Pharmaceutical Biology 1998, Vol. 36, No. 2, pp.


1388-0209/98/3602-0103$12.00 © Swets & Zeitlinger

J.-R. Ioset1, Andrew Marston1, Mahabir P. Gupta2 and Kurt Hostettmann1*
1Institut de Pharmacognosie et Phytochimie, Université de Lausanne, BEP, CH-1015 Lausanne, Switzerland 2Center for Pharmacognostic Research on Panamanian Flora (CIFLORPAN), Apartado 10767, College of

Pharmacy, University of Panama, Panama, Republic of Panama

Three antifungal xanthones have been isolated from the dichloromethane root extract of Marila laxiflora (Guttiferae). Their structures were established by spectrometric (UV, EI mass spectrometry, 1H and 13C nmr) and chemical methods. In addition, other compounds were isolated from the methanol extract: a fourth xanthone, rhamnetin, betulinic acid, and a derivative of benzoic acid.

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

MATERIALS AND METHODS Plant Material The plant was collected in August 1994 in Llano Carti, San Blas Islands, Panama. A voucher specimen is deposited at the National Herbarium of Panama (Florpan 1685) and at the Institut de Pharmacognosie et Phytochimie, Lausanne, Switzerland (No. 96013). Instrumentation and Chromatographic Materials TSP-MS and EI-MS: Finnigan MAT TSQ-700 triple stage quadropole instrument. Purity of the compounds was checked by HPLC with a Nova-Pak RP-18 column (4 µm; 150 ϫ 3.9 mm i.d.; Waters). 1H and 13C nmr spectra were measured in DMSO-d6 at 200.06 and 50.30 MHz, respectively. TMS: int. standard. UV spectra were recorded in MeOH. TLC: silica gel 60 F254 Al sheets (Merck) and diol HPTLC plates (Merck). CPC: CCC-1000 high speed countercurrent chromatograph (Pharma-Tech Research Corp.). CC: diol (40–63 µm; 440 ϫ 13 mm i.d.; Merck), Sephadex LH-20 (400 ϫ 13 mm i.d.; Pharmacia), MPLC: homepacked Lichroprep RP-18 column (15–25 µm; 460 ϫ 16 mm i.d.; Merck) and silica gel (40–63 µm; 450 ϫ 60 mm i.d.; Merck). LPLC: Lobar Lichroprep RP-18 (40–63 µm; 270 ϫ 25 mm i.d.; Merck). UV: Varian DMS 100S UVVIS spectrophotometer. Isolation Procedures Dried and ground roots (135 g) were successively extracted at room temperature with CH2Cl2 and MeOH to afford 2.46 and 8.53 g of extract, respectively. A portion of the CH2Cl2 extract (1.56 g) was separated

INTRODUCTION The Guttiferae are rich in polyphenols, including xanthones and flavonoids. Xanthones have been found to possess many biological properties, such as inhibition of monoamine oxidase enzyme (MAO) and antifungal activities (Rocha et al., 1994). As a part of our investigation into the bioactive compounds of Panamanian plants (Rahalison et al., 1993), Marila laxiflora (Guttiferae) was chosen for study because HPLC/UV analysis of a dichloromethane extract of the roots showed the presence of xanthones. This extract also had activity against the plant pathogenic fungus Cladosporium cucumerinum in a TLC autobiographic assay (Homans & Fuchs, 1970). M. laxiflora Rusby belongs to the Kielmeyeroideae, a sub-family of the Guttiferae (Hegnauer, 1966). It is a shrub distributed in lower altitude areas of Panama, Bolivia and Guatemala (D’Arcy, 1987). Although many different constituents have been isolated from the Guttiferae

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. cucumerinum in the TLC bioassay and yielded compounds 2 (4.3 mg) and 3 (2.1 mg) after separation by diol opencolumn chromatography using petrol ether-CHCl3 (1:1). Compound 1 (12.5 mg) was isolated from fr. 3 after gel filtration on Sephadex LH-20 with CHCl3MeOH (1:1). Gel filtration of fr. 1 on Sephadex LH-20 (CHCl3-MeOH 1:1), followed by diol open-column chromatography (petrol ether-EtOAc 4:1) and gel filtration on Sephadex LH-20 (CHCl3-MeOH 1:1) gave compound 4 (10.4 mg). The methanol extract was eluted on a silica gel MPLC column with a CHCl3MeOH (9:1 →6:1) step-gradient, followed by CHCl3MeOH-H2O (5:2:0.25→6.5:3.5:0.5) and finally MeOH to give 7 frs. (A–G). Fr. C was separated into 4 frs (1–4) by MPLC on RP-18 using a step-gradient of MeOH-H2O (5:95 →100:0). Fr. 4 was then submitted to purification on Sephadex LH-20 with MeOH to yield 3 mg of 5. Fr. D was also eluted on a MPLC RP18 column with a step-gradient of MeOH-H2O (20:80→30:70→40:60) to give 7 frs. (1–7). Fr. 4 was then separated on LPLC RP-18 (MeOH-H2O 35:65→50:50→100:0) and purified by Sephadex LH20 with MeOH to yield compound 6 (2.0 mg). Gel filtration of fr. E on Sephadex LH-20 (MeOH) gave 8 frs. (1–8). Fr. 4 was then dissolved in MeOH and purified by gel filtration (Sephadex LH-20; MeOH) to yield 15.5 mg of compound 7. 5-Hydroxy-1-methoxyxanthone (1) Amorphous orange powder. UV ␭max nm: 205, 244, 304, 349; ϩ NaOMe: 206, 240, 260, 314, 376; ϩ AlCl3: 206, 245, 304, 353; ϩ AlCl3 ϩ HCl: 205, 245, 304, 353; ϩ NaOAc: 210, 242, 308, 355; ϩ NaOAc ϩ H3BO3: 208, 245, 304, 353. EI-MS m/z (rel. int.): 243 (22), 242 [M]ϩ (100), 241(24), 214 (12), 213 (68), 212 (14), 196 (28), 184 (10). Methylation of 1 Compound 1 (3 mg) was methylated with diazomethane to give 1,5-dimethoxyxanthone (2 mg) (1a) as an amorphous orange powder. UV ␭max nm: 208, 244, 301, 349; ϩ NaOMe: 206, 244, 301, 349; ϩ AlCl3: 208, 244, 301, 349; ϩ AlCl3 ϩ HCl: 208, 244, 301, 350; ϩ NaOAc: 211, 244, 301, 349; ϩ NaOAc ϩ H3BO3: 217, 244, 301, 350. EI-MS m/z (rel. int.): 257 (69), 256 [M]ϩ (100), 227 (20), 210 (14).

1,5-Dihydroxyxanthone (2) Yellow powder. UV ␭max nm: 205, 248, 312, 369; ϩ NaOMe: 206, 243, 261, 318, 399; ϩ AlCl3: 205, 235, 270, 300, 311, 350, 435; ϩ AlCl3 ϩ HCl: 205, 235, 270, 297, 309, 343, 432; ϩ NaOAc: 209, 248, 315, 370; ϩ NaOAc ϩ H3BO3: 209, 248, 312, 370. EI-MS m/z (rel. int.): 229 (14), 228 [M]ϩ (100). 1,6-Dihydroxy-5-methoxyxanthone (3) Yellow powder. UV ␭max nm: 204, 238, 313, 359; ϩ NaOMe: 205, 233, 263, 364; ϩ AlCl3: 205, 215, 234, 256, 345, 413; ϩ AlCl3 ϩ HCl: 214, 235, 255, 339, 413; ϩ NaOAc: 208, 262, 363; ϩ NaOAc ϩ H3BO3: 209, 311, 356. EI-MS m/z (rel. int.): 259 (13), 258 [Mϩ] (39), 242 (21), 216 (14), 215 (100), 187 (16).

RESULTS AND DISCUSSION The air-dried roots of M. laxiflora were extracted successively with dichloromethane and methanol. Bioautography of the dichloromethane extract showed antifungal activity against C. cucumerinum. Using HPLC coupled with UV and thermospray (TSP) mass spectrometry (Wolfender et al., 1993), evidence was obtained for the presence of xanthones in this extract (Fig. 1). The extract was first fractionated by centrifugal partition chromatography (CPC), following the activity by TLC bioassay. Subsequent gel filtration on LH-20 yielded xanthone 1. Xanthones 2 and 3 were obtained by column chromatography on diol. Compound 4 was isolated after gel filtration and diol column chromatography. The methanol extract was also fractionated to provide compounds 5–7. HPLC/UV of the crude dichloromethane extract exhibited characteristic bands of xanthones for compounds 1–3 (Fig. 1). The peaks eluting at 27 and 30 min corresponded to two inactive phloroglucinols. Structure elucidation of these compounds is underway. LC-TSP mass spectroscopic analysis gave molecular ions of m/z 243 for 1, 229 for 2 and 259 for 3 which, in conjunction with nmr experiments, suggested molecular formulae of C14H10O4, C13H8O4 and C14H10O5, respectively. Structures of these xanthones were elucidated using 1H, 13C nmr technics and UV spectroscopy: 1 was attributed to 5-Hydroxy-1-methoxyxanthone, 2 to 1,5-dihydroxyxanthone and 3 to 1,6-dihydroxy-5-methoxyxanthone (buchanaxanthone). Structures 1 and 2 were confirmed by comparison with authentic samples. 5-Hydroxy-1-



methoxyxanthone was already discovered in Mammea africana (Guttiferae) (Carpenter et al., 1969) and Hypericum brasiliense (Guttiferae) (Rocha et al., 1994). 1,5-dihydroxyxanthone was previously isolated from Allanblackia floribunda (Guttiferae) (Locksley & Murray, 1971). Nmr and UV data obtained for 3 were in perfect agreement with those published for 1,6-dihy-

droxy-5-methoxyxanthone, first isolated from Garcinia buchananii Baker (Jackson et al., 1968). Besides these 3 xanthones, the dichloromethane extract yielded also betulinic acid (4), identified by nmr spectral comparison with literature data (Siddiqui et al., 1988). The structure was confirmed by LC/UV analysis with an authentic sample. Three further

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 ϫ 3.9 mm i.d.; Waters); gradient, MeOH-H2O (0.05% TFA) 30:70 → 100:0 in 30 min and 100:0 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)-1,3,5,6tetrahydroxyxanthone (5) (Jackson et al., 1966), rhamnetin (6) and 3,4-dihydroxy-benzoic acid (7) were isolated from the methanolic extract. The isolated xanthones 1–3 were shown to be responsible for the antifungal activity against C. cucumerinum in the TLC bioassay, giving inhibition of spore growth at minimum quantities of 5 µg for each xanthone on the plate. Amphotericin B, a macrocyclic antifungal agent, was active at 0.1 µg on the same plate. In dilution tests with the same microorganism (Rahalison, 1994), minimum inhibition concentration (MIC) values of 25 µg/ml and 50 µg/ml were found for 2 and 3, respectively. Compound 1 was inactive in this test up to a 100 µg/ml concentration. Amphotericin B was active at 1.25 µg/ml in the same bioassay. Compounds 4–7 were also tested but showed no activity. This is the first time that the antifungal activity of 3 has been reported and that dilution tests have been performed with xanthones 1–3, giving a better evaluation of the antifungal activity than with the autobiographic method (Rahalison, 1994).

The authors would like to thank the Swiss National Science Foundation for financial support of this work and the “Fondation Herbette” of the University of Lausanne for a travel grant to Panama.

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Accepted: September 15, 1997