Natural Products Inhibiting Candida albicans Secreted Aspartic Proteases from Tovomita krukovii

• Abstract
• Introduction
• Materials and Methods
• Plant material
• General experimental procedures
• Extraction and isolation
• Biological testing
• Results and Discussion
• Acknowledgements
• References

Abstract

Assay-guided fractionation of the ethanol extract of Tovomita krukovii resulted in the identification of four new xanthones (1 - 4) and ten known compounds (5 - 14). The structures of compounds 1 - 14 were determined by spectral data to be 3,5-dihydroxy-4-methoxyxanthone (1), 1,3,5,7-tetrahydroxy-8-isoprenylxanthone (2), 1,3,5-trihydroxy-8-isoprenylxanthone (3), 1,5,7-trihydroxy-8-isoprenylxanthone (4), 1,3,7-trihydroxy-2-isoprenylxanthone (5), 1,5-dihydroxyxanthone (6), 1,6-dihydroxy-5-methoxyxanthone (7), 1,3,5-trihydroxyxanthone (8), 1,3,6-trihydroxy-5-methoxyxanthone (9), 1,6-dihydroxy-3,5-dimethoxyxanthone (10), 1,3,7-trihydroxyxanthone (11), 3-geranyl-2,4,6-trihydroxybenzophenone (12), betulinic acid (13), and 3,4-dihydroxybenzoic acid (14). Compounds 2, 3, 12 and 13 showed inhibitory effects against Candida albicans secreted aspartic proteases (SAP) with IC₅₀ values of 15 μg/ml, 25 μg/ml, 40 μg/ml, and 6.5 μg/ml, respectively, while the other compounds were inactive. In addition, compound 12 showed activity against C. albicans, C. neoformans, S. aureus and methicillin resistant S. aureus (MRS).

Introduction

Candida albicans is an important opportunistic pathogen causing local or systemic infections in predisposed patients who are compromised immunologically or undergoing prolonged antibiotic treatment. The secreted aspartic proteases (SAP) of Candida albicans have been shown to be a major virulence factor in Candida infections [1]. Inhibition of SAP has been proposed as a new approach in the treatment of candidosis [2]. During our screening program searching for Candida albicans secreted aspartic proteases (SAP) inhibitors from higher plants, an ethanol extract of a mixture of the stem wood-stem bark of Tovomita krukovii (Guttiferae) collected in Peru was found to be active. Teas prepared from the flowers of T. brasiliensis and T. laurina have been reported to be helpful in controlling diarrhea [3], while a chloroform-soluble extract of the roots of T. brevistaminea exhibited significant cytotoxicity against the KB cell line [4]. Xanthones, which are distributed widely in the family Guttiferae (along with other secondary metabolites such as benzophenones, coumarins and betulinic acid), have been previously reported from several related Tovomita species [4]. T. krukovii, however, has not yet been investigated phytochemically or biologically.

In the present investigation, bioassay-guided fractionation of the active chloroform fraction utilizing the SAP assay led to the isolation of four new xanthones (1 - 4) and ten known compounds (5 - 14). We report herein the structure elucidation of the new xanthones and the biological evaluation of compounds 1 - 14.[*]

Materials and Methods

Plant material

The plant material of Tovomita krukovii Smith (stem wood-stem bark) was collected in Peru. A voucher specimen is deposited at the National Center for Natural Products Research, The University of Mississippi (voucher # 2553).

General experimental procedures

UV spectra were recorded on a Hewlett Packard 8435 spectrometer. IR spectra were obtained on an ATI Mattson Genesis Series FTIR spectrometer. HRESIMS were measured on a Bruker-Magnex BioAPEX 30es ion cyclotron high resolution HPLC-FT spectrometer by direct injection into an electrospray interface. The NMR spectra were recorded on a Bruker Avance DRX-400 spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C. 2D NMR were measured with standard pulse programs and acquisition parameters. Silica gel (40 μm, J. T. Baker) and RP silica gel (RP-18, 40 μm, J. T. Baker) were used for low pressure chromatography. HPLC was performed using an ODS column (column A: μ-Bondapak C18, 3.9 mm, i. d. × 300 mm, 10 μm; column B : Phenomenex, Prodigy ODS prep, 21.2 mm i. d. × 250 mm, 10 μm). TLC was performed on silica gel 60 F254 (EM Science) using CHCl₃/MeOH (9 : 1, solvent A) and benzene/EtOAc (4 : 1, solvent B) or reversed-phase KC18 F silica gel 60 (Whatman) using MeOH/H₂O (70 : 30, solvent C) and MeOH/ H₂O (80 : 20, solvent D). All microorganisms were obtained from the American Type Culture Collection. Sabouraud dextrose broth (prepared as labelled and filtered sterilized) and dextrose were purchased from Difco Labs (Detroit, MI). The fluorescent renin substrate was purchased from Molecular Probes (# R-2931, Eugene, OR). All other chemicals (yeast extract, bovine serum albumin, pepsin and pepstatin A) were obtained from Sigma (St Louis, MO). Fluorescence was measured on a Cytofluor 2350 Fluorescence Measurement System using CytoCalc^TM software (version 2.00.06, PerSeptive Biosystems, Inc., Framingham, MA).

Extraction and isolation

The powdered material (500 g) was percolated with 95 % EtOH (3000 ml × 5) and the alcoholic extracts were combined and evaporated to dryness (48 g, IC₅₀ = 25 μg/ml). Part of the ethanolic extract (40 g) was detannified by passing it over a polyamide column and washing with MeOH. The MeOH wash (tannin-free fraction, 12 g, IC_{50 =} 10 μg/ml) was suspended in MeOH/H₂O (9 : 1) (150 ml), and then partitioned successively with hexane (100 ml × 3, 1.3 g), CHCl₃ (100 ml × 3, 5.8 g), EtOAc (100 ml × 3, 1.5 g), and n-BuOH (100 ml × 3, 1.2 g). The activity resided in the chloroform fraction (IC₅₀ = 25 μg/ml). Part of the chloroform fraction (4.0 g) was chromatographed over a silica gel column (250 g) using mixtures of EtOAc/toluene of increasing polarity (4 : 1, 3 : 2, each of 2500 ml). Fractions were monitored by TLC and similar fractions combined to afford fractions A (100 - 800 ml, 100 mg), B (800 - 900 ml, 256 mg, IC₅₀ = 25 μg/ml), C (900 - 1200 ml, 1260 mg, IC₅₀ = 20 μg/ml), D (1200 - 1300 ml, 160 mg), E (1300 - 1700 ml, 200 mg), F (2200 - 2400 ml, 56 mg), G (2500 - 3000 ml, 72 mg), H (4000 - 4200 ml, 485 mg) and I (4400 - 4600 ml, 1374 mg). Fraction B was refractionated on a silica gel column (60 g) using CHCl₃ and CHCl₃/MeOH, 9 : 1 (900 ml, each) to give a total of 90 fractions (20 ml, each). Fractions 66 - 70 contained 13 (50 mg), and fractions 19 and 20 were combined and purified by HPLC (column A, MeOH/H₂O, 50 : 50, 1 ml/min, UV 254 nm) to give 6 (1.5 mg, t_R 41 min) and 7 (1.7 mg, t_R 43 min). Part of fraction C (600 mg) was chromatographed over a silica gel column (100 g) eluting with CHCl₃/EtOAc (9 : 1, 2000 ml) to give 13 (250 mg), C₁ (400 - 700 ml, 180 mg) and C₂ (1300 - 1500 ml, 120 mg). C₁ was rechromatographed over a low pressure ODS column (40 g) (MeOH/H₂O, 30 : 70, 50 : 50, 70 : 30, 100 : 0, each 100 ml) to give C₃ (120 - 180 ml, 50 mg), C₄ (220 - 230 ml, 45 mg), C₅ (270 - 280 ml, 30 mg) and C₆ (280 - 300 ml, 25 mg). HPLC purification of C₂ (column A, MeOH/H₂O, 80 : 20, 1 ml/min, UV 312 nm) gave 12 (36 mg, t_R 14 min); C₄ (column A, MeOH/H₂O, 65 : 35, 1 ml/min, UV 365 nm) gave 10 (2.6 mg, t_R 17.2 min); and C₅ (column B, MeOH/H₂O, 70 : 30, 3 ml/min, UV 312 nm) gave 3 (2 mg, t_R 81 min) and 5 (5 mg, t_R 83 min). Part of fraction E (60 mg) was separated by a low pressure ODS column (50 g) (MeOH/H₂O, 50 : 50, 70 : 30, each 100 ml) to give 11 (100 ml, 8 mg), E₁ (105 - 150 ml, 20 mg) and E₂ (154 - 200 ml, 30 mg). E₁ was further purified by HPLC (column B, MeOH/ H₂O, 90 : 10, 3 ml/min, UV 254 nm) to give 4 (3.5 mg, t_R 33 min). Refractionation of fraction F using a low pressure ODS column (15 g) (MeOH/H₂O, 50 : 50, 70 : 30, 100 : 0, each 100 ml) gave 20 fractions (15 ml, each). Of these, fraction 11 contained 14 (5 mg), and fraction 12 was purified by HPLC (column B, MeOH/H₂O, 70 : 30, 3 ml/min, UV 254 nm) to give 8 (1.5 mg, t_R 29 min) and 9 (1.5 mg, t_R 31 min). Finally, fraction G was applied onto a silica gel column (60 g) (CHCl₃/MeOH, 14 : 1, 200 ml) to give 9 fractions (20 ml, each). Fraction 5 (15 mg) was separated by a low pressure ODS column (15 g) (MeOH/H₂O, 70 : 30) to yield 1 (91 - 106 ml, 5 mg), and fraction 8 (25 mg) was purified by HPLC (column A, MeOH/H₂O, 70 : 30, 1 ml/min, UV 254 nm) to yield 2 (7 mg, t_R 11.0 min).

3,5-Dihydroxy-4-methoxyxanthone (1): Yellow powder, m. p. 189 - 190 °C (MeOH), Rf 0.26 and 0.20 (silica gel, solvents A and B, respectively), 0.64 (reversed-phase KC₁₈ F, solvent C), UV (MeOH): λ_max (log ε) = 206 (3.34), 240sh (3.48), 258 (3.57), 376 nm (2.73), IR (KBr): ν_max = 3437, 1622, 1586, 1494, 1289, 1071 cm^-1, ¹H-NMR (DMSO-d ₆): δ = 3.80 (3H, s, OCH₃-4), 7.14 (1H, t, J = 7.7 Hz, H-7), 7.21 (1H, dd, J = 7.7, 1.6 Hz, H-6), 7.26 (1H, d, J = 9.0 Hz, H-2), 7.36 (1H, d, J = 9.0 Hz, H-1), 7.48 (1H, dd, J = 7.7, 1.6 Hz, H-8), HRESIMS: m/z = 259.0593 {calcd for [M (C₁₄H₁₀O₅ + 1)], 259.0601}, ¹³C- NMR (Table [1]).

1,3,5,7-Tetrahydroxy-8-isoprenylxanthone (2): Yellow powder, m. p. 115 - 116 °C (MeOH), R_f 0.15 and 0.14 (silica gel, solvents A and B, respectively), 0.25 (reversed-phase KC₁₈ F, solvent C), UV (MeOH): λ_max (log ε) = 208 (3.21), 256 (3.08), 312 (2.88), 363 nm (2.56), IR (KBr): ν_max = 3479, 3157, 1637, 1610, 1561, 1494, 1351, 1318, 1164, 1061, 971 cm^-1, ¹H-NMR (DMSO-d ₆): δ = 1.60 (3H, s, CH₃-14), 1.76 (3H, s, CH₃-15), 4.01 (2H, d, J = 6.2 Hz, H-11), 5.18 (1H, br t, H-12), 6.10 (1H, d, J = 1.6 Hz, H-2), 6.24 (1H, d, J = 1.6 Hz, H-4), 6.74 (1H, s, H-6), 13.61 (1H, s, OH-1), HRESIMS : m/z = 329.0983 {calcd for [M(C₁₈H₁₆O₆ + 1)], 329.1020}, ¹³C-NMR (Table [1]).

1,3,5-Trihydroxy-8-isoprenylxanthone (3): Yellow powder, m. p. 129 - 130 °C (MeOH), R_f 0.27 and 0.36 (silica gel, solvents A and B, respectively), 0.17 (reversed-phase KC₁₈ F, solvent C), UV (MeOH): λ_max (log ε) = 210 (3.67), 260 (3.83), 305 (3.44), 376 nm (3.05), IR (KBr): ν_max = 3349, 2917, 1638, 1603, 1485, 1434, 1318, 1157, 1075, 815 cm^-1, ¹H-NMR (DMSO-d ₆): δ = 1.60 (3H, s, CH₃-14), 1.76 (3H, s, CH₃-15), 4.00 (2H, d, J = 6.2 Hz, H-11), 5.17 (1H, br t, H-12), 6.13 (1H, s, H-2), 6.27 (1H, s, H-4), 7.26 (1H, d, J = 9.0 Hz, H-6), 7.29 (1H, d, J = 9.0 Hz, H-7), 13.31 (1H, s, OH-1), HRESIMS: m/z = 313.1046 {calcd for [M(C₁₈H₁₆O₅ + 1)], 313.1070), ¹³C-NMR (Table [1]).

1,5,7-Trihydroxy-8-isoprenylxanthone (4): Yellow powder, m. p. 147 - 148 °C (MeOH), Rf 0.37 and 0.36 (silica gel, solvents A and B, respectively), 0.42 (reversed-phase KC₁₈ F, solvent D), UV (MeOH): λ_max (log ε) = 206 (3.26), 252 (3.13), 302 (2.86), 380 nm (2.79); IR (KBr): ν_max = 3398, 1637, 1603, 1503, 1316, 1157, 1068, 816 cm^-1, ¹H-NMR (DMSO-d ₆): δ = 1.61 (3H, s, CH₃-14), 1.78 (3H, s, CH₃-15), 4.02 (2H, d, J = 6.4 Hz, H-11), 5.19 (1H, br t, H-12), 6.69 (1H, dd, J = 8.0, 1.7 Hz, H-2), 6.81 (1H, s, H-6), 6.92 (1H, dd, J = 8.0, 1.7 Hz, H-4), 7.58 (1H, t, J = 8.0 Hz, H-3), 13.43 (1H, s, OH-1), HRESIMS: m/z = 313.1038 {calcd for [M(C₁₈H₁₆O₅ + 1)], 313.1070}, ¹³C-NMR (Table [1]).

1,3,7-Trihydroxy-2-isoprenylxanthone (5): Yellow powder, m. p. 216 - 217 °C (MeOH) [lit. [5], 217 - 218 °C (MeOH)], Rf 0.28 and 0.37 (silica gel, solvents A and B, respectively), 0.44 (reversed-phase KC₁₈ F, solvent D), ¹H-NMR, ¹³C-NMR and MS: literature [5].

1,5-Dihydroxyxanthone (6): Yellow powder, m. p. 197 - 198 °C (MeOH) [lit. [6], 194 - 196 °C)], Rf 0.67 and 0.43 (silica gel, solvents A and B, respectively), 0.63 (reversed-phase KC18 F, solvent C), ¹H-NMR, ¹³C-NMR and MS: literature [7].

1,6-Dihydroxy-5-methoxyxanthone (buchanaxanthone) (7): Yellow powder, m. p. 242 - 243 °C (MeOH) [lit. (8), 243 - 246 °C (CHCl₃)], Rf 0.69 and 0.45 (silica gel, solvents A and B, respectively), 0.61 (reversed-phase KC18 F, solvent C), ¹H-NMR and MS : literature [8], ¹³C-NMR (Table [1]).

1,3,5- Trihydroxyxanthone (8): Yellow powder, m. p. 304 - 305 °C (MeOH) [lit. [9], 302 - 305 °C (decomp., aq. MeOH)], R_f 0.65 and 0.41 (silica gel, solvents A and B, respectively), 0.62 (reversed-phase KC₁₈ F, solvent C), ¹³C-NMR and MS : literature [9], [10].

1,3,6- Trihydroxy-5-methoxyxanthone (9): Yellow powder, m. p. 291 - 292 °C (MeOH) [lit. [11], 290 - 291 °C (MeOH)], R_f 0.65 and 0.41 (silica gel, solvents A and B, respectively), 0.62 (reversed-phase KC₁₈ F, solvent C), ¹H-NMR and MS : literature [11], ¹³C-NMR (Table [1]).

1,6-Dihydroxy-3, 5-dimethoxyxanthone (10): Yellow powder, m. p. 192 - 193 °C (MeOH) [lit. [12], 190 - 192 °C (MeOH)], R_f 0.58 and 0.39 (silica gel, solvents A and B, respectively), 0.53 (reversed-phase KC₁₈ F, solvent C), ¹H-NMR, ¹³C-NMR and MS : literature [11], [12].

1,3,7-Trihydroxyxanthone (gentisein) (11): Yellow powder, m. p. 299 - 300 °C (MeOH) [lit. [13], 302 - 304 °C (EtOH) and [14], 315 - 319 °C (EtOAc)], R_f 0.19 and 0.26 (silica gel, solvents A and B, respectively), 0.49 (reversed-phase KC₁₈ F, solvent C), ¹H-NMR, ¹³C-NMR and MS: literature [10], [13], [14].

3-Geranyl-2,4,6-trihydroxybenzophenone (12): Yellow powder, m. p. 197 - 198 °C (MeOH), R_f 0.36 and 0.27 (silica gel, solvents A and B, respectively), 0.29 (reversed-phase KC₁₈ F, solvent D), UV (MeOH): λ_max (log ε) = 216 (3.87), 252 sh, 312 nm (3.53), IR (KBr): ν_max = 3452, 2333, 1627, 1313, 1157, 1062 cm^-1, ¹H-NMR (CDCl₃): δ = 1.60 (3H, s, CH₃-8″), 1.67 (3H, s, CH₃-9″), 1.80 (3H, s, CH₃-10″), 2.08 (4H, br, H-4″, 5″), 3.36 (2H, d, J = 6.5 Hz, H-1″), 5.06 (1H, br t, H-6″), 5.26 (1H, br t, H-2″), 5.94 (1H, s, H-5), 7.48 (2H, m, H-3’, 5′), 7.54 (1H, m H-4′), 7.62 (2H, m, H-2′, 6′), 10.28 (1H, s, OH-2), ¹³C-NMR (CHCl₃-d): δ = 16.4 (C-10″), 17.9 (C-8″), 21.8 (C-1″), 25.8 (C-9″), 26.6 (C-5″), 39.9 (C-4″), 96.2 (C-5), 104.8 (C-1), 106.8 (C-3), 121.8 (C-2″), 124.0 (C-6″), 128.1 (C-3′, 5′), 129.1 (C-2′, 6′), 132.2 (C-4′, 7″), 139.1 (C-3″), 140.5 (C-1′), 159.7 (C-6), 161.1 (C-2), 163.0 (C-4), and 198.2 (C = O), HRESIMS: m/z = 367.1826 {calcd for [M(C₂₃H₂₆O₄ + 1)], 367.1839}.

Betulinic acid (13): Colorless needles, m. p. 291 - 292 °C (MeOH), [α]²⁰ _D: + 11 (pyridine, c = 0.60), R_f 0.57 and 0.52 (silica gel, solvents A and B, respectively).; identified by comparison with an authentic sample (TLC, m. p. and mixed m. p.) and the reported ¹H-NMR and ¹³C- NMR data [15].

3,4-Dihydroxybenzoic acid (14): Colorless needles, m. p. 192 - 193 °C (MeOH), [lit. [16], 193 - 195 °C (acetone)], identified by comparison (¹H-NMR, ¹³C-NMR, MS) with those reported [16].

Biological testing

Induction of SAP production: The procedure of Capobianco et al. [17] was used to prepare the secreted aspartic proteases (SAP)-rich extract. Briefly, Candida albicans ATCC 10 231 were grown overnight at 30 ^oC in Sabouraud dextrose broth. The cells were washed once with 10 mM phosphate-buffered saline (pH 7.0), resuspended in 15 ml inducing medium (0.2 % yeast extract, 0.2 % bovine albumin, 2.0 % dextrose), and incubated for 8 h at 30 ^oC. The SAP-rich supernatant was collected, sterilized by filtration, aliquoted and stored at -80 ^oC until needed.

SAP inhibition assay: Using 50 mM sodium citrate buffer (pH 4.5) as the diluent, the final optimum substrate and SAP extract concentrations were determined to be 30 μM and a 1 : 286 dilution of the SAP-rich supernatant, respectively. The final volume of the reaction mixture (sample, diluted SAP extract, substrate) was 100 μl. Blanks which contained ≤ 4 % v/v DMSO were also included. Immediately adding substrate the fluorescence at 360ex/530em was measured. The plate was incubated for 1 h at 37 ^oC and then fluorescence was measured again. The increase in fluorescence over the incubation time was determined and expressed as % fluorescence compared to a positive control, which contained no sample but the same DMSO concentration.

Crude extracts were initially tested at one concentration of 200 μg/ml to identify active extracts (≥ 80 % inhibition). Active extracts were confirmed by retesting at three concentrations (50, 10, and 2 μg/ml). Solvent partitions and chromatographic fractions were tested at three concentrations to guide isolation. Isolated compounds were tested at eight concentrations and % fluorescence was plotted versus concentration. Prism software from GraphPad (San Diego, CA) was used to fit the resulting data to sigmoidal curves and determine the concentration that inhibited 50 % of activity (IC₅₀). The aspartic protease inhibitor pepstatin A was used as a positive control in these assays.

Pepsin inhibition assay: Optimum final pepsin and substrate concentrations were determined to be 10 units/ml and 30μM, respectively. The assay was performed as above except that pepsin was substituted for the SAP extract and a 50 mM sodium citrate buffer (pH 4.0) was used as the diluent.

Antimicrobial assay: Staphylococcus aureus ATCC 29 213 and methicillin-resistant S. aureus ATCC 43 300 (MRS) were stored on Eugon (Difco, Detroit) agar slants at 4 °C until needed. Susceptibility testing was performed using a modified version of the National Committee for Clinical Laboratory Standards [18]. Cells were transferred to 5 ml Eugon broth (BBL, Maryland) and incubated at 37 °C. Overnight cultures were diluted with Eugon broth (1 : 50) for the antimicrobial assay. Test compounds are dissolved in DMSO, serially-diluted using normal saline, and transferred to 96 well microtiter plates. The inoculum is added to achieve a final volume of 200 μl and final concentrations of 50 μg-0.02 μg/ml. Drug [Tetracycline at 5.0 to 0.002 μg/ml (Sigma, St Louis)] as well as growth and blank (media only) controls were added to each test plate. Plates are read turbidimetrically at 630 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont) prior to and after incubation (24 h at 37 °C). Percent growth is calculated and plotted vervus concentration to afford the IC₅₀/MIC. The procedures used for Candida albicans, Cryptococcus neoformans have been described in our previous paper [19].

Results and Discussion

Repeated chromatography of the active chloroform fraction of T. krukovii resulted in the isolation of ten known compounds (5 - 14): 1,3,7-trihydroxy-2-isoprenylxanthone (5) [5], 1,5-dihydroxyxanthone (6) [7], 1,6-dihydroxy-5-methoxyxanthone (7) [8], 1,3,5-trihydroxyxanthone (8) [9], [10], 1,3,6-trihydroxy-5-methoxyxanthone (9) [11], 1,6-dihydroxy-3,5-dimethoxyxanthone (10) [12], 1,3,7-trihydroxyxanthone (11) [10], [13], [14], 3-geranyl-2,4,6-trihydroxybenzophenone (12) [20], betulinic acid (13) [15], and 3,4-dihydroxybenzoic acid (14) [16], which were identified by comparison of their melting points, ¹H-NMR, ¹³C-NMR and MS with literature data.

The molecular formulae of compounds 1 (C₁₄H₁₀O₅), 2 (C₁₈H₁₆O₆), 3 (C₁₈H₁₆O₅), and 4 (C₁₈H₁₆O₅) were deduced by HRESIMS, which showed molecular ion peaks at m/z [M+H]⁺ 259.0593, 329.0983, 313.1046, and 313.1038, respectively. The IR spectra of compounds 1 - 4 displayed absorption bands at 3340 - 3480 cm^-1 for hydroxy groups and 1620 - 1640 cm^-1 for conjugated carbonyl functions. The UV spectra of compounds 1 - 4 exhibited four absorption bands characteristic of xanthones [21]. The oxygenation patterns of compounds 1 - 4 were determined by ¹H- and ¹³C-NMR spectroscopy (Table [1]). In the ¹H-NMR spectra, a low field singlet (δ = 13.3 - 13.6) confirmed the presence of a chelated hydroxy group in compounds 2 - 4. Signals in the regions δ = 1.60 - 1.78 (6H), 4.00 - 4.02 (2H), and 5.17 - 5.19 (1H), in compounds 2 - 4, were typical of a 3, 3-dimethylallyl unit. The downfield value of the 2H proton doublet (δ = 4.00 - 4.02) indicated the placement of the unit at C-8, peri to the carbonyl [22], which was also confirmed by HMBC correlation (Fig. [1]).

In the ¹H-NMR spectrum of compound 1, the signal at δ = 3.80, which correlated to the ¹³C-NMR resonance at δ = 61.0 in the HMQC spectrum was attributable to a methoxy group. Its ¹³C-NMR downfield value supported its location in a disubstituted environment [23]. In addition, the HMQC showed that the proton signals at δ = 7.14, 7.21, 7.26, 7.36 and 7.48 correlated to the ¹³C-NMR resonances at δ = 123.5, 119.4, 113.7, 124.9 and 114.3, respectively. The oxygenation pattern of compound 1 was determined by ¹H- and ¹³C-NMR spectroscopy. A pair of ortho-coupled aromatic protons at δ = 7.36 (1H, d, J = 9.0 Hz, H-1) and δ = 7.26 (1H, d, J = 9.0 Hz, H-2) was attributable to the protons on ring A. The B-ring protons of the 3,4,5-trisubstituted xanthone 1 exhibited an ABM system with signals at δ = 7.48 (1H, dd, J = 7.7, 1.6 Hz, H-8), 7.14 (1H, t, J = 7.7 Hz, H-7) and 7.21 (1H, dd, J = 7.7, 1.6 Hz, H-6). The ¹³C-NMR signals were unambiguously assigned using HMQC and HMBC NMR correlations (Fig. [1]). The structure of compound 1 was thus established as 3,5-dihydroxy-4-methoxyxanthone.

In compound 2, a pair of meta-coupled aromatic protons at δ = 6.10 (H-2) and 6.24 (H-4) was indicative of a 1,3-disubstituted A-ring, while the signal at δ = 6.74 (1H, s) was attributed to the H-6 of ring B. The ¹³C-NMR signals were unambiguously assigned using HMQC and HMBC NMR correlations (Fig. [1]). The structure of 2 was thus determined to be 1,3,5,7-tetrahydroxy-8-isoprenylxanthone.

The ¹H-NMR spectrum of compound 3 showed a pair of meta-coupled aromatic protons at δ = 6.13 (H-2) and 6.27 (H-4), characteristic of a 1,3-disubstituted A-ring. The B-ring protons of the 1,3,5,8-tetrasubstituted xanthone 3 exhibited a pair of ortho-coupled aromatic protons at δ = 7.26 (1H, d, J = 9.0 Hz, H-6) and δ = 7.29 (1H, d, J = 9.0 Hz, H-7). The ¹³C-NMR signals were unambiguously assigned using HMQC and HMBC NMR correlations (Fig. [1]). The structure of 3 was established as 1,3,5-trihydroxy-8-isoprenylxanthone.

In compound 4, an ABM system was observed in the ¹H-NMR spectrum with signals at δ = 6.69 (1H, dd, J = 8.0, 1.7 Hz, H-2), 6.92 (1H, dd, J = 8.0, 1.7 Hz, H-4) and 7.58 (1H, t, J = 8.0 Hz, H-3). This indicated that A-ring was substituted at C-1. A signal at δ = 6.81 (1H, s) was assigned to the H-6 proton of ring B. The ¹³C-NMR signals were unambiguously assigned using the HMQC and HMBC NMR correlations (Fig. [1]). The structure of 4 was proven to be 1,5,7-trihydroxy-8-isoprenylxanthone.

Based on the ¹H-, ¹³C-NMR and HRESIMS data, compound 12 was established as 3-geranyl-2,4,6-trihydroxybenzophenone (E-isomer) [18], isolated previously from Helichrysum monticola (Compositae) as a mixture of 2″,3″-E and Z isomers. The ¹³C- NMR assignments were unambiguously achieved using HMQC and HMBC correlations and were reported here for the first time.

Compounds 1 - 14 were evaluated against Candida albicans secreted aspartic proteases. The results showed that compounds 2, 3, 12 and 13 had inhibitory effects against Candida albicans secreted aspartic proteases with estimated IC₅₀ values of 15, 25, 40 and 6.5 μg/ml, respectively. Pepstatin A was used as a positive control (IC₅₀ of 0.002 μg/ml). Compounds 2, 3, 12 and 13 were also active against the general aspartic protease pepsin (IC₅₀ of 10, 10, 6 and 5 μg/ml, respectively). This indicates that the activities of these compounds were non-selective to SAP. Although the activities of compounds 2, 3, 12, and 13 do explain the activities of fractions B and C, they do not account for the total activity of the initial crude extract.

The known benzophenone 12 was also found to have antimicrobial activity against C. albicans, C. neoformans, S. aureus and MRS with IC₅₀/MIC of 4.0/25 μg/ml, 1.5/3.13 μg/ml, 1.5/3.13 μg/ml and 2.0/3.13 μg/ml, respectively. Amphotericin B, the positive control for C. albicans and C. neoformans, had an IC₅₀/MIC of 0.04/0.078 μg/ml and 0.3/0.625 μg/ml, respectively. Tetracycline was used as a positive control for S. aureus and MRS with an IC₅₀/MIC of 0.15/0.3125 μg/ml for both organisms.

Acknowledgements

The authors would like to thank Dr. Chuck Dunbar for performing the HRESIMS analysis, Ms. Sharon Sanders, Ms. Belynda Smiley, and Ms. Marinda Logan for biological testing and technical assistance. This work was supported in part by the United States Department of Agriculture, Agricultural Research Service Specific Cooperative Agreement No, 58 - 6408 - 7-012, and by the National Institute of Health, Institute of Allergy and Infectious Diseases, Division of AIDS, Grant #AI42500.

References

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  Search in Google Scholar
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  PubMedSearch in Google Scholar

Dr. Hala N. ElSohly

National Center for Natural Products Research

School of Pharmacy

University of Mississippi

University

MS 38677

USA

Email: helsohly@olemiss.edu

Fax: +1 662-915-7989

References

• 1 Hoegl L, Ollert M, Korting H C. The role of Candida albicans secreted aspartic proteinase in the development of candidoses.  Journal Molecular Medicine. 1996;  74 135-42
  PubMedSearch in Google Scholar
• 2 Hoegl L, Korting H C, Klebe G. Inhibitors of aspartic proteases in human diseases; molecular modeling comes of age.  Pharmazie. 1999;  54 319-29
  PubMedSearch in Google Scholar
• 3 Schultes R E. In Botanical museum leaflets. New York; Harvard University Press 1983: 29
  Search in Google Scholar
• 4 Seo E .-K, Wall M E, Wani M C, Navarro H, Mukherjee R, Farnsworth N R. Cytotoxic constituents from the roots of Tovomita brevistaminea .  Phytochemistry. 1983;  52 669-74
  PubMedSearch in Google Scholar
• 5 Garcia-Cortez D A, Young M CM, Marston A, Wolfender J L, Hostettmann K. Xanthones, triterpenes and a biphenyl from Kielmeyera coriacea .  Phytochemistry. 1998;  47 1367-74
  CrossrefPubMedSearch in Google Scholar
• 6 Jackson B, Locksley H D, Scheinmann F. Extractives from Guttiferae. Part v. Scriblitifolic acid, a new xanthone from Calophyllum scriblitifolium Henderson and Wyatt-Smith.  Journal Chemical Society (C). 1967;  22 785-99
  PubMedSearch in Google Scholar
• 7 Rocha L, Marston A, Kaplan M AC, Stoeckli-Evans H, Thull U, Testa B, et al. An antifungal γ-pyrone and xanthones with monoamine oxidase inhibitory activity from Hypericum brasiliense .  Phytochemistry. 1994;  36 1381-5
  CrossrefPubMedSearch in Google Scholar
• 8 Jackson  B, Locksley H D, Moore I, Scheinmann F. Extractives from Guttiferae. Part IX. The isolation of buchanaxanthone and two related xanthones from Garcinia buchananii Baker. Journal Chemical Society (C) 1968: 2579-83
  Search in Google Scholar
• 9 Locksley H D, Murray I G. Extractives from Guttiferae. Part XIX. The isolation and structure of two benzophenones, six xanthones and two biflavonoids from the Heartwood of Allanblackia floribunda Oliver.  Journal of Chemical Society (C). 1971;  1332-40
  PubMedSearch in Google Scholar
• 10 Frahm A W, Chaudhuri R K. ¹³C NMR spectroscopy of substituted xanthones-II ¹³C NMR spectral study of polyhydroxyxanthones.  Tetrahedron. 1979;  35 2035-8
  CrossrefPubMedSearch in Google Scholar
• 11 Ghosal S, Chaudhuri R K, Nath A. Chemical constituents of Gentianaceae IV: New xanthones of Canscora decussata .  Jounal of Pharmaceutical Sciences. 1973;  62 137-8
  PubMedSearch in Google Scholar
• 12 Wolfender J L, Hamburger M, Msonthi J D, Hostettmann K. Xanthones from Chironia krebsii .  Phytochemistry. 1991;  30 3625-29
  CrossrefPubMedSearch in Google Scholar
• 13 Kitanov G, Achtardjiev C. Isolierung von Gentisein aus Hypericum degenii Bornm.  Pharmazie. 1979;  34 447-8
  PubMedSearch in Google Scholar
• 14 Atkinson J E, Gupta P, Lewis J R. Some phenolic constituents of Gentiana lutea .  Tetrahedron. 1968;  24 1507-11
  CrossrefPubMedSearch in Google Scholar
• 15 Ikuta A, Itokawa H. Triterpenoids of Paeonia japonica callus tissue.  Phytochemistry. 1988;  27 2813-5
  CrossrefPubMedSearch in Google Scholar
• 16 Zhang Z Z, Koike K, Guo D A, Li C L, Zheng Z H, Jia Z H, . et al . Studies on the chemical constituents of Yunnan wintergreen root Gaultheria yunnanensis (II).  Zhongcaoyao. 1999;  30 167-9
  PubMedSearch in Google Scholar
• 17 Capobianco  J O, Lerner C G, Goldman R C. Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.  Analytical Biochemistry. 1992;  204 96-102
  CrossrefPubMedSearch in Google Scholar
• 18 National Committee for Clinical Laboratory Standards 1 997. Methods for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically. Approved Standard M7-A4, vol. 17, #2. National Committee for Clinical Laboratory Standards Wayne, PA; 4th ed
• 19 Li X C, ElSohly H N, Nimrod A C, Clark A M. Antifungal jujubogenin saponins from Colubrina retusa .  Journal of Natural Products. 1999;  62 674-7
  CrossrefPubMedSearch in Google Scholar
• 20 Bohlmann F, Zdero C. Neue Geranylphloroglucin-Derivate aus Helichrysum monticola .  Phytochemistry. 1980;  19 683-4
  CrossrefPubMedSearch in Google Scholar
• 21 Hostettmann K, Hostettmann M. Xanthones. Methods in Plant Biochemistry. In: Harborne JB, editor vol. 1 London; Academic Press 1989: 493-508
  Search in Google Scholar
• 22 Waterman P G, Hussain  R A. Major xanthones from Garcinia quadrifaria and Garcinia staudtii stem barks.  Phytochemistry. 1982;  21 2099-101
  CrossrefPubMedSearch in Google Scholar
• 23 Miura  I, Hostettmann K, Nakanishi K. Carbon-13 NMR of naturally occurring xanthone aglycones and glycosides.  Nouveau Journal de Chimie. 1978;  2 653-7
  PubMedSearch in Google Scholar

Dr. Hala N. ElSohly

National Center for Natural Products Research

School of Pharmacy

University of Mississippi

University

MS 38677

USA

Email: helsohly@olemiss.edu

Fax: +1 662-915-7989