An antifungal compound from Solanum nigrescens

ELSEVIER

Journal of Ethnopharmacology

43 (1994) 173-177

An antifungal compound from Solanum nigrescens Xian-guo He”, Ursula Moceka, Heinz G. Floss*ta, Armando CBceresb, Lidia Gir6nb, Helen Buckleyc, Gerard Cooney’, Joanne Mannsc, Bary W. Wilsond ‘Department of Chemistry, University of Washington. Seattle. WA 98195. USA blJniversity of San Carlos. Guatemala City, Guatemala ‘Temple University Schools of Pharmacy and Medicine. Philadelphia, PA 19140, USA dBartelle Pa@ Northwest Laboratories, Richland, WA 99352, USA (Received 19 December 1992; revision received 25 October 1993; accepted 5 April 1994)

Abstract

The antifungal activity of Solanum nigrescens extracts has been traced to the presence of a spirostanol glycoside, cantalasaponin-3. Keywords: Solanum nigrescens; Spirostanol trometry

glycoside; Candido albicans; Cryptococcus neoformans; Fab mass spec-

1. Introduction The plant

Solanum nigrescens Mart

and

Gal.

(Solanaceae) is used as a folk medicine in Guatemala for the treatment of certain superficial fungal infections, particularly vaginitis (Giron et al., 1988). In the present study we examined the effects of the hydroalcoholic extracts of S. nigrescens on cultures of Candida albicans and Cryptococcus neoformans and carried out a bioassay-directed

* Corresponding author, present address: Department of Chemistry BG-IO, University of Washington, Seattle, WA 98195, USA; Fax 206 543 8318.

fractionation of the extract which resulted in the isolation and identification of the active antifungal principle. 2. Materials and methods 2.1. Plant extract

The aqueous ethanol extract of the plant S. nigrescens was supplied by Farmaya Company in

Guatemala. The plant extract was prepared by a standard method of maceration of the dry powdered leaves of field-collected plant material with 50% EtOH as described elsewhere (Caceres et al., 1987). The final ethanol content in the extract was 45%.

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2.2. Determination of antifungal activity

Standard 96-well microtiter plates were used for minimum inhibitory dilution (MID) studies. SAB medium (glucose 2%, agar 2%, peptone 1%) was used for growth of Ca. albicans and Cr. neoformans. Dilutions of the plant extract in SAB medium ranged from 1:1 to 1:1024 and were incubated with a suspension of Cr. neoformans at 30°C for 24 h. MID was defined as the lowest dilution before visual turbidity occurred due to colony growth. Control wells contained inoculated SAB with 45% ethanol dilutions. Aliquots of the well dilutions were plated on SAB after 24 to 48 h of incubation and examined for growth. Fungicidal activity against Cr. neoformans and Aspergillus fumigatus was assessed by incubation of the fungus with the extract and removing samples at 1,2,3,4,12, and 24 h. Samples were spread onto SAB agar plates and incubated at 30°C for 48 h. Colony counts were determined in triplicate for each sample to establish the mean reduction of Cr. neoformans colonies per time period of exposure to extract. 2.3. Determination of cytotoxicity Sheep erythrocytes. Ten-fold serial dilutions of the extract were made in phosphate-buffered saline. A total volume of 0.8 ml for each dilution was placed in an Eppendorf tube. A negative control tube (containing saline only) and a positive control tube (containing tap water) were also included in the analysis. Fresh sheep erythrocytes were added to each tube, (250 000 cells per tube) to give a final volume of 1 ml. Solutions were incubated at 37°C for 30 min and then all tubes were centrifuged for 5 min. The degree of hemolysis was determined by reading the optical density of the supernatant at 405 nm. Negative control tubes exhibited no hemolysis. All dilutions of the extract from 1:1 to 1:1000 exhibited a similar degree of hemolysis as the positive control. Humanfibroblast cells. Using a 96-well plate, 0.1 ml of a suspension of viable libroblast cells (L 929 ATCC) was mixed with serial dilutions of the extract to give a final volume of 0.2 ml per well. The serial dilutions ranged from 1:2 to 1:1024. Well contents were incubated at 37°C in 5% CO2 for 30 min and then washed twice with Hanks balanced

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salt solution (HBSS). A tetrazolium dye (MTT) was added to the washed wells which were reincubated at 37°C in 5% COz for 3 to 6 h. After lysis with SDS, 100 ~1 of acidic isopropanol was added and the optical density read at 570 nm. The cells remained viable at a 1:64 dilution of the extract. At a 1:32 dilution, approximately 64% of the cells remained viable after incubation for 30 min. 2.4. Assay for activity-directed isolation of antifungal principle For the bioassay-guided isolation of the active principle possessing antifungal activity the zone of inhibition was measured in a punch well diffusion assay on agar plates seeded with Ca. albicans. The strain of Ca. albicans used was kindly provided by Prof. F. Schoenknecht, University of Washington Medical Center, Seattle. Agar plates were prepared as follows. Nutrient agar (3 g, 1.5%, Difco) was dissolved in 100 ml distilled water, and autoclaved for 15 min. After cooling, the fungal culture was inoculated into this agar solution which was then poured into 4 Petri dishes. Assay samples were dissolved in 0.5 ml MeOH. 2.5. Isolation of active principle The 50% EtOH extract (250 ml) of the plant material was concentrated in vacua to remove EtOH, followed by partition between EtOAc (3 x 150 ml) and aqueous phase (130 ml). The aqueous fraction showed positive bioassay results and was further extracted with CHCIJ (150 ml) and with CHC13/ MeOH (1: 1, 2 x 150 ml). The CHCls/MeOH fraction showed positive bioassay results and was concentrated to a small volume (30 ml). Upon standing at room temperature a yellow powder (50 mg) precipitated which showed bioactivity. This material was further purified by column chromatography. Fifteen fractions of 50 ml each were collected, and those showing positive bioassay results (fractions l-3) were pooled and concentrated to a small volume (5 ml). A pale yellow powder (10 mg) precipitated. It was dissolved in 10 ml of warm MeOH and decolorized with a small amount of charcoal. The solution was concentrated to 5 ml and kept at room temperature to give a white powder (9 mg) displaying antifungal activity.

X. He et al. 1 J. Ethnopharmacol. 43 (1994) 173-177

2.6.Chemical characterization and determination of structure

Melting points were determined on a Melt-Temp apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-370 digital polarimeter. Column chromatography was carried out in a 220 x 18 mm glass column (silica gel, 100-200 mesh, Sigma). A solvent mixture of CHC13/Me0H/Me2CO/H20 (3:3:2:0.5) was used as eluent. TLC was carried out on silica gel (E. Merck), developing with the same solvent mixture and visualizing compounds by exposure to iodine vapor. Analytical HPLC was performed on an RP-18 column (250 x 4.6 mm, i.d. 10 pm) using a Beckman Programable Solvent Module 116 and an ANSPEC L-3000 multi channel photo detector at 210 mm. The mobile phase was MeOH/HzO (9: 1). ‘H- and 13C-NMR spectra were recorded in pyridine-d5 on an IBM AF-300 FT-NMR spectrometer. Data are reported as follows: chemical shift (ppm), integration, multiplicity, coupling con-

stants (Hz), The fab mass spectra were obtained with a VG 70 SEQ mass spectrometer operating at 70 eV. Compound 1 (Fig. 1): Colorless powder from MeOH, mp. 259-261”C, [cu]25 p -45.40 (C5H5N, c = 1.29). hrfabms: 1034.5297 (CSOHs2022).fabms, m/z (rel. int): 1073 [M + K]+ (10.8), 1057 [M + Na]+ (40.2), 1035 [M + H]+ (30.6), 903 [M + H 132]+ (20.8), 741 [M + H - 132-162]+ (30-O), 579 (C33H5508) [M + H - 132 - 2 x 162]+ (100.00). ‘H-NMR: 6 0.67 (6H, m, 18-Me, 27-Me), 0.83 (3H, s, 19-Me), 1.13 (3H, d, J = 6.8 Hz, 21-Me), 4.85 (lH, d, J = 7.1 Hz, H-l of glu), 5.15 (lH, d, J = 7.8 Hz, H-l ofglu),5.2(1H,d, J=7.5Hz,H-1 ofgal),5.54 (lH, d, J = 7.4 Hz, H-l of xyl). r3C-NMR: 6 37.3, 30.0, 78.7, 34.9, 44.8, 29.0, 32.5, 35.4, 54.5, 35.9, 21.4, 40.2, 40.8, 56.5, 31.9, 81.2, 63.0, 16.6, 12.4, 42.0, 15.0, 109.2, 32.2, 29.0, 3o.b, 66.9, 17.3 (aglycone, C-l to C-27), 102.6,81.2,73.2,79.9,76.2, 60.7 (galactosyl C-l to C-6), 104.8,70.5,87.0, 70.5, 77.6, 62.6 (glucosyl C-l to C-6), 105.0, 75.7, 78.7,

1 OH

Fig. 1. Compound

175

1: cantalasaponin-3.

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71.2,78.7,63.2(glucosylC-l’toC-6’), 77.6, 70.8, 67.3 (xylosyl C-l to C-5).

Ethnopharmaeol.

1048,751,

3. Results and discussion The antifungal activity of the aqueous ethanol extract of the dried leaves of S. nigrescens was assessed in vitro against three organisms. The MID of the extract was 1:152 for Cr. neoformans and 1:256 for Ca. albicans; against A. fumigatus (dispersed in Tween 80) no antifungal activity was observed. Fungicidal activity was demonstrated by exposing Cr. neoformans to the extract for different lengths of time, then plating aliquots and counting colony numbers after 48 h of incubation. The rate of Cr. neojixwans colony reduction at MID concentrations of extract was rapid. No colonies grew on SAB agar from the inoculum that had been exposed to 1:256 extract for 6 h. No colonies grew after 4 h exposure to the 1:128 dilution. Cytotoxicity of the extract was examined against sheep erythrocytes and human tibroblastoma cells. Hemolysis of the erythrocytes was observed at all dilutions of the extract ranging from 1:1 to 1:1000 to a similar degree as in a positive control of tap water, whereas the negative control containing only saline exhibited no hemolysis. The fibroblastoma cells remained viable at a 164 dilution of the extract. At a 1:32 dilution approximately 64% of the cells remained viable after incubation for 30 min. It is not surprising that the extract demonstrates hemolysis of sheep erythrocytes at dilutions down to 1:1000. This assay of cytotoxic activity is extremely sensitive to a wide range of chemical compounds and this effect of the extract may be due to any number of components within the preparation. Reevaluation of the active antifungal principle in the extract will be necessary to determine if the hemolysis is due to the compound itself or some other constituent. The libroblast analysis demonstrates that the extract is not cytotoxic to human cells within the minimum inhibitory dilution range. This indicates that the active ingredient of the extract may have potential use as a systemic agent in the treatment of opportunistic fungal infections in humans. Bioassay-guided fractionation of the extract led

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to the isolation of a single compound (1) responsible for the antifungal activity. Compound 1 was insoluble in H20, EtOH, CHCls or EtOAc, sparingly soluble in warm MeOH, very soluble in DMSO and pyridine. It showed one spot on silica gel TLC (R, 0.6) and one peak on HPLC (fret 2.5 min). The compound was found to have a molecular weight of 1034, as directed by the pseudomolecular ions at m/z 1073,1057,1035 corresponding to [M + K]+, [M + Na]+ and [M + H]+ ions, respectively in its fabms spectrum. By hrfabms the molecular formula was established as CsoHszOu. The ion peaks at m/z 903, 741, 579 arise from the loss of terminal pentose and hexose, respectively; they correspond to [M + H - xylose]+, [M + H - xylose - glucose]+, [M + H - xylose - 2 x glucose]+. The mass spectra were in close agreement with those for a compound, cantalasaponin-3, reported in the literature. This compound had been isolated from the unrelated plant, Agava canfala (Pant et al., 1986), although no biological activity was reported. Subsequently, the same authors (Pant et al., 1987) reported the isolation of additional cantalasaponins from A. cantala, one of which, cantalasaponin-7, showed weak molluscicidal activity. Although an authentic sample of cantalasaponin could not be procured, the identity of the two compounds was firmly established by a comparison of the ‘H- and “C-NMR spectra and the optical rotation of compound 1 with the data reported in the literature (Pant et al., 1986). Triterpenoid glycosides (Mahato et al., 1988) have been reported to have a variety of biological activities (Pfandler and Stoll, 1991), but so far very few have exhibited antifungal activity. In view of the increased incidence and broader spectrum of fungal infections and the scarcity of agents currently available to the physician, there is an evident need for the development of new and more effective antifungal drugs. Hence, the discovery of the fungicidal activity of cantalasaponin-3 is of obvious interest. The compound is evidently not cytotoxic to human tibroblastoma cells at effective antifungal doses. Hemolysis may be an obstacle to its systemic use, depending on whether the observed hemolytic activity of the extract resides in cantalasaponin-3 or in some other constituents. Lack of material prevented us from resolving this question.

X. He et al. /J.

Elhnopharmacol. 43 (1994)

Acknowledgements This work was supported by grants from the National Institutes of Health (AI 20264 to HGF) and from Tecna Corporation, San Bernardino, CA. We are indebted to Prof. F. Schoenknecht and his collegues at the UW Medical Center for useful advice and assistance in the establishment of the antifungal bioassy.

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vaginitis in Guatemala and clinical trial of a Solanwn nigrescens preparation. Journal of Ethnopharmacology 22, 307-313. Ckxes, A., Giron, S.M., Alvarado, S.R. and Tort-es, M.F. (1987) Screening of antimicrobial activity of plants popularly used in Guatemala for the treatment of dermatomucosal diseases. Journal of Ethnopharmacology 20, 223-237.

Mahato, S.B., Sarkar, S.K. and Poddar, G. (1988) Triterpenoid Saponins. Phytochemistry 27, 3037-3067. Pant, G., Sati, O.P., Miyahara, K. and Kawasaki, T. (1986) A spirostanol gfycoside from Agave canraia. Phytochemislry 25, 2895-2896.

References

Pant, G., Sati, O.P., Miyahara, K. and Kawasaki, T. (1987) Search for molluscicidal agents: saponins from Agave cantala. International Journal of Crua’e Drug Research 25,35-38.

Giron, L.M., Aguilar, GA., Cziceres, A. and Arroyo, G.L. (1988) Anticandicidal activity of plants used for the treatment of

Pfander, H. and Stall, H. (1991) Terpenoid glycosides. Natural Product Reports 8, 69-95.