Doi:10.1016/j.phytochem.2006.10.009

Saponins from Allium minutiflorum with antifungal activity Elisa Barile a, Giuliano Bonanomi b, Vincenzo Antignani b, Behzad Zolfaghari c, S. Ebrahim Sajjadi c, Felice Scala b, Virginia Lanzotti a,* a Dipartimento di Scienze e Tecnologie Agroalimentari, Ambientali e Microbiologiche, Universita` del Molise, Via F. De Sanctis, 86100 Campobasso, Italy b Dipartimento di Arboricoltura, Botanica e Patologia vegetale, Universita` degli Studi di Napoli ‘‘Federico II’’ Via Universita` 100, 80055 Portici Napoli, Italy c Department of Pharmacognosy, Isfahan University of Medical Sciences, Hezar Jerib Avenue, 73461 Isfahan, Iran Received 31 July 2006; received in revised form 4 October 2006 Three saponins, named minutoside A (1), minutoside B (2), minutoside C (3), and two known sapogenins, alliogenin and neoagigenin, were isolated from the bulbs of Allium minutiflorum Regel. Elucidation of their structure was carried out by comprehensive spectroscopicanalyses, including 2D NMR spectroscopy and mass spectrometry. The structures of the new compounds were identified as (25R)-furost-2a,3b,6b,22a,26-pentaol 3-O-[b-D-xylopyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D-galactopyranosyl] 26-O-b-D-glucopyr- anoside (1), (25S)-spirostan-2a,3b,6b-triol 3-O-b-D-xylopyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D-galactopyranoside (2),and (25R)-furost-2a,3b,5a,6b,22a,26-esaol 3-O-[b-D-xylopyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D-galactopyranosyl] 26-O-b-D-glucopyranoside (3). The isolated compounds were evaluated for their antimicrobial activity. All the novel saponins showed a sig-nificant antifungal activity depending on their concentration and with the following rank: minutoside B > minutoside C ) minutoside A.
No appreciable antibacterial activity was recorded. The possible role of these saponins in plant–microbe interactions is discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Allium; Saponins; Furostanol-type; Spirostanol-type; Minutosides; Antifungal activity; Structure–activity relationships A–C (1–3), along with the known sapogenins, alliogeninand neoagigenin. The stereostructure of these compounds Saponins are a major family of secondary metabolites was elucidated by extensive NMR techniques and chemical that occur in a wide range of plant species methods. Moreover, we evaluated the possible involvement ). These compounds, called phytoanticipins of these saponins in resistance of A. minutiflorum to ), are present constitutively in plants and seem to be involved in plant disease resistance because of their well-known antimicrobial activity In our work on the discovery of bioactive saponins from Allium species (), we examined Allium minu- Bulbs of A. minutiflorum were air dried under controlled tiflorum Regel. This species is an Iranian bulbous perennial temperature (22–25 °C) and exhaustively extracted with plant known as wild onion and used for food preparation.
hexane, CHCl3, CHCl3–MeOH (9:1) and MeOH. Both We isolated and determined the structure of three new CHCl3–MeOH (9:1) and MeOH extracts were separated furostanol and spirostanol saponins, named minutosides by MPLC and HPLC techniques, affording the new com-pounds minutoside A (76.5 mg kgÀ1), B (83.5 mg kgÀ1), and C (9.3 mg kgÀ1), together with the sapogenins neoagi- Corresponding author. Tel.: +39 0874 404649; fax: +39 0874 404652.
E-mail address: (V. Lanzotti).
genin (99.0 mg kgÀ1) and alliogenin (2.1 mg kgÀ1).
0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.10.009 E. Barile et al. / Phytochemistry 68 (2007) 596–603 Minutoside A (1, isolated as an amorphous solid in relatively high yield, showed a molecular formula 50H84O25, deduced by high-resolution FAB MS mea- surements, and confirmed by 13C NMR data ( indicated a glycoterpene nature of the com- pound. In fact, the 1H NMR spectrum showed two tertiary methyls (d0.87 and 1.09), two secondary methyls (d0.98 and 1.02), and four anomeric (d4.25, 4.40, 4.63 and 4.95) in addition, a diagnostic signal at d 113.9, indicating the presence of a hemiacetal carbon and suggesting a furostane Chart 1. Chemical structures of minutoside A (1), minutoside B (2), and Combined analysis of 2D COSY and HOHAHA spectra of 1 allowed to detect six spin systems, two belonging toaglycone moiety, and the remaining four to four sugar res-idues. Consequently, each proton has been related to the directly bonded carbon through a HSQC spectrum.
1H NMR data of the aglycone portions of minutosides A (1), B (2), and C Concerning the aglycone, the first spin system started from ring A and extended up to ring E protons, while the second spin system included the side chain protons (C-23–C-27). The HMBC cross peaks, reported in allowed to connect the two spin systems through the qua- ternary C-22 and to build up the structure of the aglycone moiety as furostan-2,3,6,22,26-pentaol. The diaxial cou- pling between H-3 and H-5, detected in a ROESY spec- trum (indicated a cis orientation among them and a b-configuration of the hydroxyl at C-3. The a-orientation C NMR data of the aglycone portion of 1, 2, and 3 (125 MHz, CD3OD) E. Barile et al. / Phytochemistry 68 (2007) 596–603 Table 31H and 13C NMR data of the Sugar Portion of 1 and 2 (data extracted from 1), and 3 (500 MHz and 125 MHz, CD3OD) 1 with the usual B/C trans, C/D trans, D/E cis, and C- On the basis of those data, the stereostructure of the aglycone has been determined as depicted in formula.
Finally, the stereochemistry at C-22 has been assigned as a by accurate analysis of NMR data, and comparison withother compounds previously described Fig. 1. Selected HMBC (H ! C) and ROESY (H M H) correlations furostanol saponins (compound 1, left in aqueous solution overnight atroom temperature, gave rise to the equilibrated mixture of 2-OH has been determined by the ROESY cross-peak of of the two hemiacetals at C-22 (22a-OH and 22b-OH, H-2 with H3-19, while a b-orientation of 6-OH has been defined by the small coupling constants observed for H-6 Concerning the saccharide portion, the analysis started (d4.05, bs) indicative of its equatorial nature. The 25R ste- with the association of the four anomeric protons (d 4.25, reochemistry of the side chain was deduced by the reso- 4.40, 4.63 and 4.95) with the relevant anomeric carbon sig- nances of protons and carbons at C-25, C-26 and C-27, nals (d 104.9, 102.8, 104.5 and 104.3, respectively), through and by the vicinal couplings between H-25, and the two the HSQC experiment. The nature of the single monosac- H-26, in comparison with the literature data ( charides and their sequence has been determined by com- ). The following ROESY correlations (), H-11/ bined analysis of 2D COSY, HOHAHA, HSQC, and H3-19, H-11/H3-18, H-9/H-14, H-14/H-16, H-16/H-17, HMBC experiments. Starting from the anomeric proton and H-17/H3-21 completed the relative stereochemistry of of each sugar unit, all the proton signals within each spin E. Barile et al. / Phytochemistry 68 (2007) 596–603 system were recognized by COSY and HOHAHA spectra, C-5. In particular, this last carbon revealed to be an oxy- and then connected to the relevant carbon by HSQC spec- genated quaternary atom (d74.9) and, in addition, the a- trum. Then, HMBC and ROESY spectra gave key infor- orientation of OH-5 has been defined by the downfield mation on the glycosidic linkages. The data obtained shift observed for H1-a as compared to corresponding indicated that all sugars were in the pyranose form, three resonance of 1 (d 2.28 in 3 vs d 2.18 in 1). These evi- being hexoses and one pentose. Combined analysis of the dences defined the structure of 3 as (25R)-furost- coupling constants of each spin system, taken by 1H NMR spectrum or by 1H subspectrum of 2D HOHAHA, 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D-galactopyranosyl]- together with informations on the spatial proximity of pro- tons obtained by 2D ROESY (e.g. diaxial couplings Minutoside C possesses as structural feature an OH-5a between H-1I–H-3I and H-3I–H-5I) allowed the identifica- that is rare among furostanol saponins. Its aglycone moiety tion of two glucoses, one galactose, and one xylose. All has been recently found in two saponins isolated from A.
sugars were determined as b-anomers on the basis of the large JH-1–H-2 coupling constants. Furthermore, ROESY differing from minutoside C in the saccharide chain linked (H-4I/H-1II, H-3II/H-1III) and HMBC (C-4I/H-1II, C-3II/ H-1III) correlations permitted the sequence deduction of a Along with these compounds we isolated two sapoge- trisaccharide chain, that has been placed at C-3 of the agly- cone on the basis of ROESY (H-3/H-1I) and HMBC (C-3/ H-1I) cross peaks. Finally, the remaining b-glucose has The five isolated compounds were tested for their anti- been located at C-26 by considering the HMBC C-26/H-1IV microbial activity against a number of fungal and bacterial microorganisms. All saponins showed a significant anti- To confirm the nature of the sugar units and to deter- fungal activity depending on their concentration and, gen- mine their absolute configuration, 1 has been subjected to erally, the following rank was observed: minutoside acid hydrolysis (1 N HCl), followed by trimethylsilylation C > neoagigenin > alliogenin ) minuto- and GC analysis on a chiral column in comparison with side A These results indicate that antifungal both series of glucose, galactose, and xylose. By this proce- activity of minutosides B and C in some cases (i.e. Tricho- dure, the sugars were identified to belong to the commonly derma sp.) may be comparable to that of common natural found D-series. This procedure has been applied to all new isolated compounds. All these data indicated the structure of 1 as (25R)-furost-2a,3b,6b,22a,26-pentaol 3-O-[b-D- in the tested fungi, such as hyphal swelling and changes xylopyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b- in the rate of sporulation were evident (data not shown).
D-galactopyranosyl] 26-O-b-D-glucopyranoside.
Minutoside B, that is one of the most abundant in the plant Minutoside B (2, ), isolated as an amorphous tissue (83.5 mg kgÀ1), displayed the highest antifungal solid, constituted the major compound of the saponin frac- tion. It showed a molecular formula of C44H72O19, deduced On the basis of our results, some structure–activity rela- by high-resolution FAB MS measurements, and confirmed tionships within this class of antifungal agents were estab- by 13C NMR data ) which differed from 1 in lished. In particular, comparison of the chemical structure the absence of a hexose moiety. Analysis of 2D NMR spec- of the spirostanol saponin minutoside B with the corre- tra of 2 indicated the same trisaccharide chain as 1, com- sponding furostanol saponin minutoside A, indicated the posed by b-Gal, b-Glc, and b-Xyl. Differences between 1 importance of a spirostanol-type aglycone for the anti- and 2 were found in the aglycone portion, that in 2 has fungal activity. This is confirmed by the observation that been easily determined as neoagigenin by NMR analysis neoagigenin, the sapogenin of minutoside B, also showed a considerable activity. A positive effect of a hydroxyl group at C-5 was evident by comparing the activities of Finally, interglycosidic linkages have been determined minutosides A and C, which differ from each other by by key correlations in the HMBC spectrum which the presence of the 5-OH group on C. This structural fea- allowed the elucidation of the chemical structure of com- ture is, thus, responsible of the higher activity showed by pound 2 as (25S)-spirostan-2a,3b,6b-triol 3-O-b-D-xylo- the furostanol saponin minutoside C. This observation is pyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D- in contrast with a previous study reporting the absence of any antifungal activity for the furostanol saponins from Minutoside C (3), C50H84O26 by high-resolution FAB MS, was isolated in low yield. It was disclosed to be the The two strains of the antagonistic fungus Trichoderma 5-hydroxy analogue 1. This has been first suggested by harzianum were much more sensitive than the fungal patho- MS data, being the m/z value of minutoside A 16 amu gens to minutosides B and C, and neoagigenin less when compared to 3. Indeed, inspection of 1D and This result is consistent with previous findings which report 2D NMR data revealed that these two molecules differed a high sensitivity of Trichoderma spp. to saponins from uniquely for the chemical shifts of C/H atoms around Medicago sativa and Panax quinquefolius ( E. Barile et al. / Phytochemistry 68 (2007) 596–603 instead, was the less sensitive to all saponins ).
One of the mechanism by which saponins display an anti- microbial activity is based on their ability to form com-plexes microorganisms. This causes damages in the membrane and the consequent collapse of cells (). Tolerance of P. ultimum could be related with the lack of sterols in the membrane of oomycetes. In addi- tion, all five saponins did not show any appreciable antimi- crobial activity towards the selected bacteria (data not shown). Sensitivity of bacteria to saponins has often been although comparative studies between fungiand bacteria showed that bacteria are less sensitive in gen- The mechanisms underlying saponins antibacterial The potent antifungal activity displayed by A. minutiflo- rum saponins, especially minutoside B, suggests that these preformed compounds, alone or in combination, may actas chemical barriers to fungal attacks. This hypothesis is also supported by the high content in the bulbs of both minutoside B and neoagigenin (). However, many fungi may attack plants by producing enzymes that degrade saponins into non-toxic molecules ( example, the fungus Gaeumannomyces graminis var. avenae is able to infect oat plants because it secretes avenacinase, an extracellular enzyme that detoxifies avenacins, the oat has also been found in the fungus Armillaria mellea, a plant pathogen that is able to degrade the antifungal isoflavone genistein into non-toxic metabolites ().
From this point of view, the high sensitivity of the two T. harzianum strains to A. minutiflorum saponins could depend on the low or null ability of this fungus to detoxify these compounds. It is known that Trichoderma, though it infects roots, is not pathogenic to plants because it limits infection to the superficial cell layers ( Saponins and/or other plant antimicrobial com-pounds could be involved in limiting Trichoderma from further invading the plant tissue. More studies are needed Optical rotations were measured on a Perkin–Elmer 192 polarimeter equipped with a sodium lamp (589 nm) and 10- cm microcell. High-resolution ESIMS experiments were performed on an Applied Biosystem API 2000 triple–quad- rupole mass spectrometer. The spectra were recorded by infusion into the ESI source using MeOH as solvent.
GC–MS analysis was performed on a Carlo Erba instru- E. Barile et al. / Phytochemistry 68 (2007) 596–603 ment. FTIR spectra were run on a Perkin–Elmer 1600 spec- ond one, instead, was purified by HPLC using H2O– trometer in KBr. 1H and 13C NMR spectra were recorded MeOH (2:8) as eluent, affording the known sapogenin neo- on a Varian Unity Inova spectrometer at 500.13 and 125.77 MHz, respectively. Chemical shifts were referred to the residual solvent signal (CD3OD: dH 3.31, dC 49.0).
The bulb MeOH extract (50.0 g) was partitioned The multiplicities of 13C NMR resonances were determined between n-BuOH and H2O in order to remove sugar com- by DEPT experiments. 1H connectivities were determined pounds. The organic layer was filtered and then concen- by using COSY and HOHAHA experiments; the 2D trated under vacuum giving a crude extract (35.6 g), HOHAHA experiments were performed in the phase-sensi- which was chromatographed by MPLC on a RP-18 column tive mode (TPPI) using the MLEV-17 (mixing time 125 ms) using a linear gradient solvent system from H2O to MeOH.
sequence for mixing. One-bond heteronuclear 1H–13C con- Preliminary NMR studies of the eluates revealed that only nectivities were determined with 2D HSQC pulse sequence one fraction contained saponin compounds. This fraction, with an interpulse delay set for 1JCH of 130 Hz. Two and eluted with H2O–MeOH (35:65) was further purified by three bond heteronuclear 1H–13C connectivities were deter- HPLC on a semipreparative C18 column with the mobile mined with 2D HMBC experiments, optimised for 2–3JCH phase H2O–MeOH (45:55), giving the pure new compound of 8 Hz. Nuclear Overhauser effect (NOE) measurements were performed by 2D ROESY experiments. Medium pres-sure liquid chromatography (MPLC) was performed on a Bu¨chi 861 apparatus using LiChroprep RP-18 (40–63lm)columns. Prep. TLC on SiO2 with BuOH:H2O:CH3COOH 60:25:15 (BAW) for development was used. Spots were pyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D- visualized with cerium sulphate in 2 N H2SO4. HPLC in galactopyranosyl] 26-O-b-D-glucopyranoside (1); Yield: isocratic mode was performed on a Varian apparatus equipped with a RI-3 refractive index detector [semiprepar- (c = 0.1 MeOH); IR (KBr) mmax 3410, 2930, 1150, ative l-Bondapack C18 column (7.8 mm · 300 mm, i.d.)].
1045 cmÀ1; 1H NMR data, see 13CNMR data, see . HRFABMS (negative ion): Wild samples of A. minutiflorum were collected in the Ardestan (2200 m), Isfhan provinces, Iran, in June 2003 and identified by Prof. S. Zarre, Department of Biology,University of Tehran. A voucher specimen (No. 1146) has been deposited at the Department of Pharmacognosy, (1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D-galactopy- Isfahan University of Medical Sciences.
ranoside (2); Yield: 150.4 mg; colorless amorphous solid;[aŠ25 À 35:2 (c = 0.1 MeOH); IR (KBr) m 1150, 1043 cmÀ1; 1H NMR data, see ; 13CNMR data, see . HRFABMS (negative ion): found The bulbs were air-dried immediately after collection, m/z 904.4728 [MÀH]À; calculated for C44H72O19 m/z under controlled temperature (22 °C) and without exposure of light, giving a dry weight of 1.8 kg. They were finelyhand-cut and then exhaustively extracted, at room temper- ature, with the following solvents in this order: n-hexane,CHCl3, CHCl3–MeOH (9:1), and MeOH. Each solvent extraction stage was conducted for 1 day and was repeated pyranosyl-(1 ! 3)-O-b-D-glucopyranosyl-(1 ! 4)-O-b-D- four times using 3 l of solvent, under stirring.
galactopyranosyl] 26-O-b-D-glucopyranoside (3); Yield: The CHCl3–MeOH (9:1) extract of bulbs was concen- 16.7 mg, colorless amorphous solid; ½aŠ25 À 41:7 (c = 0.1 trated under vacuum to afford a crude organic extract MeOH); IR (KBr) mmax 3400, 2934, 1159, 1048 cmÀ1; 1H (15.0 g), which was chromatographed by MPLC on a RP-18 column using a linear gradient solvent system from HRFABMS (negative ion): found m/z 1100.5313 H2O to MeOH. Preliminary NMR analyses of the eluates [MÀH]À; calculated for C50H84O26 m/z 1100.5332.
let us to select two interesting fractions eluted with H2O–MeOH (1:9) and MeOH 100%. The first one was chro- 3.7. Determination of sugar absolute configurations matographed by HPLC on a semipreparative C18 columnwith the mobile phase H2O–MeOH (3:7) to give the new A solution of each isolated compound (1 mg) in 1 N compounds 2 (150.4 mg, tR = 19.0 min) and 3 (16.7 mg, HCl (0.25 ml) was stirred at 80 °C for 4 h. While cooling, tR = 14.0 min) along with the known sapogenin alliogenin the solution was concentrated under a stream of N2. The E. Barile et al. / Phytochemistry 68 (2007) 596–603 (Trisil-Z) and pyridine (0.1 ml) and the solution was stirred incubated at 25 °C and after 48 h the inhibition was at 60 °C for 5 min. The solution was dried with a stream of N2, and the residue was separated by water and CH2Cl2(1 mL, 1:1). The CH2Cl2 layer was analyzed by GC (All-tech L-Chirasil-Val column, 0.32 mm · 25 m; temperatures for injector and detector, 200 °C; temperature gradient sys-tem for the oven, 100 °C for 1 min and then raised to This work was supported by MIUR Grant PRIN 180 °C; rate 5 °C/min). Peaks of the hydrolysate of 1, 2, 2004038183/002. We thank Prof. Zarre, University of Teh- and 3 were detected at 10.98, 13.98, and 14.66 min in the ran, for identifying the plant material. Mass and NMR ratio of 1:1:2 for 1 and 3 and in the ratio of 1:1:1 for 2.
spectra were recorded at the ‘‘Centro Interdipartimentale Retention times for authentic samples after being treated di Analisi Strumentale’’, Universita` di Napoli Federico II.
simultaneously with Trisil-Z were 10.98 (D-xylose) and11.05 (L-xylose), 13.98 (D-galactose) and 13.75 (L-galac-tose), Co-injection of each hydrolysate with standard D-xylose, D-galactose, and D-glucose gave single peaks.
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