Identification of Antifungal Compounds from Ferula persica. var. persica.

Abstract

In this investigation, the antifungal activities of chloroform and water extracts of Ferula persica. var. persica. Willd. roots were studied using conventional disk diffusion method. The chloroform extract showed antifungal activity at concentrations tested. Bioassay-guided fractionation study of the chloroform extract by preparative thin-layer chromatography (PTLC) detected two antifungal fractions that were effective against some fungi. Using conventional spectroscopy methods, the active fractions were identified as t-butyl 3-[(1-methylpropyl)dithio]-2-propenyl malonate (persicasulfide A) (1) and t.-butyl 3-[(1-methylthiopropyl)dithio]-2-propenyl malonate (persicasulfide B) (2), recently isolated from Ferula persica. var. latisecta.. These compounds showed the most potent antifungal activity with MICs of ≤ 62.5 µg/ml against filamentous fungi.

Keywords:

• Antifungal
• Ferula persica
• persicasulfide A
• persicasulfide B
• roots
• sulfur compounds

Introduction

Life-threatening fungal infections have increased dramatically in immunocompromised patients during the past decades (Ghannoum, 1997). Antimycotic agents currently available are limited either by their low effectiveness or by their toxicity due to the prolonged treatments often required, which can become expensive (Frexia et al., 1998). Therefore, there is a constant need for more effective fungicidal agents. In our program searching for antifungal agents, we have selected an extract prepared from the roots of Ferula persica. var. persica. Willd. (Umbelliferae). Members of the genus Ferula. are widespread throughout central Asia. The roots of F. persica. are used for the treatment of diabetes in folk medicine (Afifi & Abu-Irmaileh, 2000). No report on the antifungal effect of this species has been published in literature. This paper reports the bioactivity-guided fractionation of the chloroform extract of F. persica. var. persica. roots, leading to the isolation of two antifungal active compounds (Fig. 1). The minimum inhibitory concentrations (MICs) of each compound and a reference drug (fluconazole) were determined on eight species of microorganisms: Aspergillus flavus. (PTCC 36006), Aspergillus fumigatus. (PIM 112), Aspergillus niger. (PIM), Candida albicans. (ATCC 14053), Cryptococcus neoformans. (Kf 33), Saccharomyces cerevisiae. (PLM 454), Neurospora crassa. (PLM 454), and Tricophyton mentagrophytes..

Figure 1 Structures of persicasulfide A (1) and persicasulfide B (2).

Materials and Methods

Plant collection

Ferula persica. Willd. var. persica. was collected in May 2002 from north of Tehran, Iran, at an altitude of 2000 m and was identified. A voucher specimen of the plant (no. 6523-TEH) was deposited in the Herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences (Iran).

Extraction, chromatography, and spectroscopy

The roots of the plant were air-dried at room temperature and pulverized. The chloroform and water extracts were prepared by maceration of the powder for 72 h at room temperature. Bioassay-guided fractionation was carried out by preparative thin-layer chromatography (PTLC) on silica gel (60 F₂₅₄, Merck, Germany) using petroleum ether/ethyl acetate (2:1) as the solvent system (Iranshahi et al., 2003). The fractions were visualized under UV at 254 nm and eluted using chloroform. The pure compounds were identified using conventional spectroscopy. ¹H NMR and ¹³C NMR spectra were measured in CDCl₃ with tetramethylsilane (TMS) as an internal standard using a Varian 400 Unity plus. spectrometer (Aldrich Chemie, Steinheim, Germany).

Antifungal activity

The antimycotic activity of chloroform and water extracts of F. persica. roots were first studied against the following strains: Aspergillus niger. PIM, Aspergillus flavus. PTCC 36006, Candida albicans. ATCC 14053, and Cryptococcus neoformans. Kf 33 (Barry & Thornsberry, 1991). Test plates (diameter: 15 cm) were prepared with Sabouraud dextrose agar (SDA) medium and inoculated on the surface with a cell suspension in sterile dissolution of 0.9% saline in the case of yeast or with a spore suspension in Tanquay buffer in the case of filamentous fungi. In both cases, the concentration was adjusted to 10⁶ CFU/ml. Paper disks containing different concentrations of extracts (1, 2, 4, and 8 mg) were applied over the test plates. The diameters of the growth inhibition zones were measured after incubation at 30°C for 48 h. All the assays were carried out in triplicate. In order to identify antifungal fractions, approximately 5 mg of each TLC fractioned compound was dissolved in n.-hexane. The presence of antifungal activity was determined using disk diffusion bioassays with A. niger. PIM and 500 µg compound per disk. Disks containing n.-hexane were used as negative control in all experiments.

Disk diffusion susceptibility test

In order to investigate the MICs of the chloroform extract and its bioactive compounds, a collection of test organisms of fungi was used (see Table 2). All test strains were obtained from the Laboratory of Microbial Quality Control, Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences. Organisms were tested on SDA. Excess moisture was absorbed for 10 min prior to application of 6-mm paper disks containing twofold dilutions of chloroform extract and its active fractions in n.-hexane. The MIC was defined as the lowest drug concentration resulting in a clear zone of growth inhibition around the disk after conventional incubation period (Pettit et al., 2003). Paper disks containing different concentrations of fluconazole (Sigma Chemical Co, St. Louis, MO, USA) were applied over the test plates as comparative positive control.

Results and Discussion

Table 1 shows the antimycotic effect of F. persica. var. persica. extracts against test organisms. The chloroform extract of this species showed antifungal activity, whereas aqueous extract did not show any antimycotic activity at certain concentrations. TLC analysis of the chloroform extract showed at least 9 compounds, which were visualized under UV at 254 nm. The antifungal activities of the each of the fractions were tested for A. niger. by a disk diffusion method. The bioactivity-guided fractionation of this extract led to the isolation of two compounds (1, 2) (see Fig. 1) with similar activity. The active components of the root extract involved antifungal activity had R_f = 0.62 and R_f = 0.55 on PTLC, respectively. The structures of the compounds was confirmed by ¹H NMR, ¹³C NMR spectra. The spectral data (¹H NMR, ¹³C NMR) of these antifungal compounds revealed that these compounds were t.-butyl 3-[(1-methylpropyl)dithio]-2-propenyl malonate (persicasulfide A) and t.-butyl 3-[(1-methylthiopropyl)dithio]-2-propenyl malonate (persicasulfide B), previously isolated from Ferula persica. var. latisecta. (Iranshahi et al., 2003).

Table 1.. Antifungal activity of the chloroform and water extracts of Ferula persica. var. persica. roots.

	Growth inhibition zone diameter^a.
	Chloroform extract (mg)				Water extract (mg)
Organisms (strain number)	1	2	4	8	1	2	4	8
Aspergillus niger. (PIM)	12	14	17	19	–	–	–	–
Aspergillus flavus. (PTCC 36006)	10	12	15	17	–	–	–	–
Candida albicans. (ATCC 14053)	–	–	–	–	–	–	–	–
Cryptococcus neoformans. (Kf 33)	–	8	9	10	–	–	–	–

−, negative. ^a.Given in mm, mean value n. = 3.

Persicasulfide A (1): yellow oil. ¹H NMR (CDCl₃): δ = 0.98 (t., 3H, J. = 7.3 Hz, MeCH₂), 1.3 (d., 3H, J. = 6.8 Hz, MeCH), 1.53 (s., 9H, CH₃), 1.51–1.74 (m., 2H, MeCH₂CH), 2.8 (m., 1H, CHS), 2.9 (s., 2H, COCH₂CO), 4.58 (dd., 2H, J. = 6.4 Hz, 1.2 Hz, CH₂O), 5.97 (dt., 1H, J. = 14.8 Hz, 6.4 Hz, = CH─CH2), 6.35 (dt., 1H, J. = 14.8 Hz, 1.2 Hz, SCH =). ¹³C NMR (ppm): C₁ (11.4), C₂ (28.8), C₃ (48), C₄ (20), C₅ (132.9), C₆ (122.5), C₇ (63.7), C₈ (170.4), C₉ (44), C₁₀ (169.7), C₁₁ (79.2), C₁₂ (26.6).

Persicasulfide B (2): yellow oil. ¹H NMR (CDCl3): δ = 1.0 (t., 3H, J. = 7.3 Hz, MeCH₂), 1.53 (s., 9H, CH₃), 1.61–1.79 (m., 2H, MeCH₂CH), 2.2 (s., 3H, CH₃S), 2.9 (s., 2H, COCH₂CO), 3.7 (dd., 1H, J. = 8.8 Hz, 5 Hz, CHS), 4.6 (d., 2H, J. = 6.4 Hz, CH₂O), 5.97 (dt., 1H, J. = 15.2, 6.4 Hz, CH=CHCH2), 6.4 (d., 1H, J. = 15.2 SCH =). ¹³C NMR (ppm) C₁ (11.3), C₂ (27.7), C₃ (60.5), C₄ (14.5), C₅ (132.1), C₆ (123.4), C₇ (63.6), C₈ (170.3), C₉ (43.9), C₁₀ (169.6), C₁₁ (79.1), C₁₂ (26.5).

The inhibitory effect of these newly isolated compounds (Iranshahi et al., 2003) against numerous test organisms is shown in Table 2. As shown in Table 2, persicasulfide A and persicasulfide B represent the most potent antifungal candidates with MICs of ≤ 62.5 for filamentous fungi (Aspergillus spp.., T. mentagrophytes., and N. crassa.) or 1000 µg/ml for C. neoformans.. However, these sulfur-containing derivatives from F. persica. roots had no activity at 1000 µg/ml against the strains C. albicans. and S. cerevisiae. (Table 2). The crude extract of F. persica. exhibited weaker activity against all test strains of filamentous fungi or yeasts (MICs ≥ 250 µg/ml),and in particular against C. albicans. or S. cerevisiae. even at 0.8% concentration (i.e., 8000 µg/ml).

Table 2.. Minimum inhibitory concentrations (MICs, µg/ml) of the tested compounds after 2 days (n. = 3).

		MICs (µg/ml)
No.	Species	Chloroform extract	Persicasulfide A	Persicasulfide B	Fluconazole
1	Candida albicans. ATCC 14053	>8000	>1000	>1000	10
2	Aspergillus niger.PIM	250	<62.5	<62.5	10
3	Aspergillus fumigatus. PIM 112	500	62.5	62.5	>40
4	Aspergillus flavus. PTCC 36006	250	<62.5	<62.5	40
5	Trichophyton mentagrophytes.	500	62.5	62.5	20
6	Neurospora crassa.	250	<62.5	<62.5	ND
7	Cryptococcus neoformans. Kf 33	4000	1000	1000	40
8	Saccharomyces cerevisiae. PLM 454	>8000	>1000	>1000	10

ND, not determined.

Acknowledgments

This research was supported by a grant from the Pharmaceutical Sciences Research Center and Vice-Chancellor for Research, Tehran University of Medical Sciences, Tehran, Iran. We also appreciate scientific assistance from the Tehran University Working Group on Natural Products and Medicinal Plants (TUNPMP).