Journal of Chemical Ecology, Vol. 23, No. 7, 1997

ANTIFUNGAL XANTHONES FROM Calophyllumbrasiliensis HEARTWOOD1

RICARDO REYES-CHILPA,*-' MANUEL JIMENEZ-ESTRADA,and ELIZABETH ESTRADA-MUNIZ

Instituto de Quimica, Universidad National Autonoma de MexicoCiudad Universilaria

Coyoacan. 04510 Mexico, DF

(Received October 14, 1996; accepted March 17, 1997)

Abstract—The heartwood of the tropical tree Calophyllum brasiliensis isknown to be highly resistant to fungi and termites. To determine whetherresistance to wood-rotting fungi could be caused by bioactive secondarymetabolites, a chemical and biological study was carried out. Hexane, ace-tone, methanol, and water extracts were prepared. The yield of the extractsranged from 0.04% (hexane) to 4.81% (acetone). Methanol, acetone, andwater extracts (5 mg/ml = 0.5%) inhibited the mycelial growth of the brownrot fungus Postia placenta by 83%, 59%, and 2 1 % , respectively. Chroma-tographic separation of the acetone and methanol extracts afforded fiveprenylated xanthones: 6-desoxyjacareubin (I) , 1,5-dihydroxy-2-(3,3-dimethyl-allyl)-3-methoxy-xanthone (II), jacareubin ( I I I ) and l,3,5-trihydroxy-2-(3,3-dimethylallylj-xanthone (IV) and l,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl)-xanthone (V). Xanthones III, IV, and especially V, were the most abundantconstituents of both extracts and inhibited at 0.25 mg/ml the mycelial growthof P. placenta. Inhibitory activity ranged from 55.5% (V) to 68.8% (III andIV mixture). Acetylation of xanthones did not induce a sharp change in theextent of fungistasis compared with parent compounds. The above resultssuggest that C. brasiliensis xanthones actually play a defensive role againstwood decay fungi

Key Words—Xanthones, Calophyllum brasiliensis, heartwood, antifungalactivity, Postia placenta, Guttiferae, wood, fungi, extractives.

*To whom correspondence should be addressed.'Contribution 1264 from Instituto de Quimica, UNAM. Presented in part at the 22nd InternationalSymposium on Natural Products Chemistry, Mexico City, May 6-7, 1996.

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0098-033l/97/0700-l90l$l2.50/0© 1997 Plenum Publishing Corporation

1902 REYES-CHILPA, JIMENEZ-ESTRADA, AND ESTRADA-MUNIZ

INTRODUCTION

The genus Calophyllum (Guttiferae) is composed of about 130 species confinedto the warm humid tropics of the world. In the Western Hemisphere, one of themost widely distributed species is Calophyllum brasiliensis Camb. This largetree is found in tropical forests from southern Mexico to Brazil. It can reach 40m in height and 1.3 m diameter at breast height. The timber is used for con-struction, flooring, and furniture (Chudnoff, 1984, Ortega-Escalona et al., 1991).Experimental tests indicate that C. brasiliensis heartwood is highly resistant tothe brown rot fungus Lenzites trabea (Torelli, 1982) and to the subterraneantermites Reticulitermes flavipes and Coptotermes formosanus (Carter andCamargo, 1983); it has also been rated as resistant to the white rot fungusCoriolus versicolor (Torelli, 1982).

Natural resistance to fungi and termites is primarily attributed to the contentof secondary metabolites present in heartwood, since these compounds fre-quently exhibit antifungal (Gdmez-Garibay et al., 1990; Scheffer and Cowling,1966) and antitermitic (McDaniel 1992; Scheffrahn, 1991; Reyes-Chilpa et al.,1995) properties. In addition, extraction of secondary metabolites with organicsolvents and water renders heartwood susceptible to wood-destroying organisms(Deon 1983; Reyes-Chilpa et al., 1987). In this paper we report the isolationof five xanthones (I-V) from Calophyllum brasiliensis heartwood. We alsoreport the antifungal activity of heartwood extracts and compounds III, IV, andV against the brown rot fungus Postia placenta.

METHODS AND MATERIALS

Biological Material. Calophyllum brasiliensis heartwood was obtained froma tree collected in the Lacandona Rain Forest, State of Chiapas, Mexico(Barcenas-Pazos, 1995). Vouchers and wood samples are deposited at the Insti-tute of Ecology A.C. Herbarium (XAL) in Xalapa, Mexico. Postia placentafungus, strain Mad 698, was obtained from the Forest Products Laboratory,Madison, Wisconsin.

Isolation of Compounds. Heartwood shavings (711.5 g) were extracted atroom temperature with hexane, acetone, methanol, and water. Extracts wereconcentrated under reduced pressure. After preparative thin-layer chromatog-raphy (pTLC), the hexane extract yielded /3-sitosterol. Part of the acetone extract(28 g) was subjected to column chromatography (silica gel) eluting with hexane,acetone, and mixtures of these solvents. Fractions 9-16 eluted with hexanc-acetone (9:1) yielded a yellow powder (5 mg) that was identified as 6-dcsoxy-jacareubin (I) (Figure 1). Fractions 17-20, eluted with hexane-acetone (9: 1),yielded a yellow powder (12 mg), identified as l,5-dihydroxy-2-(3,3-dimeth-

ANT1FUNGAL XANTHONESs 1903

FIG. 1. Natural and transformed xanthones from Calophyllum brasiliensis heartwood.

ylallyl)-3-methoxyxanthone (II). Fractions 21-24, eluted with hexane-acetone(8:2), yielded a yellow powder (790 mg) that was further identified as a mixtureof jacareubin (HI) and 2-(3,3-dimethyllallyl)-l,3,5-trihydroxyxanthone (IV).Preparative TLC of this mixture yielded a pure sample of IV. Fractions 30-43,eluted with hexane-acetone (7.5:2.5), yielded 2-(3,3-dimethylallyl)-l,3,5,6-tetrahydroxyxanthone (V) as a yellow powder (3.3 g).

Part of the methanol extract (2.5 g) was subjected to column chromatog-raphy (silica gel) and eluted with hexane, ethyl acetate, and mixtures of thesesolvents. Fractions 12-18, eluted with a 9:1 mixture, yielded a mixture ofcompounds III and IV (86 mg). Fractions 26-39, eluted with 8:2 mixture,

1904 REYES-CHILPA, JIMENEZ-ESTRADA, AND ESTRADA-MUNIZ

afforded V (516 mg). Structures of compounds were elucidated from their IR,UV, 'H NMR and MS spectroscopic data.

Transformation of Compounds. Compound V (500 mg) was acetylated withanhydrous acetic in pyridine at room temperature for 24 hr. The reaction wasstopped with the addition of water giving a solid that was filtered and washedwith 10% HC1 and water. The solid (391 mg, yield 56%) was crystallized fromCH2Cl2/MeOH and identified as 2-(3,3-dimethylallyl)-l-hydroxy-3,5,6-triace-tylxanthone (Va). A mixture of III and IV (150 mg) was also acetylated aspreviously described, and the reaction products were separated by CC. Firstfractions yielded l,3,5-triacetyl-2-(3,3-dimethylallyl)-xanthone (IVa) (8 mg),while the latter fractions yielded 6 mg of a mixture of triacetyljacareubin (IIIa)and l',2'-dihydro-5,6-diacetyljacareubin (IIIb).

Bioassays. The effects of extracts (5 mg/ml = 0.5% w/v) and isolatedcompounds (0.25 mg/m = 0.025%) on the mycelial growth of the fungus Postiaplacenta were examined as described by Reyes-Chilpa et al. (1997). The extractswere dissolved in acetone, methanol, or water and incorporated into the growthmedium (malt-agar 1.5%). The isolated compounds were dissolved in acetone.Controls containing each solvent were run simultaneously. Phenol (Sigma) wasalso tested in the same way for comparison. Finally, to test whether fungalmetabolism could modify xanthones in vitro, we redissolved agar from plateswith compound V and extracted the solution several times with ethyl acetate.The organic phase was then subjected to pTLC.

RESULTS

Effects of Extracts

The highest yield of soluble metabolites was obtained with acetone (4.81 %),while the poorest was achieved with hexane (0.04%) (Table 1). At the concen-tration tested (5 mg/ml = 0.5% w/v), three extracts showed fungistatic activityagainst Postia placenta (Table 1). The methanol extract was the most active,inhibiting the mycelial growth by 83.6%. Acetone and water extracts were lessactive, inhibiting mycelial growth by 59% and 21.9%, respectively. Differencesbetween extracts were statistically significant. The hexane extract was not testedbecause of its low yield.

Compounds Isolated

Chromatographic separation of the acetone and methanol extracts yieldedfive prenylated xanthones: 6-desoxyjacareubin (I), l,6-dihydroxy-2(3,3-di-methylallyl)-3-methoxyxanthone (II), jacareubin (III), l,3,5-trihydroxy-2-(3,3-dimethylallyl)-xanthone (IV), and l,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl)-

ANTIFUNGAL XANTHONES 1905

TABLE 1. INHIBITION OF Postia placenta MYCELIAL GROWTH BY C. brasiliensisHEARTWOOD EXTRACTS (5 mg/ml)

Extract

ControlHexaneAcetoneMethanolWater

Yield (%)

0.044.810.920.13

Growth (cm)"

4.88 ± 0.16"nt

2.00 ± 0.00*0.80 ± 0.14'3.81 ± 0.06''

Inhibition (%)

0.0 + 3.2nt

59.0 + 0.083.6 ± 2.821.9 ± 1.2

"Mean ± standard deviation of three replicates five days after innoculation. Least significant dif-ference > 0.26; values followed by a different letter are significantly different at P = 0.05 (Tukey's( test), nt: not tested.

xanthone (V). Their 'H NMR data are shown in Table 2. Besides these com-pounds, spectroscopic evidence also suggested the presence of l',2'-dihydro-5,6-diacetyljacareubin (Illb) along with the diacetyl derivative of jacareubin(IIIa). The mass spectrum of IIIa showed surplus peaks at 412 m/z (19%), 370(15%), and 328 (31%), which accounted for the molecular ion [C22H20O8] + ,and the loss of one and two C2H2O fragments, respectively. 'H NMR of IIIaalso suggested residual IIIb, considering two small triplets at 2.7 and 1.85 ppmassigned to methylene protons at the 1' and 2' positions. The origin of compoundIIIa as a natural product or as an artifact was not determined.

Xanthones III, IV, and especially V were the most common constituentsof both the acetone and methanol extracts. The yields of compound V were11.7% and 20.6%, respectively; while the mixture of compounds HI and IVaccounted for 2.8% and 3.4%, respectively.

6-Desoxyjacareubin (I). Yellow powder, mp 214-215°C (reported 212-214°C; Jackson et al., 1967, 1969). EMIE 70 eV (m/z): 310 M+ (21.3%)[C18H1405], 295 (100%) [M+-CH3], 257 (4.1%) [M + -C4H7], 147 (11.5%).

l,5 - Dihydroxy - 2 - (3,3-dimethylallyl) - 3- methoxyxanthone (II).Yellow powder, mp 254-255°C (reported 242-244°C; Sen et al., 1981). EMIE 70 eV(m/z): 326 M+ (53.3%) [CI9HI8O5], 311 (41.6%) [M + -CH3J, 283 (73.3%)[M+-C3H7], 271 (100%) [M + -C4H7], 258 (10%), 241 (11.6%).

/,3,5-Triacetyljacaraubein (IIIa). Yellow powder, mp 169-170°C. UVXmax [MeOH], nm (e): 239 (3496), 292 (3584), 327 (1493). EMIE 70 eV(m/z): 410 M+ (50%) [C22H18O8], 395 (93%) [M + -CH3] = A, 353 (97%)[A-C2H20) + , 311 (100%) [A-2C2H20] + , 43 (42%) [C2H3O] + .

l,3,5-Trihydroxy-2-(3,3-dimethylallyl)-x anthone(IV). Yellow powder, m p288-290°C (reported 280-281 °C; Gunasekera et al., 1977). EMIE 70 eV

1906 REYES-CHILPA, JIMENEZ-ESTRADA, AND ESTRADA-MUNI/.

ANTIFUNGAL XANTHONES 1907

(IM/Z): 312 M+ (55%) [C18H16O5], 297 (35%) [M+-CH3], 269 (60.8%)[M + -C3H7], 257 (100%) [M + -C4H7], 244 (10.8%).

l-Hydroxy-2-(3,3-dimethylallyl)-3,5,6-triacetylxanthone (IVa). Yellowpowder, mp 121-123°C. UV Xmax [MeOH], nm (e): 360 (6879), 304(17894),239(60534). IR i>max (KBr): 2925, 1774, 1641, 1614 (C = C), 1436. EMIE 70eV (m/z): 396 M+ (80.3%) [C22H20O7] + , 353 (51.7%) [M+-C2H3O] = A, 341(34.8%) [M+-C4H7] = B, 311 (69.6%) [A-C2H3O] + , 299 (100%) [B-C2H3O] + ,269 (37.5%) [CI5H8O4] + , 257 (66%) [C14H8O4] + , 55 (20%) [C4H7| + , 43 (25%)[C2H30] + .

l,3,5,6-Tetrahydroxy-2-(3,3-ditnethylallyl)-xanthone (V). Yellow powder,mp 260-262°C (reported 255-257°C; Jackson et al., 1966). EMIE 70 eV(m/z): 328 M+ (54%) [C18H,6O6] + , 313 (30.3%) [M+-CH3|, 285 (66.6%)[M+-C3H6], 273 (100%) [M+-C4H71, 260 (13.5%)

l-Hydroxy-2-(3,3-dimethylallyl)-3,5,6-triacetylxanthone (Va). Rectangularyellow prisms, mp 193-196°C. IR vmax (CHC13): 2917, 1785, 1649, 1611,1452, 1373, 1260, 1161, 116, 1087. EMIE 70 eV (m/z): 454 M+ (100%)[C24H22O9] + , 411 (32.2%) [M+-C2H3O] = A, 399 (44%) [M + -C4H7j + = B,368 (60%) [A-C2H30] + , 357 (68.6%) [B-C2H2O] + , 355 (10.1%), 327 (62.7%),315 (61.8%), 273 (50%), 272 (32.2%), 69 (15.2%), 43 (33%).

Effects of Xanthones

The natural xanthones III, IV, and V showed fungistatic properties againstP. placenta when tested at 0.25 mg/ml. Phenol showed fungicidal activity atthe same concentration. Differences among the control, xanthones, and phenolwere statistically significant by ANOVA tests (Table 3). Under our conditions,the natural xanthones exhibit similar inhibitory activity, ranging from 55.8%(V) to 67.3% (III and IV). Acetylation of xanthones did not induce a sharpchange in the extent of fungistasis compared with parent compounds. At best,the derivatives showed a 8% increase (Va) or decrease (IIIa and IVa) in activity.Significant differences among natural and acetylated xanthones could not bedetected (Table 3).

The effect of increasing concentrations of xanthone V on P. placenta growthwas also examined. Concentrations of 0.5 and 1.0 mg/ml inhibited the mycelialgrowth by 64.4 and 74.6%, respectively. This last value is significantly differentfrom the 0.25 mg/ml treatment (Table 3). Inhibition caused by xanthone V atthe highest concentration was not significantly different from phenol at 0.25 mg/ml. Therefore it seems that V is about four times less potent than phenol. Atthe end of the experiments, all the agar plates treated with V were pooled andextracted to examine if this compound had suffered any transformation. Onlyone compound could be reisolated and purified in good yield (71%); it wasidentified as V according to 'H NMR data.

1908 REYES-CHILPA, JIMENEZ-ESTRADA, AND ESTRADA-MUNIZ.

TABLE 3. INHIBITION OF Postia placenta MYCELIAL GROWTH BY C, brasiliensisXANTHONES AND PHENOL"

Compound (mg/ml)

ControlV (1.00)V (0.50)V (0.25)IV (0.25)IIIa and IVa (0.25)Va (0.25)III and IV (0.25)Phenol (0.25)

Growth (cm)

4.53 ± 0.33"1.15 ± 0.41M

1.60 ± 0.14''2.00 ± 0.42'"'1.95 ± 0.40'"'1.85 ± 0.07'"'1.68 ± 0.16'11.48 ± 0.28''0.60 ± 0.00"'

Inhibition (%)

00.0 ± 7.274.6 ± 9.064.6 ± 3.055.8 ± 9.256.9 ± 8.859.1 ± 1.562.9 ± 3.567.3 ± 6.1

100.0 ± 0.0

"Mean of three replicates + standard deviation six days after innoculation. Least s ignif icant differ-ence >0.59; values followed by a different letter are significantly different at P = 0.05 (Tukey'sI test).

DISCUSSION

The heartwood of Calophyllum species contains xanthones and neoflavonoids,while the leaves possess coumarins, benzopyrans, and triterpenes (Ampofo andWaterman, 1986; Patil et al., 1993). Xanthones isolated from this genus can besimple or modified, especially with prenyl (3,3-dimethylallyl) -derived sub-stituents. Our results indicate that 2-prenylated xanthones (I, II, HI, IV, andV) are the main constituents of C. brasiliensis heartwood. All of these com-pounds exhibit an 1,3,5-trioxygenated substitution pattern. In addition, com-pound V has an extra hydroxyl on C-6. While compounds I, III, IV, and Vhave been previously isolated from other Calophyllum species, compound IIhas only been obtained from Garcinia mangosta hulls (Sen et al., 1981). Com-pound IIIb has not been reported as a natural product; so it is possible that itcould be an artifact produced during the acetylation procedure.

The natural xanthones III, IV, and V showed fungistatic activity againstthe brown rot fungus Postia placenta (Table 3). Xanthones III, IV, and espe-cially V, were the most abundant constituents of both the acetone and methanolextracts. For the heartwood sample here analyzed, these compounds representat least 0.65% (w/w). Compound V alone accounts for 0.53%. C. brasiliensisheartwood resistance against wood decay fungi thus appears to depend mostlyon these compounds, especially V. It was previously reported that 1,3,5,6-tetrahydroxyxanthone isolated from Madura pomifera heartwood inhibits the

ANTIFUNGAL XANTHONES 1909

growth of the wood rotting fungi Gleophyllum trabeum and Trametes versicolor(Schultz et al., 1995). This compound is the biogenetic precursor of V andshowed an IC50 (50% inhibition of radial mycelial growth) greater than 200ppm with both fungi (Schultz et al., 1995). In our case, compound V, at asimilar concentration (0.25 mg/ml = 250 ppm), inhibited the growth ofP. placenta by 55.8% (Table 3). It is noteworthy that at the same concentrationthe antifungal activity of C. brasiliensis heartwood xanthones was lower thanthat exhibited by synthetic phenol. Schultz et al. (1995) also observed thatM. pomifera heartwood compounds were less active than commercial fungicides.

Some other xanthones have been shown to be inhibitory to phytopathogenicfungi. For instance 1,5-dihydroxyxanthone, 6-desoxyjacareubin, 5-hydroxy-l-methoxyxanthone (Rocha et al., 1994), and l,3,5-trihydroxy-2-methoxyxan-thone (Pinto et al., 1994) inhibited the growth of Cladosporium curcumerinum.The former compound was also active against Trichophyton mentagrophytes(Pinto et al., 1994). On the other hand, four 3-OMe substituted xanthones wereinactive against C. curcumerinum (Rodriguez et al., 1995; Pinto et al., 1994),suggesting that free hydrogen or hydroxyl at this position might be essential forantifungal activity. Nevertheless, blocking of hydroxyls (including that on C-3)by acetylation of V or the mixture of HI and IV did not induce a significantchange in fungistasis as compared with parent compounds (Table 3).

Several xanthones have been recently reported as antioxidants and freeradical scavengers (Minami et al., 1994, 1995). These properties are importantconsidering that wood degradation by brown rot fungi involves secretion offungal H2O2 and its interactions with wood Fe2+ ions (Kirk, 1983). From thisperspective, it is possible to hypothesize that during fungal attack xanthonescould first be oxidized, thus delaying degradation of structural polymers. Pre-liminary evidence indicates that compound V was not oxidized in vitro byP. placenta, but the presence of Fe2+ ions was not assured in this system. Weare currently studying oxidative metabolism of V under controlled conditions.

Acknowledgments—Research was supported by grant NI214996 DGAPA-UNAM. The authorsare grateful to Fernando Ortega-Escalona and Guadalupe Barcenas Pazos for providing C. brasilieniswood, to Dr. Terry Highley for donation of the fungus, and to Dr. Ana Luisa Anaya Lang for herfacilities for culturing it.

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