Polyphenol derivatives from bioactive butanol phase of the Tunisian narrow-leaved asphodel (Asphodelus tenuifolius Cav., Asphodelaceae)

1 Laboratory of Application of Resources and Natural Substances Chemistry to the Environment, Faculty of Sciences of Bizerta, 7021, Jarzouna, Bizerta, Tunisia. 2 Research Unit 12-04, Applied Chemistry and Environment, Faculty of Sciences of Monastir, 5000 Monastir, Tunisia. 3 Laboratory of Botany and plant Ecology, Faculty of Sciences of Bizerta, 7021, Jarzouna, Bizerta, Tunisia. 4 Laboratory of Bactereology, University Hospital F. Bourguiba, 5000 Monastir, Tunisia. 5 Laboratory of Parasitology-Mycology, University Hospital F. Bourguiba, 5000 Monastir, Tunisia. 6 Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, I-80131, Napoli, Italy.


INTRODUCTION
The use of medicinal plants to treat illness and to preserve human health presumably predates the first recorded history.In the modern era, chemists and biologists are highly interested in studying the medicinal potential of natural extracts aiming at the discovery of useful drugs.
As a contribution to the chemical and biological studies of Medicinal plants growing in Tunisia, the present work deals with the investigation of the narrow-leaved asphodel, Asphodelus tenuifolius Cav.(Asphodelaceae), one of the seven species within the Asphodelus L. genus grown in Tunisia (Cuénod et al., 1954).Some authors judged A. tenuifolius Cav. to be either a variety or a subspecies of the fistulosus asphodel (Asphodelus fistulosus var.tenuifolius (Cav.)Baker (Le Floc'h et al., 2010) or A. fistulosus subsp tenuifolius (Cav.)Trab).Later, it has been shown, on the basis of biometric and genetic criteria, that A. tenuifolius and A. fistulosus L. are clearly two independent species (Ruíz Rejón et al., 1990;Díaz Lifante, 1991).
A. tenuifolius Cav. is an annual or a biennial plant with fibrous roots, a low stem, all leaves radical, fistulous and narrow with a length of 3 to 7 cm.Flowers are clearly bell-shaped and fructiferous pedicels are articulated (Cuénod et al., 1954).This small plant is widely used for various culinary purposes.
The leaves are either boiled or cooked in oil, the seeds are crushed and mixed with flour to make bread and the young shoots are added raw to food to enhance the taste.This plant is little appreciated as pasture.In Egypt, the seeds are reported to be diuretic and are eaten with yogurth (A guide to Medicinal Plants in North Africa, 2005).In vitro antimicrobial activities of crude extracts from Indian-herbal medicinal A. tenuifolius have been studied.Benzene extract exhibited good antibacterial activity against Proteus mirabilis and a very good susceptibility to Klebsiella pneumonia and Pseudomonas aeruginosa (Panghal et al., 2011).
Antifungal activities of petroleum ether, benzene, chloroform, ethyl acetate and methanol extracts of the Indian A. tenuifolius were tested against three fungal species showing potential antimicrobial activities (Menghani et al., 2012).Previous phytochemical studies led to the isolation of Asphorodin 1, a triterpenoidal diglycoside showing a potent inhibitory activity against the enzyme lipoxygenase (LOX) (Safder et al., 2009).In the frame of our ongoing project aimed at contributing to the valorization of the Tunisian flora by searching new natural products possessing beneficial biological activities, the present work has been focused on the chemical and biological investigation of butanol phase obtained from the organic extract of A. tenuifolius Cav.growing spontaneously in Tunisia.Thus, we report here the isolation and the characterization of trans N the isolation and the Faidi et al. 551 characterization of trans N -feruloyltyramine (1), luteolin (2), luteolin-7-O-β-D-glycopyranoside (3), apigenin (4) and chrysoeriol (5), isolated for the first time from butanol extract of A. tenuifolius Cav.

Plant
Aerial parts of the narrow-leaved asphodel plant were collected during flowering period at the beginning of March, 2011 from the Kairouan region, center of Tunisia.The plant was identified by Dr. Ridha El Mokni, a member at the Laboratory of Botany and plant Ecology, Faculty of Sciences of Bizerta, Jarzouna, Bizerta, Tunisia, where a voucher specimen [AT (TC, Kair.) 2011-017] has been deposited.

General material
1 H (400 MHz) and 13 C (100 MHz) nuclear magnetic resonance (NMR) spectra were measured on a varian Inova spectrometer.Chemical shifts were referenced to the residual solvent signal (CDCl3: δH 7.26, δC 77.0 or CD3OD: δH 3.34, δC 52.0).Homonuclear 1 H connectivities were determined by the COSY experiment.Electrospray ionization mass spectrometry (ESI-MS) spectra were performed on a LCQ Finnigan MAT mass spectrometer.Medium pressure liquid chromatography was performed on a Büchi apparatus using a silica gel (230 to 400 mesh) column; HPLC were achieved on a Knauer apparatus equipped with a refractive index detector.LUNA (Phenomenex) columns were used.

Isolation and identification
Dried aerial parts of A. tenuifolius Cav.(1.5 kg) were extracted with methanol at room temperature three times to afford 150 g of crude extract after evaporation in vacuum of the solvent.The methanol extract was dissolved in water then successively extracted with CH2Cl2, EtOAc and butanol.Butanol phase (8.5 g) was subjected to column chromatography packed with silica gel 60 eluted with a solvent gradient of increasing polarity from hexane/EtOAc 1:1 to EtOAc and then to methanol.79 fractions of 250 ml were collected and then joined into 27 groups (A1 to A27) on the basis of analytical thin-layer chromatography.

Isolation of N-feruloyltyramine (1)
The group A19 (171 mg) was subjected to HPLC separation on normal phase using a mixture of hexane/EtOAc (35:65) and affording eight subfractions (F1 to F8).Subfraction F5 (7 mg) was further purified via HPLC using same solvent to afford 1 mg of compound 1 as a white solid.
*Corresponding author.E-mail: saoussenhammami@voila.fr.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Isolation of luteolin (2)
Group of fractions A17 (107 mg) provided 20 mg of compound 2 as a yellow amorphous powder after precipitation in Ethyl acetate.

Isolation of acetylated derivative of luteolin glycoside (3)
70 mg from group A26 (158 mg) were acetylated using acetic anhydride in pyridine at room temperature.The acetylated fraction thus obtained was chromatographed on normal phase HPLC eluted with n-hexane/EtOAc (4:6) mixture (3 ml/min) to give eight subfractions G1 to G8.The most polar one (G8) was further subjected to a second HPLC purification eluted with n-Hexane/EtOAc (2:8) mixture (0.6 ml/min) to afford 1.1 mg of compound 3 as a yellow amorphous powder.

Antifungal assay
Disc diffusion method was employed during the preliminary antifungal screening of crude extracts.Test strains suspension of 1 Mc Farland was prepared from fresh cultures.Plates were aseptically streaked with the tested micro-organisms and allowed to dry for a few minutes.Sterile filter paper Whatman discs (6 mm of diameter) were impregnated with 20 µl of crude extract solution, were then aseptically placed on the inoculated Sabouraud chloramphenicol plates.The plates were therefore incubated during 24 h at a temperature of 37°C.Tests were carried out in triplicates.
The presence of a clear circular zone around the sample impregnated disc was used as an indicator of antifungal activity.
The results were recorded by measuring inhibition diameter zones in mm.Disc impregnated with the solvent was used as negative control.For comparative purposes, standard drug fluconazole (40 µg/disc) was used as a positive control (Hammami et al., 2013).

Determination of minimum inhibitory concentration
Overnight broth cultures were adjusted to yield approximately 1 × 10 6 CFU/ml of bacteria.The Minimal inhibitory concentrations (MIC) were determined on the basis of the broth microdilution assay using liquid cultures in 96 well microplates from measuring bacterial growth.A sample from each extract (200 μl) was added to four wells of the first column of each plate and then serially diluted with dimethyl sulfoxide (DMSO) (10%) solution as doubling dilutions up to the well number eight of first column dilution factor (1:1).Each well was then inoculated with 50 ml of inocula.Four wells of one column from each plate were inoculated just with conidial suspension without any extract (positive control).Broth medium was used as a negative control.The microplates were incubated for 24 h at 37°C (clinical and Laboratory Standard Institute., 2008;Mousavi and Raftos, 2012).

Antifungal and antimicrobial effects of crude extracts
The three crude phases of the organic extract (methylene chloride, ethyl acetate and butanol) from aerial parts of A. tenuifolius Cavan.were evaluated for antimicrobial activity against some intestinal and skin bacterial pathogens (S. aureus, E. faecalis, E. coli and P. aeruginosa) and four Candida species: C. albicans, C. prapsilosis, C. glabrata and C. krusei using dilution and disc diffusion methods, respectively.The results illustrated in Table 1 indicated that butanol extract produced the strongest activity against the Gramnegative bacteria E. coli and P. aeruginosa (MIC = 0.729 and 0.156 mg/ml -1 , respectively).Also the other phases of the organic extract from aerial parts of A. tenuifolius Cavan.showed sig-nificant antimicrobial activity against P. aeruginosa, well known for its involvement in nosocomial infections and frequent resistance to antibiotics.
The disc diffusion antifungal assays showed that four Candida species were very sensitive to the polar butanol extract solution at a concentration of 50 mg/ml, thus inhibition zones were between 14 for C. albicans and 20 mm for C. krusei.Most important is the high bioactivity of butanol polar extract against C. krusei which seems more significant than that of the standard antibiotic fluconazole (Table 2).Overall, antimicrobial tests suggested that active compounds are mainly polar and dissolve in butanol.These results are consistent with those of some previous studies, indicating that the inhibitory activity is pathogen specific anddependent on the solvent, concentration of the crude drug and also on rate of diffusion and that alcohols are the most appropriate solvents for extraction of antimicrobial substances (Ahmed et al., 1998;Moorthy et al., 2013).
Encouraged by the results of antimicrobial tests, the butanol extract was subjected to extensive purification and five major polyphenols were isolated in the pure state and characterized through comparison of their spectroscopic data with those reported in the literature.
In compound 2, 1 H NMR spectrum of exhibited protons of ABX system at δ 7.40 (dd, J 1 = 8.8 Hz, J 2 = 2 Hz); 7.39 (d, J = 2Hz) and 6.91 (d, J = 8.8Hz) of 1,3,4-trisubstituted phenyl unit, one singlet at δ 6.55 attributed to proton H 3 of flavonoids and two meta-coupled doublets at δ 6.42 and 6.21 (J = 2 Hz) characteristic of protons H 8 and H 6 from A ring of 5,7-dihydroxyflavonoid.These NMR data were in accordance with luteolin skeleton (Figure 2) isolated earlier from A. fistulosus L. and now found for the first time in A. tenuifolius Cav.(Owen et al., 2003).Luteolin has been previously described for its anti-inflammatory effects and its high inhibitory activity against synthesis of both thromboxane and leukotriene (Odontuya et al., 2005).
In compound 3, luteolin glycoside has been identified via its acetylated derivative 3a (Figure 3). 1 H NMR spectrum displayed signals from luteolin skeleton acetylated on C 5 and a series of signals resonating between δ 4.00 and 5.40 ppm attributable to acetylated sugar moiety, whose signals and coupling constants indicated the presence of a glucopyranose unit.Thus, comparison of these data with those of the literature (Xizhi et al., 2011) allowed the identification of the structure of luteolin glucoside for the first time in A. tenuifolius Cavan.Luteolin and luteolin glucoside have been mentioned for their antidiabetic effects through inhibition of αglucosidase and α-amylase (Elhawary et al., 2011).
In  (Ersoz et al;2002) allowed us to assign the structure of the 5,7,4'-trihydroxyflavone apigenin to compound 4 (Figure 4).Antianxiety activity of apigenin have been previously studied by Suresh Kumar et al. (2006), indicating that this phenolic derivative exhibited significant anxiolytic activity in mice using elevated plus maze model of anxiety (Kumar and Sharma, 2006).
In compound 5, ESI-MS gave ion peak at m/z 299 attributable to pseudomolecular anion [M-H] in agreement with a molecular formula of C 16 H 12 O 6 .Analysis of its 1 H NMR spectrum revealed characteristic protons of one methoxy group at δ 3.98, the H-3 signal at δ 6.62 and aromatic protons at δ 6.45 (1H, d, J = 2 Hz); δ 6.20 (1H, d, J = 2 Hz); δ 6.95 (1H, d, J = 8.8 Hz); δ 7.52 (1H, d, J = 8 Hz) and δ 7.50 (1H, brs).These data pointed to the structure of a methylated derivative of luteolin 2. Comparison of these data with those of flavonoids skeletons (Kang et al., 2010) allowed to propose the structure of chrysoeriol (Figure 5) isolated for the first time from A. tenuifolius Cav.anti-infective agents is strongly needed.In the frame of our research program aimed at exploiting the potential of Tunisian endemic flora, we have demonstrated that extracts obtained from the spontaneous plant A. tenuifolius Cav., widely used as culinary ingredient, has a consistent activity against some bacterial and fungal strains.The analysis of the considerably potent antimicrobial butanol phase revealed that it was mainly composed by polyphenols and in this paper we have described in detail those described for the first time from this species.

DISCUSSION
Flavonoids have been reported to possess many biological and medicinal activities including enzyme inhibition, anti-inflammatory, cytotoxic-antitumor and others.However, flavonoid-rich plants have also been extensively used for their antimicrobial activities (Cushnie and Lamb, 2005) and the best example is given by propolis, whose antimicrobial (antibacterial and antifungal) properties have been unambiguously attributed to its flavonoid content (Grange and Davey, 1990).Several hypotheses have been made for the mechanism of antibacterial action of flavonoids but the inhibition of nucleic acid synthesis and the alteration of the membrane function seem to be the better demonstrated mechanisms (Cushnie and Lamb, 2005).Although we have not separately tested the antimi-crobial activity of the flvonoids isolated from A. tenuifolius Cav., on the basis of the above considerations, their involvement in the determination of the activity of the extract seems very likely.On the other hand, it is unlikely that a single flavonoid would be the sole responsibility of the evidenced activity, while the entire flavonoid fraction, exerting a combined and possibly synergistic effect, can be identified as the active part of the plant.

Conclusion
On the basis of the data reported in the present study and those in the literature, five phenolic derivatives were isolated for the first time from antimicrobial bioactive butanol extract of the narrow-leaved asphodel (A.tenuifolius Cav., Asphodelaceae) growing spontaneously in Tunisia.The compounds were identified as follows: trans-N-feruloyltyramine (1), luteolin (2), luteolin-7-O-β-Dglycopyranoside (3), apigenin (4) and chrysoeriol (5).These compounds may be mainly responsible for the antimicrobial activity of the butanol extract.However, further studies into the activity of pure isolated compounds are needed to evaluate the potential health and food protecting benefits.

Table 1 .
MIC of crude extracts from aerial parts of Asphodelus tenuifolius Cav. using the dilution assay.

Table 2 .
Antifungal activity of organic extracts from aerial parts of A. tenuifolius Cav. using the disc diffusion method.