬘⬘⬘ 5⬘⬘⬘ 6⬘⬘⬘
102.1 71.7 72.2 73.7 69.2 17.5
␦H
HMBCb
6.28 d Ž9.8. 8.53 d Ž9.8.
C-2, C-4a C-2, C-8a
7.35 s
C-4a, C-6
7.84 d Ž2.5. 7.23 d Ž2.5. 5.02 d Ž7.5. 3.64 dd Ž9.5, 7.5. 3.50 t Ž9.5. 3.44 t Ž9.5. 3.55 m 3.62 dd Ž12.0, 4.5. 4.07 dd Ž12.0, 3.0. 4.70 d Ž1.5. 3.80 dd Ž2.0, 1.5. 3.69 dd Ž2.0, 8.5. 3.40 t Ž8.5. 3.82 dd Ž6.5, 8.5. 1.21 d Ž6.5.
C-7, C-6 C-6 C-5, C-2⬘⬘, C-3⬘⬘
C-6⬘⬘, C-2⬘⬘⬘rC-3⬘⬘⬘
C-5⬘⬘⬘, C-4⬘⬘⬘
a
Chemical shift values are in ppm and J values in Hz presented in parentheses. All signals were assigned by 1D TOCSY and 2D DQF-COSY, HSQC, and HMBC studies. b Selected long-range connectivities observed in the HMBC spectrum.
negative ions gave a molecular anion wM-Hxy at mrz 509 and a fragmentation pattern ascribable to the sequential loss of a deoxyhexose Ž146 mass units. and a hexose Ž162 mass units.. Its UV spectrum was characteristic of a linear type of furanocoumarin w4x and its IR absorption indicated the presence of an aromatic ring and an ␣ ,-unsaturated lactone. The 1 H-NMR spectrum showed signals characteristic of a furanocoumarin ŽTable 1. and almost superimposable on those of bergapten except for the absence of ᎐OMe group at C-5. Accepting the formula of bergaptol Ž5-hydroxypsoralen. as the basic structural moiety, it was necessary to account for a substitution of composition C 12 H 20 O 9 . The 1 H-NMR spectrum indicated the presence of two anomeric proton signals at ␦ 5.02 Ž d, J s 7.0 Hz. and 4.70 Ž d, J s 1.5 Hz., overlapped signals typical of protons on a sugar between ␦ 3.40 and 4.07, and a high field signal at ␦ 1.21 Ž3 H, d, J s 6.5 Hz. characteristic of a Me of a deoxyhexose. The 13 C-NMR spectrum exhibited 23 signals of which 11 were assigned to the 5-hydroxypsoralen moiety and 12 to the disaccharide portion by the aid of direct and long-range correlations observed in the HSQC and HMBC spectra ŽTable 1.. 5-O-Glycosylation was suggested from the upfield shift of C-5 Ž ␦
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152.6. and from C-4a Ž ␦ 108.8. and C-6 Ž ␦ 116.4. resonances shifted downfield with respect to those of bergapten. A combination of high field mono and two-dimensional NMR techniques, such as 1D TOCSY, 2D DQF-COSY, HSQC and HMBC indicated the sugar moiety to be ␣-L-rhamnopyranosyl-Ž1 ª 6.--D-glucopyranoside. Even at high field Ž600 MHz. the 1D sugar spectral region of 1 was complex as most of the shifts were overlapped and found between ␦ 3.40 and 4.07. The isolated anomeric proton signals resonating in the uncrowded region of the spectrum Ž ␦ 5.02 and 4.70. and the low frequency protons at ␦ 1.21 have been the starting point for the 1D TOCSY experiment. Because of the selectivity of the multistep coherence transfer, the 1D TOCSY subspectra of the single monosaccharide could be extracted from the crowded overlapping region and attributed to one set of coupled protons. 1D TOCSY subspectrum obtained irradiating at ␦ 5.02 showed a set of coupled protons at 4.07, 3.64, 3.62, 3.55, 3.50 and 3.44 which were recognized as belonging to the hexose unit. The second deoxyhexose unit was deduced in the same manner by irradiating at ␦ 1.21 and obtaining a subspectrum showing a set of coupled protons at ␦ 3.40, 3.69, 3.80, 3.82 and 4.70 ŽTable 1.. The 2D DQF-COSY spectrum established the proton sequence from H-1 to H-6 within each sugar fragment. The HSQC spectrum correlated each proton resonance to the corresponding carbon resonance led to the identification of the sugars as glucopyranose and rhamnopyranose w6x. The absence of any glycosylation shift for the carbon resonances of rhamnopyranosyl suggested this sugar to be terminal while glycosylation shift of approximately 6 ppm was observed for C-6⬘⬘ of the inner glucopyranosyl. Finally, the unambiguous confirmation of the interglycosidic linkage and of the glycosylation at C-5 of the coumarin moiety was obtained from the HMBC spectrum which showed correlation peaks between the anomeric proton signal of glucopyranose Ž ␦ 5.02. and C-5 Ž ␦ 152.1. resonance of bergaptol moiety and between the anomeric proton signal of rhamnopyranose Ž ␦ 4.70. and C-6⬘⬘ of glucose Ž ␦ 68.20. indicating the glucosyl moiety was linked to the furanocoumarin aglycon. The chemical shift, multiplicity of signals, the value of the proton coupling constants in the 1 H-NMR spectrum, as well as the C-2, C-3 and C-5 resonances in the 13 C-NMR spectrum, indicated the -configuration of glucopyranosyl and the ␣-configuration of rhamnopyranosyl unit w6x. Therefore, 1 was ␣-L-rhamnopyranosyl-Ž1 ª 6.--Dglucopyranosyl-bergaptol.
References w1x Gupta M.P. Phytochemistry of plant used in traditional medicine. Oxford: ED. Hostettman K., Marston A., Maillard M., Hamburger K. University Press Oxford, 1995: 359᎐398. w2x Orellana SL. Indian medicine in highland Guatemala Albuquerque. University of New Mexico Press, 1987:200᎐220. w3x Terreaux C, Maillard M, Stoeckli-Evans H, Gupta MP, Downum KR, Quirke JME, Hostettmann K. Phytochemistry 1995;39:645. w4x Kuster RM, Bernardo RR, Da Silva AJR, Parente JP, Mors WB. Phytochemistry 1994;36:221.
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De Tommasi N, Rastrelli L, De Simone F, Cumanda J, Pizza C. Phytochemistry 1996;42:163. Vilegas W, Sanommiya M, Rastrelli L, Pizza C. J Agric Food Chem 1999;47:403. Bodenhausen E, Ruben D. J Magn Reson 1986;69:185. Martin GE, Crouch RC. J Nat Prod 1991;54:1. Davis DG, Bax A. J Am Chem Soc 1985;107:7198.
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