ABSTRACT

Three antifungal xanthones have been isolated fromthe dichloromethane root extract of

Marila laxiflora(Guttiferae). Their structures were established byspectrometric (UV, EI mass spectrometry, 1H and 13Cnmr) and chemical methods. In addition, other com-pounds were isolated from the methanol extract: afourth xanthone, rhamnetin, betulinic acid, and aderivative of benzoic acid.

INTRODUCTION

The Guttiferae are rich in polyphenols, including xan-thones and flavonoids. Xanthones have been found topossess many biological properties, such as inhibitionof monoamine oxidase enzyme (MAO) and antifungalactivities (Rocha et al., 1994). As a part of our inves-tigation into the bioactive compounds of Panamanianplants (Rahalison et al., 1993), Marila laxiflora (Gut-tiferae) was chosen for study because HPLC/UVanalysis of a dichloromethane extract of the rootsshowed the presence of xanthones. This extract alsohad activity against the plant pathogenic fungus Cla-dosporium cucumerinum in a TLC autobiographicassay (Homans & Fuchs, 1970). M. laxiflora Rusbybelongs to the Kielmeyeroideae, a sub-family of theGuttiferae (Hegnauer, 1966). It is a shrub distributedin lower altitude areas of Panama, Bolivia andGuatemala (D’Arcy, 1987). Although many differentconstituents have been isolated from the Guttiferae

(Bennett & Lee, 1989), the genus Marila has not yetbeen studied phytochemically.

MATERIALS AND METHODS

Plant MaterialThe plant was collected in August 1994 in Llano Carti,San Blas Islands, Panama. A voucher specimen isdeposited at the National Herbarium of Panama (Flor-pan 1685) and at the Institut de Pharmacognosie etPhytochimie, Lausanne, Switzerland (No. 96013).

Instrumentation and Chromatographic MaterialsTSP-MS and EI-MS: Finnigan MAT TSQ-700 triplestage quadropole instrument. Purity of the compoundswas checked by HPLC with a Nova-Pak RP-18 column(4 µm; 150

3 3.9 mm i.d.; Waters). 1H and 13C nmrspectra were measured in DMSO-d6 at 200.06 and50.30 MHz, respectively. TMS: int. standard. UV spec-tra were recorded in MeOH. TLC: silica gel 60 F254 Alsheets (Merck) and diol HPTLC plates (Merck). CPC:CCC-1000 high speed countercurrent chromatograph(Pharma-Tech Research Corp.). CC: diol (40–63 µm;440 3 13 mm i.d.; Merck), Sephadex LH-20 (400 313 mm i.d.; Pharmacia), MPLC: homepacked Lichro-prep RP-18 column (15–25 µm; 460 3 16 mm i.d.;Merck) and silica gel (40–63 µm; 450 3 60 mm i.d.;Merck). LPLC: Lobar Lichroprep RP-18 (40–63 µm;270 3 25 mm i.d.; Merck). UV: Varian DMS 100S UV-VIS spectrophotometer.

Isolation ProceduresDried and ground roots (135 g) were successivelyextracted at room temperature with CH2Cl2 and MeOHto afford 2.46 and 8.53 g of extract, respectively. Aportion of the CH2Cl2 extract (1.56 g) was separated

Pharmaceutical Biology 1388-0209/98/3602-0103$12.001998, Vol. 36, No. 2, pp. 103–106 © Swets & Zeitlinger

ANTIFUNGAL XANTHONES FROM ROOTS OF

MARILA LAXIFLORA

J.-R. Ioset1, Andrew Marston1, Mahabir P. Gupta2 and Kurt Hostettmann1*

1Institut de Pharmacognosie et Phytochimie, Université de Lausanne, BEP, CH-1015 Lausanne, Switzerland2Center for Pharmacognostic Research on Panamanian Flora (CIFLORPAN), Apartado 10767, College of

Pharmacy, University of Panama, Panama, Republic of Panama

Keywords: Antifungal activity, Guttiferae, Marila laxiflora,roots, xanthones.

* Author to whom correspondence should be addressed.

by CPC using petrol ether-EtOAc-MeOH-H2O(1:1:1:1, upper phase as mobile phase) giving frs 1–3.Fr. 2 was shown to be fungicidal against C. cucumer-inum in the TLC bioassay and yielded compounds 2(4.3 mg) and 3 (2.1 mg) after separation by diol open-column chromatography using petrol ether-CHCl3

(1:1). Compound 1 (12.5 mg) was isolated from fr. 3after gel filtration on Sephadex LH-20 with CHCl3-MeOH (1:1). Gel filtration of fr. 1 on Sephadex LH-20(CHCl3-MeOH 1:1), followed by diol open-columnchromatography (petrol ether-EtOAc 4:1) and gel fil-tration on Sephadex LH-20 (CHCl3-MeOH 1:1) gavecompound 4 (10.4 mg). The methanol extract waseluted on a silica gel MPLC column with a CHCl3-MeOH (9:1

→6:1) step-gradient, followed by CHCl3-MeOH-H2O (5:2:0.25→6.5:3.5:0.5) and finally MeOHto give 7 frs. (A–G). Fr. C was separated into 4 frs(1–4) by MPLC on RP-18 using a step-gradient ofMeOH-H2O (5:95→100:0). Fr. 4 was then submittedto purification on Sephadex LH-20 with MeOH toyield 3 mg of 5. Fr. D was also eluted on a MPLC RP-18 column with a step-gradient of MeOH-H2O(20:80→30:70→40:60) to give 7 frs. (1–7). Fr. 4 wasthen separated on LPLC RP-18 (MeOH-H2O35:65→50:50→100:0) and purified by Sephadex LH-20 with MeOH to yield compound 6 (2.0 mg). Gel fil-tration of fr. E on Sephadex LH-20 (MeOH) gave 8 frs.(1–8). Fr. 4 was then dissolved in MeOH and purifiedby gel filtration (Sephadex LH-20; MeOH) to yield15.5 mg of compound 7.

5-Hydroxy-1-methoxyxanthone (1)Amorphous orange powder. UV lmax nm: 205, 244,304, 349; 1 NaOMe: 206, 240, 260, 314, 376; 1

AlCl3: 206, 245, 304, 353; 1 AlCl3 1 HCl: 205, 245,304, 353; 1 NaOAc: 210, 242, 308, 355; 1 NaOAc 1H3BO3: 208, 245, 304, 353. EI-MS m/z (rel. int.): 243(22), 242 [M]1 (100), 241(24), 214 (12), 213 (68), 212(14), 196 (28), 184 (10).

Methylation of 1Compound 1 (3 mg) was methylated with dia-zomethane to give 1,5-dimethoxyxanthone (2 mg) (1a)as an amorphous orange powder. UV lmax nm: 208,244, 301, 349; 1 NaOMe: 206, 244, 301, 349; 1

AlCl3: 208, 244, 301, 349; 1 AlCl3 1 HCl: 208, 244,301, 350; 1 NaOAc: 211, 244, 301, 349; 1 NaOAc 1H3BO3: 217, 244, 301, 350. EI-MS m/z (rel. int.): 257(69), 256 [M]1 (100), 227 (20), 210 (14).

1,5-Dihydroxyxanthone (2)Yellow powder. UV lmax nm: 205, 248, 312, 369; 1NaOMe: 206, 243, 261, 318, 399; 1 AlCl3: 205, 235,270, 300, 311, 350, 435; 1 AlCl3 1 HCl: 205, 235,270, 297, 309, 343, 432; 1 NaOAc: 209, 248, 315,370; 1 NaOAc 1 H3BO3: 209, 248, 312, 370. EI-MSm/z (rel. int.): 229 (14), 228 [M]1 (100).

1,6-Dihydroxy-5-methoxyxanthone (3)Yellow powder. UV lmax nm: 204, 238, 313, 359; 1NaOMe: 205, 233, 263, 364; 1 AlCl3: 205, 215, 234,256, 345, 413; 1 AlCl3 1 HCl: 214, 235, 255, 339,413; 1 NaOAc: 208, 262, 363; 1 NaOAc 1 H3BO3:209, 311, 356. EI-MS m/z (rel. int.): 259 (13), 258[M1] (39), 242 (21), 216 (14), 215 (100), 187 (16).

RESULTS AND DISCUSSION

The air-dried roots of M. laxiflora were extracted suc-cessively with dichloromethane and methanol. Bioau-tography of the dichloromethane extract showedantifungal activity against C. cucumerinum. UsingHPLC coupled with UV and thermospray (TSP) massspectrometry (Wolfender et al., 1993), evidence wasobtained for the presence of xanthones in this extract(Fig. 1). The extract was first fractionated by centrifu-gal partition chromatography (CPC), following theactivity by TLC bioassay. Subsequent gel filtration onLH-20 yielded xanthone 1. Xanthones 2 and 3 wereobtained by column chromatography on diol. Com-pound 4 was isolated after gel filtration and diol col-umn chromatography. The methanol extract was alsofractionated to provide compounds 5–7.

HPLC/UV of the crude dichloromethane extractexhibited characteristic bands of xanthones for com-pounds 1–3 (Fig. 1). The peaks eluting at 27 and 30 mincorresponded to two inactive phloroglucinols. Structureelucidation of these compounds is underway. LC-TSPmass spectroscopic analysis gave molecular ions of m/z243 for 1, 229 for 2 and 259 for 3 which, in conjunctionwith nmr experiments, suggested molecular formulae ofC14H10O4, C13H8O4 and C14H10O5, respectively. Struc-tures of these xanthones were elucidated using 1H, 13Cnmr technics and UV spectroscopy: 1 was attributed to5-Hydroxy-1-methoxyxanthone, 2 to 1,5-dihydroxyx-anthone and 3 to 1,6-dihydroxy-5-methoxyxanthone(buchanaxanthone). Structures 1 and 2 were confirmedby comparison with authentic samples. 5-Hydroxy-1-

104 J.-R. IOSET ET AL.

methoxyxanthone was already discovered in Mammeaafricana (Guttiferae) (Carpenter et al., 1969) andHypericum brasiliense (Guttiferae) (Rocha et al., 1994).1,5-dihydroxyxanthone was previously isolated fromAllanblackia floribunda (Guttiferae) (Locksley & Mur-ray, 1971). Nmr and UV data obtained for 3 were inperfect agreement with those published for 1,6-dihy-

droxy-5-methoxyxanthone, first isolated from Garciniabuchananii Baker (Jackson et al., 1968).

Besides these 3 xanthones, the dichloromethaneextract yielded also betulinic acid (4), identified bynmr spectral comparison with literature data (Siddiquiet al., 1988). The structure was confirmed by LC/UVanalysis with an authentic sample. Three further

MARILA LAXIFLORA ANTIFUNGAL XANTHONES 105

Fig. 1. LC-TSP-MS of the dichloromethane extract of the roots of Marila laxiflora (Guttiferae) with UV and mass spectra of xanthones 1–3.HPLC: column Nova-Pak RP-18 (4 µm; 150 3 3.9 mm i.d.; Waters); gradient, MeOH-H2O (0.05% TFA) 30:70 → 100:0 in 30 min and100:0 for 5 min (1 ml/min). TSP: vaporizer, 100°C; source, 280°C; ammonium acetate buffer (0.5 M, 0.2 ml/min); positive ion mode.

known compounds, 2-(3,3-dimethylallyl)-1,3,5,6-tetrahydroxyxanthone (5) (Jackson et al., 1966), rham-netin (6) and 3,4-dihydroxy-benzoic acid (7) wereisolated from the methanolic extract.

The isolated xanthones 1–3 were shown to beresponsible for the antifungal activity against C. cuc-umerinum in the TLC bioassay, giving inhibition ofspore growth at minimum quantities of 5 µg for eachxanthone on the plate. Amphotericin B, a macrocyclicantifungal agent, was active at 0.1 µg on the same plate.In dilution tests with the same microorganism (Rahali-son, 1994), minimum inhibition concentration (MIC)values of 25 µg/ml and 50 µg/ml were found for 2 and3, respectively. Compound 1 was inactive in this test upto a 100 µg/ml concentration. Amphotericin B wasactive at 1.25 µg/ml in the same bioassay. Compounds4–7 were also tested but showed no activity.

This is the first time that the antifungal activity of 3has been reported and that dilution tests have been per-formed with xanthones 1–3, giving a better evaluationof the antifungal activity than with the autobiographicmethod (Rahalison, 1994).

ACKNOWLEDGEMENTS

The authors would like to thank the Swiss National ScienceFoundation for financial support of this work and the “Fon-dation Herbette” of the University of Lausanne for a travelgrant to Panama.

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Accepted: September 15, 1997

106 J.-R. IOSET ET AL.