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. 2018 Nov 3;20:e00291. doi: 10.1016/j.btre.2018.e00291

Waste residues from Opuntia ficus indica for peroxidase-mediated preparation of phenolic dimeric compounds

Ana Laura Sánchez-Carvajal a, Sergio Alatorre-Santamaría b, Gerardo Valerio-Alfaro a, Liliana Hérnández-Vázquez c, Arturo Navarro-Ocaña d,⁎
PMCID: PMC6288046  PMID: 30568885

Graphical abstract

graphic file with name fx1.jpg

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Keywords: Opuntia ficus cladodes, Food waste residues, Peroxidase, Oxidative coupling, Phenolic compounds, Dimerization reactions

Highlights

  • •

    Peroxidase enzymatic activity in wastes from local edible plant material.

  • •

    Reutilization of a highly-abundant waste material that otherwise would be discarded.

  • •

    Specific enzymatic activity towards phenolic compounds.

  • •

    Dimerization of important phenolic compounds with important biological activity.

Abstract

A methodology to detect peroxidase activity in Opuntia ficus indica cladodes waste extracts was performed and then used towards phenolic compounds. The extracts were able to dimerize three different molecules. Dimeric compounds were produced with yields ranging from 11% to 55%. The influence of H2O2 concentration was also tested, finding better yields when the peroxide-to-substrate ratio was 1:1. Some water-miscible solvents were used trying to increase overall yields, but no-significant positive results were found. In fact, one of them, THF, seemed to inhibit dimerization reaction. Hence, we have tested an alternative natural peroxidase source obtained from the wastes of a local highly-consumed vegetable and studied their enzymatic activity towards the preparation of biologically active, valuable compounds.

1. Introduction

Peroxidases (EC 1.11.1.7) are oxidoreductases that catalyze the oxidation of a diverse group of organic compounds using hydrogen peroxide as the ultimate electron acceptor. They are known for a variety of commercial applications and have been used in large scale commercial processes [1,2]. Peroxidases are readily available enzymes characterized by high stability, activity and low substrate specificity that do not require additional cofactors. Therefore, they can be used isolated, mainly from the roots of horseradish plant as main commercial source. The biocatalytic oxidation of guaiacol (o-methoxyphenol) and related compounds generated a variety of oligophenols (dimers to pentamers), and some other oxidation products [3].

The radical character of intermediates formed in the reactions catalyzed by peroxidases constitutes an important drawback that hampers a more extensive use of these enzymes in chemical synthesis. The direct one-electron oxidation of the substrate molecule involved in different coupling reactions leads to formation of a wide variety of polymeric products, decreased yields of target compounds and thus, making their isolation a hard task [4].

In the current study we have tested wastes of a locally available and highly consumed vegetable, Opuntia ficus indica, as an alternative source of natural peroxidases and studied their potential enzymatic activity. O. ficus indica is one of the cactus species most extensively cultivated in Mexico, due both to the nutritional values and high digestibility of its fruits and cladodes. In fact, during 2014 total national production of this vegetable was above 8 × 105 ton (SIAP, Mexico).1

Before consumption, cladodes are submitted to a process to remove the thorns produced in the meristem. This specific part of the plant is known for lignification processes in which peroxidases are highly-involved. Moreover, this waste product accounts for approximately 30% of dry weight of the cladodes and therefore it can be considered as potentially important source of peroxidases.

On the other hand, phenolic compounds, are widely distributed in plants and are present in considerable amounts in fruits, vegetables, and beverages of normal human diet [5]. Compounds such as ortho-methoxy para-substituted phenols have attracted considerable attention due to its various biological and pharmacological activities, mainly as antioxidants [6,7]. Furthermore, some ortho-coupled dimer derivatives have been reported to present better biological properties than their monomeric counterparts [[8], [9], [10], [11], [12], [13], [14], [15]].

Therefore, in the present paper we report the biotransformation of several phenolic compounds, which are well known for their antioxidant activity, with an enzymatic extract from Opuntia ficus indica waste products yielding phenolic dimeric compounds, along with some preliminary results on the influence of H2O2 concentration and the use of a co-solvent.

2. Experimental Section

All chemicals, materials, and commercial horseradish peroxidase C (HRP C) were purchased from Sigma-Aldrich™ or JT Baker™. All chemicals were of analytical grade and were used without further purification. H2O2 was used as a 30% (v/v) solution in H2O.

2.1. Plant Material

The plant material used for preparation of peroxidase crude extract was obtained from O. ficus indica young cladodes (or pads) measuring 6 to 8 cm in length, collected from local farmers in Milpa Alta (Mexico City). Thorns were removed from cladodes and then used for the preparation of enzymatic extract.

2.2. Crude enzymatic extract

Crude enzymatic extract was obtained by homogenizing 100 g of waste material with 300 ml of phosphate buffer pH 7 (100 mM). The homogenized extract was centrifuged at 5000 rpm for 5 min at 4 °C, and the supernatant was stored at -65 °C until assays were performed.

2.3. Peroxidase Activity

Activity was determined spectrophotometrically by the change in absorbance at 590 nm due to benzidine oxidation. The reaction mixture contained 200 μL benzidine solution (1% in ethanol), 10 μL of enzyme extract, 30% H2O2 solution (0.08 mM), and acetate buffer (100 mM, pH = 5.0), in a total volume of 1.2 mL. The assay was performed at 25 °C using a Lambda 2S spectrophotometer.

2.4. Protein Determination

Protein was quantified through a spectrophotometric procedure based on the dye-binding method of Bradford (1976)2 using bovine serum albumin (BSA) as standard and a commercial Bradford reagent kit: 1 mL of the reagent was mixed with enzymatic extract (10 μL) and after 15 min absorbance of the solution was measured at 595 nm.

2.5. Analytical methods

Gas chromatography-mass spectrometry analysis for conversion degree was performed on a Shimadzu GC–MS QP 5050 instrument using a DB-5 (1% methyl phenyl silicone) capillary column purchased from Alltech Associates, USA (30 m, 0.32 mm i.d., 0.25 mm film thickness), and equipped with electronic impact source (75 eV) and a quadrupole analyzer. Helium was used as the carrier gas. Conditions of injections for all cases were as follow: Temperature of injector and detector 250 °C, initial column temperature 150 °C (2 min) then raised up to 250 °C at a 10 °C min-1 rate. 13C-NMR (75 MHz) spectra were performed on a Varian Gemini 300 with tetramethylsilane (TMS) as the internal reference and CDCl3 or CD3OD used as solvents.

2.6. Preparative Peroxidase-promoted dimerizations

A standard methodology for reactions of all three phenolic compounds was set using eugenol as starting product: reaction mixture contained 100 mM acetate buffer solution (pH = 5.0), 4 × 104 Enzymatic Units (EU) from O. ficus indica peroxidase enzymatic extract, 30% H2O2 solution (100 μL) and eugenol (0.3 mM) in a final volume of 40 mL. Biotransformation reaction was incubated at room temperature for 4 h at 250 rpm on an orbital shaker, after which reaction was stopped with a suitable volume of ethyl acetate. Reaction was monitored every 30 min taking aliquots of 1 mL, extracted with 2 mL of EtOAc, and then developed through TLC (hexane:EtOAc, 8:2 or chloroform:acetic acid, 9:1). After reaction was completed, crude product was purified.

2.7. Purification of dimeric compounds

Purification was performed by preparative TLC (crude mass up to 250 mg) or column chromatography (crude mass higher than 250 mg), using a 30% EtOAc/hexane eluent system. Isolated product was analyzed by 13C NMR spectroscopy and mass spectroscopy. 3,3′-Dimethoxy-5,5′-di(prop-2-en-1-yl)biphenyl-2,2′-diol (5) was obtained from dimerization of eugenol as a yellowish oil (48% isolated yield). 13C NMR (75 MHz, CDCl3): δ = 39.9; 56.0; 110.8; 115.7; 123.2; 124.5; 131.9; 137.7; 141.1; 147.7. MS (EI, m/z): 326 (M+,100).

2.8. Biotransformations of eugenol with different concentrations of H2O2

The reaction mixture contained, in a total volume of 40 mL, 4 × 104 EU O. ficus indica peroxidase enzymatic extract, different H2O2 concentrations (0.3 mM, 0.6 mM or 0.9 mM), eugenol (0.3 mM) and a correspondent volume of acetate buffer. Reaction mixtures were incubated in continuous orbital shaking for 4 h at room temperature and 150 rpm. The enzymatic reaction mixture was extracted with ethyl acetate (1 x 200 mL), organic phase was dried over Na2SO4 and concentrated in a rotatory evaporator. The product of the reaction mixture was purified by column chromatography (hexane:ethyl acetate, 7:3). Reaction was followed through TLC taking 2 μl of the extract and developed with hexane/ethyl acetate (8:2).

2.9. Biotransformations of eugenol with various solvent-water ratios

To a reaction mixture as described above (H2O2 0.3 mM), was added a water miscible solvent (acetone, dioxane or THF, 10 mL), following a buffer:solvent ratio of (3:1). Time and work-up of crude reaction was made exactly as stated previously.

3. Results and discussion

3.1. Determination of peroxidase activity and TLC

An initial goal was to determine peroxidase activity in the thorns removed from cladodes through a standard method. The assay proved an enzymatic activity of 12.51 U/mg of protein.

In the next step oxidative dimerization of selected substrates such as ferulic acid (1), eugenol (2) and isoeugenol (3) (Fig. 1) was studied since their dimers are well known for their biological activity as antioxidants, outdoing their respective monomers in certain cases ([8,12,13,[16], [17], [18], [19]]). These structures also play an important anti-inflammatory and chemopreventive roles.

Fig. 1.

Fig. 1

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Chemical structures of selected substrates for peroxidase activity test on extracts from Opuntia ficus indica cladodes waste.

Qualitative TLC results implied peroxidase-promoted bioconversions as new products were detected for all proposed substrates and decreased intensity from starting material was observed (Fig. 2). Control experiments showed no formation of dimers in the absence of either enzymatic crude or H2O2. All experiments were compared to the biooxidation reactions performed with commercially available horseradish peroxidase (HRP) and compounds 1, 2 and 3. In all cases similar products to those formed with standard reaction were found (Fig. 2). In consequence it was decided to conduct more specific essays for each of the substrates studied.

Fig. 2.

Fig. 2

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TLC plates from preliminary dimerization reactions with enzymatic extract. TLC revealed with: ceric sulphate and UV (254 nm), respectively. A,A′: FA (10% AcOH in Chlorofom), B,B′: Eugenol (20% EtOAc in Hexane), C,C′: Isoeugenol (20% EtOAc in Hexane). In all cases: lane 1 (standard), lane 2 (blank), lane 3 (peroxidation reaction with HRP), lanes 4-7 (reaction catalyzed with extracts for 5, 10, 15 and 60 min of reaction).

3.2. Biotransformation of ferulic acid catalyzed by an Opuntia ficus indica peroxidase enzymatic extract

Compound 1, an in vivo substrate for horseradish peroxidase and other oxidoreductases, was partially consumed by the enzymatic extract yielding a major compound (Fig. 2A and 2 A’, 30%) that was purified, and its structure confirmed by 13C-NMR and MS as symmetric dilactone 4 (Fig. 3). The 13C-NMR spectrum presented only 10 well differentiated signals, that along with the MS data (M+ = 385), suggested the formation of a symmetric molecule such as 4, showing duplicate carbon signals. The data obtained were in accordance with already published studies for this compound [17].

Fig. 3.

Fig. 3

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Structure of products from dimerization reactions.

Several works report that ferulic acid dimers usually present better antioxidant activity than the monomer, due to an extended resonance that lead to more stable conjugate-transient structures that quench free radical species [12]. Even though most of the dimers have better properties, compound 4 has been reported as having lesser activity than ferulic acid [20].

Notwithstanding, when dilactone 4 is presented on its open form inhibits lipid peroxidation better than compound 1 [11], as authors claimed that antioxidant capacity in cinnamic acid derivatives is increased with more hydroxyl groups bond to the aromatic ring as well as longer, uninterrupted conjugation within the molecule. These results were recently analyzed through in-silico studies [21], and authors found same correlation between extended conjugation and reactivity towards free radicals and antioxidant activity. Similar results were found by Jia et al. [12].

Furthermore, biooxidation of ferulic acid using peroxidase from Opuntia ficus indica crude extract afforded better yield than traditional chemical catalysts applied in previous works. For example, Stafford and Brown [22] reported the synthesis of ferulic acid dilactone by means of chemical catalysts ―FeCl3 or (NH4)2S2O8/FeCl3― with yields of 18% and 20%, respectively. Additionally, in other enzymatic studies dilactone 4 was obtained as a product of peroxidase-promoted dimerization of ferulic acid using either fungus or vegetable source of the enzyme, but yields did not exceed the aforementioned range [[22], [23], [24]].

3.3. Biotransformation of eugenol 2 and isoeugenol 3 catalyzed by an Opuntia ficus indica peroxidase enzymatic extract

Dimerization of phenolic compounds presents a good application potential, as these products have been found with interesting properties when compared with their respective monomers. For example, it has been reported that dimers of eugenol, such as compound 5 (Fig. 3) showed less cytotoxicity and greater anti-inflammatory activity than its monomer 2 ([[25], [26], [27]]). Other authors claimed that eugenol and isoeugenol dimers, possess higher antitumoral activity [8], as well as promising use in antidiabetic drugs [15] and neuroprotective activity [13].

Therefore, potential application of these molecules has triggered the study of their preparation, either using chemical or enzymatic methods. For example, dimerization of eugenol was already achieved before using hydrogen peroxide and Fe (II) with yields of 20% and with Cu (I) and amines with yields between 26–79% depending of the amine used [28]. Among several other studies, Llevot et al. (2016) studied dimerization of phenolic compounds, including eugenol, using laccase-catalyzed coupling reactions with excellent yields (87–96%).

In the present study, the dimerization of o-methoxyphenols 2 and 3 led to the formation of two main compounds (Fig. 2B, 2B’, 2C and 2C’), which are already known dimers: bis-eugenol 5 (3,3´-dimethoxy-5,5´-di-2-propenyl-1,1´-biphenyl,2,2´-diol) and dehydrodiisoeugenol 6 (2-(3-methoxy-4-hydroxyphenyl)-3-methyl-5-(1-propenyl 9-7-methoxy-2,3-dihydrobenzofuran), respectively (Fig. 3). Compound 6 resulted from both phenolic and benzylic coupling exhibited by isoeugenol.

Structure of compound 5 was determined by comparison of its spectroscopic properties versus chemical arrangement, finding symmetry in the molecule as it was already observed for dilactonic diferulate. 13C-NMR spectra showed only half number of signals compared to the total amount of carbons presented in the dimer [29], while MS fragmentation pattern was identical to that suggested before for this compound by Krawczyk et al. [30]. Biocatalyzed and synthetic products were compared for its antioxidant activity on a TLC plate that was stained with DPPH reagent (Fig. 4), both substances showing the same Rf value and antioxidant qualitative activity.

Fig. 4.

Fig. 4

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Comparative TLC of dimerized eugenol through 2 (synthetic reaction) and 3 (enzymatic reaction). Eugenol standard was spotted on 1. (10% EtOAc in Hexane, stained with DPPH radical).

Preliminary yield for the dimeric oxidation of eugenol obtained in this study was 11%, which is lower than reported earlier [30]. Authors described the coupling of eugenol and isoeugenol by means of horseradish peroxidase and hydrogen peroxide affording two main dimeric compounds: 5, the most common product of ortho-oxidative coupling of eugenol at 22% yield, and 6 obtained from isoeugenol 3 at 19% yield.

On the other hand, compound 6 was synthesized using conventional chemical catalysis, in yields that range from 35 to 95%. [19,28,31]. Both antioxidant activity and the ability to quench hydroxy-radicals by this compound were tested by different research groups, [32,33] proving better anti-inflammatory properties than eugenol [31,28,19].

In the context of this work, product 6 showed the best isolated yield of the three dimerized compounds reaching 55%. Similarly, the structure of compound 6 was assigned by comparing NMR and MS data of the chemically synthesized molecule with already published information. [18,29]. To our knowledge, compound 6 has been prepared previously through enzymatic oxidative coupling by Krawczyk et al. [30] and Sarkanen and Wallis [34] with yields ranging from 20% to 65%.

It should be noted that peroxidase-catalyzed oxidation of ferulic acid, eugenol, isoeugenol and related structures had been thoroughly reported previously ([35,36,30,37,38], Llevot et al., 2016, [39,24,34,40,41], and more). Importantly, preliminary results obtained in this work suggested good enzyme selectivity in the cladode extracts towards tested substrates.

Furthermore, it appeared that the presence of guaiacol moiety was necessary to achieve such selectivity, as other cinnamic acid derivatives that were tried with this extract (e.g. p-coumaric and sinapic acids) did not present such behavior (data not shown). In fact, some authors had found specific guaiacol peroxidades from different parts of O. ficus indica species [42].

On this regard, it could not be proved if one or more enzymes were involved in the transformations. On one hand, in a prior study it was reported the isolation of only one peroxidase from this type of plant [43]. On the other, there are reports on the extraction of several protein fractions from O. ficus indica, that present peroxidase activity, thus suggesting the presence of more than one enzyme [44].

3.4. Biotransformations of eugenol (2) with different H2O2

To evaluate the effect of experimental conditions on the outcome of reaction, it was decided to work with the substrate that presented the lowest yield, compound 2. Therefore, the effect of H2O2 concentration and the use of different co-solvents were studied (Scheme 1).

Scheme 1.

Scheme 1

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Reaction conditions optimization for eugenol (2).

Since higher conversion yields were reported to be obtained when the concentration of peroxide was closer to that of the substrate [45,46] three different ratios of H2O2/Eugenol were tested: 1:1, 2:1 and 3:1 (Table 1). In all three cases the quantity of isolated product was higher. Yields for 1:1 and 2:1 ratios increased considerably from 11% (0.5:1 ratio) to 44 and 39%, respectively, whereas a 3:1 ratio yielded a lower conversion (18%), but still better than the first reported.

Table 1.

Effect of peroxide:eugenol ratio on total reaction conversion.

RATIO (H2O2:Eugenol) 0.5:1a 1:1 1:2 1:3
CONVERSION (%)b 11 48 39 18
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a

Original reaction ratio.

b

Amount of 5 isolated.

With the aim of understanding observed results, it was decided to increase the ratio of H2O2/Eugenol (8:1; 12:1 and 16:1). The higher the concentration of peroxide was used, the less product 5 was detected (data not shown), suggesting the possible inactivation of the enzyme(s) as a consequence of the increased concentration of H2O2 [47]. Nonetheless, further experiments should be performed to determine the actual influence of H2O2 on peroxidase extract used in this study.

3.5. Effect of water-miscible solvents on the biotransformations of eugenol (2)

Once concentration of H2O2 in the enzymatic reaction with eugenol was optimized, the effect of water-miscible solvents in the reaction media was evaluated. The proper reaction media modifies the solubility of reactants, intermediates and products, hence altering the reaction outcome by potentially increasing the reactive contact between the substrate and the enzyme.

Since eugenol solubility in water is rather low, three co-solvents which are polar, non-protic and water-miscible were examined. Acetone, dioxane and tetrahydrofuran (THF) readily solubilized the substrate at 1:3 (solvent:buffer) ratio (Table 2). However, in terms of reaction yield only acetone presented beneficial effect on compound 5 production, slightly increasing the yield (49%). Addition of dioxane to the reaction mixture decreased product formation (32%), while THF prevented the production of dimer. Obtained results showed no significant improvement on the bioconversion when a co-solvent was added.

Table 2.

Effect of co-solvent in the dimerization of eugenol.

CO-SOLVENT ACETONE DIOXANE THF
CONVERSION (%)a 49 32 0
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a

Amount of 5 isolated.

Although the use of these solvents is limited at various levels by pharmaceutical and food-control government agencies, the authors wanted to present a novel source of peroxidase enzyme activity from an otherwise waste material, preparing already known interesting compounds with important biological activity.

4. Conclusion

In conclusion, the crude peroxidase extract obtained from cladodes of Opuntia ficus wastes showed a remarkable activity toward a number of common phenolic compounds. Furthermore, biotransformations of ferulic acid, eugenol and isoeugenol to specific dimeric compounds were achieved. Additionally, it appeared that the presence of guaiacol moiety, common to all three compounds tested was necessary, as other cinnamic acid derivatives did not present such behavior (data not shown). Reaction yields were increased by adjusting the molar ratio between H2O2 and substrate. Usage of co-solvents did not improve reaction efficiency.

Overall, a new source of peroxidase enzymatic activity from an abundant food residue was presented and a milder methodology for the preparation of biologically active molecules was proposed.

Conflict of interest

None.

Acknowledgments

Authors would like to thank Consejo Nacional de Ciencia y Tecnología (México) (Project No. 2012 CB 180128) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (UNAM, México) (Project No. 26-IN220015) for the funding of this research.

Footnotes

1

Sistema de Información Agroalimentaria y Pesquera, Mexican Government.

2

Bradford M. Analytical Chem.1976, 72, 248-254.

Appendix A

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.btre.2018.e00291.

Appendix A. Supplementary data

The following is Supplementary data to this article:

mmc1.docx (939.7KB, docx)

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