Biogenetic Conversion of Wyerone and Dihydrowyerone into Wyerone Epoxide in Vicia faba Cotyledons and Screening of Antibacterial Activity

Background. Vicia faba is a plant that belongs to the family Fabaceae. Cotyledons of this plant produce compounds called phytoalexins as a result of fungal and bacterial infection or stress factors. *e phytoalexins are furanoacetylenic compounds.*ey include wyerone, wyerol, wyerone acid, their dihydro derivatives, and wyerone epoxide. *ese compounds have antimicrobial activities mainly against fungi. Objective. *e purpose of this study was to elucidate the biogenetic conversion of wyerone and dihydrowyerone to wyerone epoxide in V. faba and to investigate the antibacterial activities of some of these phytoalexins. Materials and Methods. Seeds of Vicia faba were used. Labelled wyerone and dihydrowyerone were obtained by treating CuCl2induced cotyledons with sodium (2-C) acetate and separated by TLC and HPLC. Labelled wyerone and dihydrowyerone were then applied to induced bean cotyledons to establish any possible interconversion to wyerone epoxide. Antibacterial activity of wyerone, wyerone acid, and wyerone epoxide was investigated by disc diffusion test against a panel of microorganisms. Zones of inhibition were reported. Results. *e radiolabeling studies showed that 12.4% of C-wyerone, and 6.01% of C-dihydrowyerone were incorporated into wyerone epoxide. *is indicates that wyerone epoxide was most probably derived from wyerone and dihydrowyerone. In addition, a new compound, 11-hydroxywyerone was isolated for the first time. Additionally, this study showed that wyerone, wyerone epoxide, and wyerone acid had no antibacterial activity against Gram-negative bacteria but were active against Gram-positive bacteria.


Introduction
Plants were found to have physical and biochemical responses towards pathogen infections.
ey evolve coordinate events by relying on their immune system to recognize and actively respond to these infections.One example of the defense mechanisms is the accumulation of antimicrobial secondary metabolites, which are called the phytoalexins [1,2].ese phytoalexins are synthesized by plants as a result of infections (fungal, bacterial, or viral) [3,4] or due to other forms of stress, which could be chemical elicitors (e.g., mercury salts, copper) or physical events like UV light [5].Fabaceae plants usually synthesize certain isoflavonoid phytoalexins, but certain species accumulate nonisoflavanoid compounds instead of or in addition to isoflavanoid [6,7].

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Journal of Chemistry in pod endocarp has led to the suggestion that wyerone may act as precursor for wyerone epoxide. is was supported by the observed conversion of wyerone to wyerone epoxide during fractionation of pod tissue [14][15][16].e biosynthesis of wyerone in Vicia faba was elucidated previously and found to be derived from acetate malonate pathway via fatty acid derivatives [17], and the conversion of wyerone to wyerone acid had been elucidated earlier [18].e aim of this study is to explore possible biogenetic conversions in V. faba cotyledons from wyerone and dihydrowyerone to wyerone epoxide.During the separation of phytoalexins, a new compound, 11-hydroxywyerone was isolated.is study excluded any fungal conversions, since such fungal transformation of wyerone derivatives has been studied extensively [14,19,20].
e antibacterial activity of phytoalexins has largely been confined to isoflavonoids from Fabaceae, with one report of this activity of wyerone in comparison with other isoflavonoid phytoalexins [21] was published.Moreover, most of the antimicrobial activity studies focused on antifungal rather than antibacterial activity [3,4].In the present study, we tried to explore more on this issue regarding three of these phytoalexins, wyerone, wyerone acid, and wyerone epoxide against five Gram-positive and two Gram-negative bacteria.e effect of wyerone acid and wyerone epoxide was not evaluated previously on these bacteria.ese bacteria studied were human and plant pathogens and a saprophyte.

Materials and Methods
2.1.Materials.Seeds of Vicia faba (broad bean, cv.Aquadulce) were procured from Sinclair McGill.2-14 C-sodium acetate, 58 mCi/mmol, was procured from New England Nuclear (NEN, Boston, MA), 14 C-Toluene (Internal liquid scintillation counting (LSC) standard) was procured from Perkin-Elmer, and Silica gel (0.5 mm, GF254 TLC plates) was procured from Merck.All other chemicals were of analytical grade and procured from Sigma-Aldrich (St. Louis, MO, USA).ATCC 19304, and the saprophyte used was Bacillus megaterium ATCC 14581.ese microorganisms were procured from the American Type Culture Collection (ATCC).Media used were procured from Oxoid (UK).Amoxicillin was a kind gift from Hikma pharmaceuticals (Amman, Jordan).

Plant Material, Feeding Techniques, and Isolation of Phytoalexins.
e phytoalexins were synthesized using the procedure described earlier by Al-Douri and Dewick [17,18].Seeds of V. faba were immersed in ethanol for 10 minutes, washed with water, soaked in dilute detergent (Quadralene, UK) for 5 minutes, sterilized by immersion with sodium hypochlorite (2%) for 5 minutes, and washed again.Damaged seeds distinguished by eye during sterilization were discarded.e healthy seeds were imbibed in distilled water for 24 hours.e seed coat was removed, and 100 g of the obtained cotyledons were separated and placed flat side up on a moist tissue paper in glass trays (Figure 2).e chemical elicitor, aqueous CuCl 2 (3 mM), was spotted onto the surface of these cotyledons using a syringe.Sufficient amount of elicitor solution was added to keep the whole surface moistened.
e glass trays containing the cotyledons were covered by a foil paper and kept at 25 °C for several days in a dark cabinet.Extra CuCl 2 was added when necessary to keep the surface moist.In our laboratory, a fiveday time period was found the best to give the highest production of phytoalexins.When the five days elapsed, the inducer solution was removed by a syringe and replaced by drops of sodium (2-14 C) acetate solution (1 mCi) as a precursor for synthesis of the phytoalexins to be studied.e cotyledons were left for another two days adding drops of distilled water to keep the cotyledons moist.After this period, the cotyledons were homogenized in a blender with 200 ml dichloromethane.is was repeated ten times after filtering the extract.Dichloromethane extract was concentrated using a rotary evaporator.

Purification of the Different Compounds.
e extract was applied on TLC chromatography (silica gel GF254; hexaneacetone, 2 : 1) to separate the zones representing the phytoalexins [17,18]. 14C-phytoalexin mixture was detected as a deep blue fluorescent band under UV254.e band was scratched and eluted by acetone.e resulting mixture was further purified by second TLC using chloroform-light petroleum (2 : 1) and then by chloroform: methanol (25 : 1).e resulting band, the purified phytoalexins, was scratched, eluted with acetone, and dried.A sample from this phytoalexins mixture was subjected to Proton NMR analysis ( 1 HNMR) to identify the structure of the separated phytoalexins.High-performance liquid chromatograph (HPLC) connected to a variable wavelength Perkin-Elmer LC55 UV detector was used to separate the labelled phytoalexins.Samples were dissolved in MeOH prior to injection.Reversed-phase columns (Spherisorb S5, ODS-2 hypersil), 25 cm × 8 mm (semipreparative), were employed with solvent methanol : acetonitrile : water (30 : 20 : 50), at flow rates of 1 ml/min or 2 ml/min [22].Radioactivity of the compounds isolated was measured by SL 4000 liquid scintillation counter with dioxin-based liquid scintillator (BDH). 14C-toluene internal standard was used for measurement of efficiencies.

Feeding Experiment of the Labelled (Wyerone and Dihydrowyerone) into the Induced Cotyledons to Produce
Wyerone Epoxide.Approximately 1 mg samples of 14 Cwyerone and 14 C-dihydrowyerone obtained from the previous experiment (Section 2.4) were added as dispersions in 1-2 mL of water with the aid of few drops of Tween 20.Each was placed on 20 g of induced V. faba cotyledons.Extraction of the cotyledons was carried out as described above to Journal of Chemistry isolate and purify the 14 C-wyerone epoxide.However, 14 C-Wyerone epoxide was detected as a small blue fluorescent band under UV254 below the band of 14 C-wyerone/ 14 Cdihydrowyerone mixture.
e band was scratched and eluted with acetone.A sample was analysed by 1 HNMR to identify the compound.In this TLC, a new band was observed under UV254.It was scratched, eluted with acetone, and purified again according to the procedure mentioned in Section 2.4.e compound was identified by 1 HNMR and 13 CNMR.Radioactivity of the compounds isolated was measured by liquid scintillation counter as described above.

Screening for Antibacterial Activity. Gram-positive and
Gram-negative bacteria were selected to evaluate the antibacterial activity of the separated phytoalexins.ese bacteria were chosen to represent plant pathogens, human pathogens, and a saprophyte.Bacterial suspensions of 0.5 McFarland (ca 1 × 10 8 CFU/ml) were prepared and diluted to get 10 6 CFU/ml.ese suspensions were each spread separately on Mueller-Hinton agar plates (9 cm diameter).
e separated phytoalexins from cotyledons of V. faba were dissolved in methanol.Blank paper discs (9 mm) were loaded gradually with phytoalexin methanolic solutions to reach a final amount of 60 µg/disc.e discs were left to dry by air.
e loaded discs were placed on the inoculated Mueller-Hinton agar plates.Discs loaded with the broadspectrum antibiotic, amoxicillin (60 µg), were used as a control, and discs loaded with methanol alone were used as negative control.e plates were incubated at 30 °C for 24 hour for all strains except Pseudomonas syringae (26 °C for 24-36 hr).Zones of inhibition were measured and reported.
e test was performed in duplicate.

Results and Discussion
3.1.Biosynthetic Conversion, Isolation, and Purification of Compounds.In the first feeding experiment using 14 C-sodium acetate, the phytoalexin mixture separated by TLC chromatography was detected as deep blue fluorescent band under UV254, with R f value 0.31. 1 HNMR analysis revealed the presence of approximately 8% of 14 C-dihydrowyerone in the mixture of 14 C-wyerone and 14 C-dihydrowyerone. is is in agreement with previous results reported in our laboratory [17,18].However, the mixture purified by TLC was reported previously to contain about 13% of the dihydro derivative [20].e resulting phytoalexin mixture was separated by semipreperative HPLC. is system was previously used for quantitative analysis of seven wyerone derivatives isolated from V. faba cotyledons as reported in the literature [22].e purified 14 C-wyerone obtained was quantified by UV spectroscopy (Perkin-Elmer 554 Spectrophotometer).It has UV absorbance λ max in ethanol at 224, 290, and 350 nm compared with those reported in the literature at 226, 291, and 351 nm (€ � 27,000) [22].e purified 14 C-dihydrowyerone was quantified by UV spectroscopy and produced a spectrum (λ max ethanol, 340, 235 nm) entirely consistent with that reported [18,22].
e quantitation of the isolated 14 C-wyerone and 14 Cdihydrowyerone was determined using the reported extinction coefficient (€ � 27,000) [22].Accordingly, the yields of the mixture of the labelled wyerone/dihydrowyerone were 20-28 mg/100 g fresh tissue of cotyledons.e biosynthesis of wyerone was proven previously to be from acetate units via acetate pathway. is was done by a series of experiments applying radiolabeled 14 C-and 13 C-isotopes techniques [17].In the present study, 14 C-sodium acetate was incorporated efficiently into wyerone (Table 1). is is in agreement with earlier reports [17,18].e radiolabel analysis of the isolated 14 C-wyerone/ 14 C-dihydrowyerone is shown in Table 2. 14 C-Wyerone Epoxide, 14 C-Wyerone Acid, and a New Compound.After the feeding experiment of 14 C-Wyerone and 14 C-dihydrowyerone into the induced cotyledons (Section 2.5), 14 C-wyerone epoxide was detected as a small blue fluorescent band under UV254 below the labelled wyerone/dihydrowyerone mixture, with R f value of 0.26.e purified 14 C-Wyerone epoxide was quantified by UV λ max 350 using an extension coefficient (€ � 27,000) [15].Radiolabel measurements of this compound are shown in Table 3.

Separation of
e results of this experiment indicated that wyerone was an effective precursor of wyerone epoxide.14 C-Wyerone acid was separated using the same solvent with R f value of 0.12.e compounds were isolated and identified using HPLC and 1 HNMR.e results of isolation and identification are consistent with previous reports [11,13].Nevertheless, Wyerone acid was isolated in order to test its antibacterial activity.e new band which was observed under UV254 below the band of 14 C-wyerone and 14 C-wyerone epoxide on TLC had an R f value of 0.21. is band was collected and purified using the same solvents used above.e resulting compound was identified as a new moiety, 11-hydroxywyerone, which is separated for the first time (Figure 1). 1 HNMR and 13 CNMR for this compound are the following: (250 MHz, CDCl 3 ): δ1.02 (3H, t, J � 7.1 Hz), 1.38-1.57(2H,m), 1.58-1.70,(3H, q/br), 3.79 (3H, s), 4.16 (1H, t, J � 7.0 Hz), 6.69 (1H, d, J � 16.5 Hz), 7.34 (1H, d, J � 3.5 Hz), 7.45 (1H, d, J � 3.5 Hz), 7.69 (1H, d, J � 16.5 Hz). e radioactivity of the new compound was measured and found to be 6.97 × 10 7 Sp.act.dpm/mM.Incorporation data showed the highest conversion of 14 C-wyerone (12.4%) into 14 C-wyerone epoxide.e results in this study indicated that it is possible that a metabolic grid existed for these compounds in V. faba, in this case, hydroxylation of the ester followed by oxidation of the secondary alcohol.
ese might be controlled by relatively specific enzymes.Supporting this hypothesis would require further feeding experiment with this new labelled compound and isolation of wyerone epoxide.e incorporation value should be higher than that in the present study.Another aspect of this hypothesis is the involvement of the enzyme which catalyzes this first reaction in the biosynthetic pathway from 11-hydroxywyerone into the epoxide, which will need further elaboration as in other studies [23].Experiments are still in progress.
e results also indicated a high incorporation (6.01%) of 14 C-dihydrowyerone into 14 C-wyerone epoxide.In each experiment, recovery of the precursor fed was considerably less than that of wyerone indicating further metabolism or degradation of the applied compound.In this case, dihydrowyerone may be transformed into wyerone epoxide quite efficiently by dehydrogenation to produce the 11, 12-alkene first, followed by the same pathway as for wyerone itself.

Antimicrobial Activity.
e antimicrobial activity of the three phytoalexins against five Gram-positive and two Gram-negative bacteria were evaluated.
ese bacteria represent human pathogens, plant pathogens, and a saprophyte (Table 4).e 60 µg amount of phytoalexin per disc was chosen to be tested based on an amount of compound versus zone of inhibition experiments performed to determine the minimum amount of phytoalexins that produced the largest zone of inhibition (data not shown).For most bacteria-phytoalexins combination, 50-60 µg/disc was the lowest amount to produce the largest zone of inhibition.Above this amount, a plateau was reached.
erefore, 60 µg/disc was selected to be tested for the three compounds against the bacteria.Amoxicillin showed the highest zone of inhibition against all the tested microorganisms.e negative control (methanol on disc) showed no inhibition zone.e three phytoalexins showed no antimicrobial activity against the Gram-negative bacteria but had noticeable activity against the Gram-positive bacteria.Assuming that the three phytoalexins have similar diffusion coefficients, then, these phytoalexins have comparable activity against Gram-positive bacteria, since at the same dose (60 µg/disc), the zones of inhibition against plant pathogens and human pathogens are very close (range between 14 and 16 mm).
e saprophyte Bacillus megaterium showed the least susceptibility to the three phytoalexins with zones of inhibition (12-13 mm).
ese results are in line with the previous report.Gnanamanickam and Mansfield [21] had shown that wyerone and some flavonoid phytoalexins were more toxic against Gram-positive than Gram-negative bacteria.Several other researches were conducted to investigate the antifungal activity of phytoalexins and only one studied their antibacterial effect [4,5,14].To the best of our knowledge, this is the first study to report antibacterial activity of wyerone acid and wyerone epoxide against the microorganisms selected in this research.ese results could open the door for developing new atypical antimicrobial agents.

Conclusion
e current study tried to elucidate the biogenetic conversion of some of the furanoacetyene phytoalexins of V. faba, particularly the biogenetic conversion among wyerone, dihydrowyerone, and wyerone epoxide.e conversions were studied through feeding experiments with labelled precursors to yield the phytoalexins.e results from the incorporation and dilution data showed that wyerone epoxide is most probably derived from wyerone.Moreover, dihydrowyerone could be a precursor to wyerone epoxide through more sequence of events, Journal of Chemistry which is explained by less incorporation value.It is not possible at this stage to surmise about the natural sequence leading to wyerone epoxide.Nevertheless, we may suggest that the epoxidation is a late process of the biogenesis starting from wyerone and dihydrowyerone, which could be passing through the new compound 11-hydroxywyerone (Figure 3).However, further work is required to elicit these steps by feeding of this new compound to the cotyledons and measuring the incorporation data to wyerone epoxide.On the contrary, the three phytoalexins showed noticeable activity against the Gram-positive bacteria with no activity against the Gram-negative bacteria chosen.is activity could be promising to explore new sources for antimicrobial agents.

Figure 3 :
Figure 3: Isolation of a new compound, 11-hydroxywyerone, during the biosynthetic conversion of wyerone into wyerone epoxide.Dotted lines: the proposed pathway in the present study.Solid line: the previously reported conversion of wyerone into wyerone acid.

Table 1 :
Mean radioactivity measurement of 14 C-labelled wyerone and 14 C-dihydrowyerone mixture from feeding experiments with sodium (2-14 C) acetate.e results are average of two trials.

Table 2 :
Mean radioactivity measurement of separated14C-wyerone and 14 C-dihydrowyerone from first feeding experiment obtained after separation by HPLC.e results are average of two trials.

Table 4 :
Mean zones of inhibition in (mm), n � 2, for the three phytoalexins and amoxicillin (positive control) tested against different bacterial genera.