Antimicrobial and Antioxidant Activities of Coumarins from the Roots of Ferulago campestris (Apiaceae)

We report the isolation of several coumarins and the stereochemical assessment of some pyranocoumarins, as well as the antibacterial and antioxidant activities of the three most abundant ones (grandivittin, agasyllin and aegelinol benzoate) isolated from the roots of Ferulago campestris collected in Sicily and of the hydrolysis product (aegelinol). Aegelinol and agasyllin showed antibacterial activity against nine ATCC and the same clinically isolated Gram-positive and Gram-negative bacterial strains. At a concentration between 16 and 125 μg/mL both coumarins showed a significant antibacterial effect against both Gram-negative and Gram-positive bacteria. In particular the ATCC strains Staphylococcus aureus, Salmonella thypii, Enterobacter cloacae and Enterobacter earogenes (MIC = 16 and 32 μg/mL for aegelinol and agasyllin, respectively) were the most inhibited. Antibacterial activity was also found against Helicobacter pylori: a dose-dependent inhibition was shown between 5 and 25 μg/mL. The antioxidant activity of the coumarins was evaluated by their effects on human whole blood leukocytes (WB) and on isolated polymorphonucleate (PMN) chemiluminescence (CL), PMA-stimulated and resting.


Introduction
Ferulago campestris (Besser) Grec., (F. galbanifera (Mill) Kock. = Ferula ferulago L.), finocchiazzo, is an annual or perennial herb with small flowers that grows in the Mediterranean area. Previous phytochemical studies on the roots of F. campestris collected in Egypt revealed the presence of monoterpene coumarins and sesquiterpene lactones [1]. Some Ferulago species have been used since ancient times in folk medicines for their sedative, tonic, digestive and aphrodisiac properties and also in the treatment of intestinal worms and haemorrhoids. Moreover, they are used against ulcers, snake bites, as well as headache and diseases of the spleen [2] and the gums obtained by incision of the roots of several species are used as spices and drugs and their use as vermifuge and for carminative disorders has been reported [3]. In continuation of our research for biologically active compounds from Sicilian medicinal plant sources [4,5], we investigated the constituents of the roots of Ferulago campestris. Several coumarins were isolated and the most abundant ones were tested for their antibacterial and antioxidant activities.

Results and Discussion
Finely ground dried roots of Ferulago campestris (Besser) Grec. were extracted with petroleum ether and dichloromethane, and the extracts were subjected to column chromatography. From the petroleum ether extract, osthol (1, Figure 1), a previously known prenylated coumarin [6] was obtained. The structure of compound 1 was elucidated using 1D and 2D NMR experiments and this data, not previously reported, are given in the Experimental section.
Furthermore, three abundant coumarins 2-4 ( Figure 1) differing in the ester moieties, were isolated and identified. These compounds were shown to have a pyran ring fused with the coumarin nucleus formed through a six endo-trig cyclization involving a prenyl chain. In fact, they showed, in their 1 H-NMR spectra, the signals for methylene protons (H-1'a and H-1'b) and an oxygenated methine (H-2') [7]. The difference among them was the presence of a senecioyl, angeloyl or benzoyl ester group, respectively.
Consequently, we decided to determinate the absolute stereochemistry by mean of Mosher's [11] and Horeau's methods [12]. The Mosher esters were prepared by the standard procedure for esterification using the S acid chloride (S-MTPA-Cl) and the R acid chloride (R -MTPA-Cl). Then the 1 H-NMR spectra were recorded and Δδ = δ(S)-δ(R) values were determined, where δ(R) are the proton chemical shifts on the alcoholic portion of the R ester (6a) and δ(S) are the proton chemical shifts of the alcoholic portion of the S ester (6b). In accordance with the selection rules, we concluded that the absolute configuration of C-2' is R.

Protons δ(S) δ(R) ∆δ
With regard to the Horeau's method, compound 5 was esterified with 2-phenylbutyric anhydride (meso). The reaction was followed by polarimeter and α 1 = -1.34 (±0.029) and α 2 = -1.54 (±0.02) were determined. In accordance with the Horeau's rule [(α 1 -1.1 α 2 ) > 0], the absolute configuration of the C-2' was thus confirmed to be (R). Therefore we concluded that structure of compound 5 is as indicated in Figure 2: The investigation of dichloromethane extract resulted in the isolation of felamidin (7, Figure 3), an isomer of aegelinol benzoate, in which a prenyl chain gave a five exo-trig cyclization resulting in a coumarin with a tetrahydrofuran fused ring. The 1 H-NMR spectrum showed the presence of two methylene protons at δ H 3.30 (2H-1', m) and of a benzoyl residual at C-3'. Felamidin (7) was previously isolated from the roots of Ferulago isaurica [13].

Antibacterial activity
The observed combined resistances are shown in Table 1. Among the four coumarins tested only aegelinol (5) and agasyllin (3) showed any significant antibacterial activity. At a concentration between 16 and 125 μg/mL both coumarins showed a significant antibacterial effect against both Gram-negative and Gram-positive bacteria. In particular, the ATCC strains Staphylococcus aureus, Salmonella thypii, Enterobacter cloacae and Enterobacter earogenes were the most inhibited, showing a Minimum Inhibitory Concentrations (MIC) of 16 and 32 μg/mL for aegelinol and agasyllin, respectively. The antibacterial activity of both coumarins was higher against Gram-negative than Gram-positive bacteria as reported previously from other plants [14,15]. In particular, we found a remarkable activity against Salmonella thypii, which is responsible for severe infections and is very often resistant to conventional antibiotics. Comparing the coumarin activities against ATCC and clinical isolated strains, we found similar effects, although the clinical isolates, except one, were resistant to more than one reference antibiotic. Antibacterial activity was also found against Helicobacter pylori: a dose-dependent inhibition was shown between 5 and 25 μg/mL (Figures 4 and 5).  This last result was of interest because Helicobacter pylori is a Gram-negative, microaerophilic, shaped bacterium that is free living in the mucous layer of the human stomach. This bacterium is spread worldwide, with a frequency ranging from 25 % in developed to 90 % in developing areas, but not all infected people develop illnesses. Infection from this bacterium leads to different clinical disorders including chronic gastritis, peptic ulcer and gastric adenocarcinoma [16]. Helicobacter pylori induced disorders depending on the inflammatory response mediated by cytokines [17].

Chemiluminescence studies
We showed the antioxidant effects of the coumarins, grandivittin (2), agasyllin (3) and aegelinol (5) on the in vitro human PMN respiratory burst. The aegelinol benzoate (4) is inactive. The antioxidant activity of the coumarins was evaluated by their effects on human whole blood leukocytes (WB) and on isolated polymorphonucleate (PMN) chemiluminescence (CL) using concentration range between from 0.01 to 100 μg/mL.
Comparing the effects of coumarins on both PMN and WB chemiluminescence emission, we found that aegelinol is the most active, followed by agasyllin; grandivittin is less active, whereas aegelinol benzoate has no activity. The coumarins showed a dose-dependent and linear inhibitory activity on isolated PMN, as well as on WB CL emission PMA-stimulated and resting, showing a faster inhibition when stimulation was done. This could depend on the fact that the coumarins act like ROS scavengers and can also interfere with cellular activation mechanisms (membrane and/or cytoplasmic receptors or enzymes) [18]. The unactivated cells could be less sensitive to this down-regulation than PMAstimulated ones. Finally, our data represent an answer to the continual demand for new antibiotics and antioxidants for the continuous emergence of antibiotic-resistant strains and the growing interest in the substitution of synthetic antioxidants with natural ones.

Experimental Section
General NMR spectra were recorded in CDCl 3 on a Bruker Avance series 300 MHz spectrometer. MS spectra were recorded on Shimadzu GCMS-QP2010 Plus spectrometer. Optical rotations were

Spectroscopic data for compounds 2-5
The physical and spectroscopic data of these compounds were in complete agreement with those reported in the literature [7].
NMR data for compound 6a. 1  Spectroscopic data for felamidin (7) The physical and spectroscopic data of this compound were in complete agreement with those reported in literature [13]. Preparation of coumarins for antibacterial assays: The coumarins were added with 5×10 −2 M stock solution in DMSO. They were diluted from 0.01 to 1000 μg/mL concentrations in sterile physiological tris buffer (pH 7.4, 0.05 M) [19] just before use.

MIC determination:
The antibacterial activity was expressed as MIC (minimum inhibitory concentration) values. The MIC was defined as the lowest concentration able to inhibit any visible bacterial growth. Bacterial strains were grown on Mueller-Hinton (MH) agar plates (DIFCO, Detroit, MI) and suspended in MH broth (DIFCO). The MIC values were measured using the broth-dilution method (MH broth) [20]. Each coumarin was tested in triplicate, the experiment was performed four times.
Anti-Helicobacter pylori activity: Helicobacter pylori was cultured as elsewere reported [21], inoculated on a Brucella Agar plate containing 10% sterile defibrinated sheep blood, 5 mL Vitox (Pharmanex) and 2 mL Skirrow (DIFCO) (medium supplements) and cultured at 37 °C for 72 h. The bacterial colonies were collected and diluted to 107 colony forming units (CFU)/mL with 0.9% NaCl saline solution. The coumarins were diluted with DMSO and then added to the liquid culture medium. The culturing medium was made of 14 g Brucella broth, 1.25 mg FeCl 3 ·6H 2 O, 500 mL bi-distilled H 2 O, 25 mL fetal calf serum, 5 mL Vitox and 2 mL Skirrow. Each bacterial suspension was added to the culturing medium obtaining 106 CFU/100 ml/well density. The mixture was incubated at 37 °C for 72 h [22,23]. Control cultures were made culturing the bacterium without and with metronidazole at 0.5 μg/mL.
Chemiluminescence studies: Luminol-dependent chemiluminescence assay represents a simple, rapid and sensitive method to identify compounds with antioxidant and anti-inflammatory activity [24].
Preparation of coumarins for chemiluminescence assays: The coumarin solutions were added with 5×10 −2 M stock solution in DMSO and, immediately before being used, diluted from 0.01 to 100 μg/mL in modified Krebs-Ringer phosphate medium (KRP) [24].
Luminol-dependent chemiluminescence assays: Chemiluminescence assays were performed following the protocol described by De Sole et al. [24] and using an automatic luminometer (Autolumat LB 953, Berthold, Wildbad, Germany). Whole blood and isolated PMN CL emission was evaluated within 3 h after venipuncture. The reaction mixtures were prepared in 4 mL polypropylene vials. For the chemiluminescence test on resting leukocytes, each vial contained 100 μL of the various coumarin concentrations, 100 μL of diluted whole blood (WB, 1:100) or isolated PMN suspension (0.5×10 6 /mL) and sufficient KRP to yield a final volume of 1.0 mL. In activated leukocyte evaluation, samples with 100 μL of 1.5 μM phorbol myristate acetate (PMA; Sigma Chemicals Co.) were prepared in the same final volume. To exclude the possibility that the coumarins quenched the activated luminol, the following controls were included: luminol and coumarins at the various concentrations in presence or absence of PMA. The reaction temperature was 37 °C and the resulting light emission on each vial was recorded for 0.5 s, over a 90 min period. CL emission was evaluated as peaks. All measurements were performed in triplicate and expressed as percent inhibition. The effects of coumarins on phagocyte viability were determined by the trypan blue dye exclusion test [27].

Statistical analysis
The following statistical protocol was established: • Construction of dose-response curves: • Regression analysis with evaluation of the linearity of dose-response curves (dose logarithm as independent variable and CL percent inhibition as dependent) was drawn for each cumarin. If response regression was linear, the angular coefficient significance was calculated to verify the null hypothesis of an angular coefficient equal to zero.
• Comparison between dose-response curves: • When the curves of the coumarins were linear, the analysis of variance applied to regression (ANOVA-R) was performed to compare the angular coefficients; if one or more curves were nonlinear, analysis of variance was carried out by comparing the coumarins at each concentration.
The results of all experiments were expressed as mean ± S.D. In all tests, values of p < 0.05 were regarded as significant.