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Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and α-lipoic acid
Journal of Biotechnology 157 (2012) 344–349
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and ␣-lipoic acid Shiva Shanker Kaki, Carl Grey, Patrick Adlercreutz ∗ Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 16 August 2011 Received in revised form 17 October 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: ␣-Lipoic acid Phenolics Antioxidant Synthesis Lipase
a b s t r a c t Chemo-enzymatic synthesis of six esters of natural phenolics and ␣-lipoic acid was carried to produce novel compounds with potential bioactivity. The synthetic route was mild, simple, and efficient with satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay and inhibition of lipid oxidation in fish oil emulsion system. Among the prepared derivatives, three compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alphatocopherol in the DPPH radical scavenging assay, where as in fish oil emulsion system, two derivatives showed activity, which was similar to the reference antioxidants. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Phenolic compounds, one of the most widely occurring groups of phytochemicals, are of considerable interest as they are reported to exhibit anti-allergenic, anti-artherogenic, anti-inflammatory, anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects (Nagendran and Samman, 2006; Lazos et al., 2011). In recent years there has been a growing interest to produce molecules by lipophilization of phenolic compounds with fatty acids and the product molecules thus produced are reported to have improved function as antioxidants for food and pharma applications (Figueroa-Espinoza and Villeneuve, 2005). The molecules thus produced can have the beneficial effects of both the parent compounds and also help in the delivery and uptake of the compounds. Also, synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertbutylhydroquinone (TBHQ) have been used widely as antioxidants in foods, but concerns over the safety of their use have led towards interests in natural antioxidants (Shahidi and Wanasundara, 1998). This has led to an increased interest in research related to modification of the natural phenolic compounds in order to produce molecules with a wide range of applications in food and pharma industries. The examples include the combination of different fatty acids and phenolics such as ferulic acid, dihydrocaffeic acid,
∗ Corresponding author. E-mail address: [email protected] (P. Adlercreutz). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.11.012
rutin, and vanillyl alcohol (Kermasha et al., 2006; Zhu et al., 2009; Viskupicova et al., 2010; Adlercreutz et al., 2011). Another important molecule which shows antioxidant and anticancer properties is lipoic acid. It has been reported that lipoic acid is used to treat various diseases such as atherosclerosis, thrombosis and diabetes. It is used as a dietary supplement (Hagen et al., 2004; Zhang et al., 2008). Recently it was reported that lipoic acid can inhibit the formation of reactive oxygen species and cyclooxygenase-1 activity which are stimulated by arachidonic acid and it may also exhibit a therapeutic benefit in treatment of platelet hyperaggregation-based diseases (Chou et al., 2010). A number of experimental and clinical studies have been carried out which showed that ␣-lipoic acid is potentially useful as a therapeutic agent in conditions related to diabetes, ischemia-reperfusion injury, heavy-metal poisoning, radiation damage, neurodegeneration and HIV infection (Packer et al., 1995). It has been reported that molecular combinations obtained by joining two biologically active molecules can result in novel compounds with enhanced biologically activities (IgglessiMarkopoulou et al., 2009; Shahidi and Zhong, 2010). Therefore, the combination of these two natural biologically active molecules into a same structure could result in the formation of novel hybrid molecules with significant biological activity. The aim of the present study was to synthesize novel esters involving phenolics and ␣-lipoic acid. Six esters of lipoic acid with natural phenolics (A–F) were chemo-enzymatically prepared. Two of those are already known compounds (C and F) and F was reported to exhibit antiproliferative effect on human colon cancer HT-29 cells superior to the compounds from which it was derived (Bernini
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et al., 2011). The earlier chemical synthesis reported involved about four to five steps and therefore, we thought it would be more advantageous to combine the functional phenolics enzymatically with ␣-lipoic acid. The enzyme catalyzed route would be more favourable compared to chemical synthesis for such biologically active compounds because of the mild reaction conditions and the selectivity of enzymes, which lead to high product yield and purity. The use of organic media for enzymatic reactions makes it possible to carry out reversed hydrolytic reaction, such as ester synthesis, very efficiently (Adlercreutz, 2000; Klibanov, 2001). Lipases are particularly useful because they are able to convert many non-natural substrates. There are several reports on modification of natural phenolics to produce new products with multifunctional properties for use in food, pharma and cosmetic industries (Villeneuve, 2007).
2.2.1. Compound A ((4-hydroxyphenyl) methyl 5-(1,2-dithiolan-3-yl) pentanoate) 4-Hydroxybenzyl alcohol (1 mmol; 124.2 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2butanone:hexane; 2:1 (v/v)). The reaction was carried out as described above and compound A was obtained as oily liquid in 68% yield (212 mg). 1 H NMR (CDCl 400 MHz): ı 1.35–1.55 (m, 2H, CH ), 1.65–1.75 3 2 (m, 4H, 2 × CH2 ), 1.9 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.2 Hz, COCH2 ), 2.5 (m, 1H, CH2 ), 3.10–3.25 (m, 2H), 3.6 (m, 1H, CH), 5.0 (s, 2H, CH2 O), 5.1 (s, 1H, OH), 6.8–6.85 (d, 2H, J = 6.8 Hz, Ph), 7.25–7.3 (2H, J = 6.8 Hz, Ph). IR (cm−1 ): 3460, 2994, 2911, 1724, 1435, 1406. HR-MS (EI, m/z) [M+Na]+ calculated for C15 H20 O3 S2 Na 335.0752; Found: 335.0997.
2. Materials and methods
2.2.2. Compound B ((4-hydroxy-3-methoxy-phenyl) methyl 5-(1,2-dithiolan-3-yl) pentanoate) Vanillyl alcohol (1 mmol; 154 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2-butanone:hexane; 1:1 (v/v)). The reaction was carried out as described above and compound B was obtained as oily liquid in 64% yield (220 mg). 1 H NMR (CDCl 400 MHz): ı 1.45–1.5 (m, 2H, CH ), 1.65–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.6 Hz, CH2 ), 2.5 (m, 1H, CH2 ), 3.05–3.15 (m, 2H), 3.55–3.6 (m, 1H, CH), 3.9 (s, 3H, OCH3 ), 5.05 (s, 2H, CH2 O), 5.65 (s, 1H, OH), 6.85–6.9 (m, 3H, Ph). IR (cm−1 ): 3427, 2929, 2854, 1724, 1515, 1452. HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O4 S2 Na 365.0857; Found: 365.0741.
2.1. Materials and enzymes ␣-Lipoic acid, vanillyl alcohol, 4-hydroxybenzyl alcohol, 4hydroxyphenylethyl alcohol, ferulic acid, acetyl chloride, IBX, diisobutyl aluminium hydride and sodium dithionite were purchased from Sigma (Sigma–Aldrich Sweden AB, Stockholm, Sweden). Immobilized lipase B from Candida antarctica (Novozym 435) was a generous gift from Novozymes A/S (Bagsvaerd, Denmark). Tuna fish oil was supplied by LYSI, Reykjavik, Iceland. All other solvents and reagents were of highest grade of purity available and were purchased from VWR International Sweden (Stockholm, Sweden). Coniferyl alcohol was synthesized from ferulic acid employing a reported protocol in which ferulic acid was converted to ethyl ferulate and consequently reduced to coniferyl alcohol using DIBAL (Quideau and Ralph, 1992). The spectral data was similar to the published data and the pure isolated compound was used for the enzymatic reaction. The structures of the purified products were determined by 1 H NMR using 400 MHz NMR (Bruker, UltraShield Plus 400, Germany). Mass spectral analysis was carried out on hybrid QStar Pulsar quadrupole time-of-flight mass spectrometer (PE Sciex Instruments, Toronto, Canada), equipped with a nano electrospray ionization (ESI) source. IR spectra were recorded on a Perkin-Elmer spectrometer. Antioxidant assays were performed using PerkinElmer Lambda Bio+ spectrophotometer. Enzymatic reactions were carried out on a thermo shaker (MKR 13, HLC BioTech, Bovenden, Germany).
2.2. Synthesis of compounds A–D as depicted in Scheme 1
2.2.3. Compound C (2-(4-hydroxyphenyl) ethyl 5-(1,2-dithiolan-3-yl) pentanoate) 4-Hydroxyphenyl ethanol (1 mmol; 138.2 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2butanone:hexane; 1:1 (v/v)). The reaction was carried out as described above and compound C was obtained as oily liquid in 80% yield (260 mg). 1 H NMR data was consistent with the previously reported data (15) and is as follows: 1 H NMR (CDCl 400 MHz): ı 1.35–1.50 (m, 2H, CH ), 1.60–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.28 (t, 2H, J = 7.6 Hz, CH2 ), 2.45–2.50 (m, 1H, CH2 ), 2.9 (t, 2H, J = 6.8 Hz, CH2 ), 3.10–3.25 (m, 2H), 3.50–3.60 (m, 1H, CH), 4.25 (t, 2H, J = 6.8 Hz, CH2 O), 4.90 (s, 1H, OH), 6.75–6.80 (d, 2H, J = 6.4 Hz, Ph), 7.10 (2H, J = 6.4 Hz, Ph). IR (cm−1 ): 3362, 2929, 2858, 1726, 1515, 1436. HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O3 S2 Na 349.0908; Found: 349.0958.
Enzymatic synthesis was carried out as follows. To a mixture of phenolic compound (1 mmol) and lipoic acid (1.2 mmol) in mixtures of 2-butanone: hexane (v/v, 6 mL) was added lipase from C. antarctica (15% based on total substrate weight). l-Cysteine (0.5%) was added to inhibit the polymerization of lipoic acid (Lawrence Lowell and Eisenberg, 2007). The reaction mixture was shaken at 25 ◦ C for 15 h. At the end of the reaction, enzyme was filtered and to the reaction mixture ethyl acetate (10 mL) was added and washed with 5% sodium bicarbonate. The aqueous phase was extracted with ethyl acetate (2 × 10 mL) and the combined organic phases were washed with water and finally with saturated sodium chloride solution and dried over anhydrous sodium sulphate. The crude product was purified on a silica gel column chromatography with ethyl acetate:hexane (2:1, v/v) to obtain the pure ester compound as an oily liquid.
2.2.4. Compound D ([(E)-3-(4-hydroxy-3-methoxy-phenyl) allyl] 5-(1,2-dithiolan-3-yl) pentanoate) Coniferyl alcohol (1 mmol; 180 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2-butanone:hexane; 2:1 (v/v)). The reaction was carried out as described above and compound D was obtained as oily liquid in 70% yield (258 mg). 1 H NMR (CDCl 400 MHz): ı 1.45–1.55 (m, 2H, CH ), 1.60–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.35 (t, 2H, J = 7.6 Hz, CH2 ), 2.5 (m, 1H, CH2 ), 3.15–3.25 (m, 2H), 3.55–3.65 (m, 1H, CH), 3.9 (s, 3H, OCH3 ), 4.7 (d, 2H, J = 6.8 Hz, CH2 O), 5.6 (s, 1H, OH), 6.15–6.2 (m, 1H, CH), 6.6 (d, 1H, J = 16 Hz, CH), 6.85–6.95 (m, 3H, Ph). IR (cm−1 ): 3433, 2932, 2855, 1724, 1594, 1450. HR-MS (EI, m/z) [M+Na]+ calculated for C18 H24 O4 S2 Na 391.1014; Found: 391.1169.
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2.3. Synthesis of compounds E and F as depicted in Scheme 2 The compounds E and F were prepared by aromatic hydroxylation of compounds A and C respectively following a reported protocol with slight modification (Bernini et al., 2008). Briefly, to the substrate (0.5 mmol) in methanol (5 mL) at 0 ◦ C was added IBX (0.6 mmol; 374 mg of 45% IBX) and the mixture was magnetically stirred until the end of the reaction time (3 h). Then, water (5 mL) and sodium dithionite (1.2 mmol; 209 mg) were added and the mixture was further stirred for 10 min at room temperature. Solvent was evaporated and the residue was solubilized in ethyl acetate (15 mL) and treated with saturated sodium bicarbonate (15 mL). The aqueous phase was extracted with ethyl acetate (2 × 15 mL) and the combined organic phases were washed with sodium chloride solution and dried over anhydrous sodium sulphate. The crude reaction product was purified by silica gel chromatography with ethyl acetate: hexane (2:1, v/v) to obtain compounds E and F as oily liquids in 65% (106 mg) and 68% (120 mg) yields respectively. 2.3.1. Compound E: (3,4-dihydroxyphenyl) methyl 5-(1,2-dithiolan-3-yl) pentanoate 1 H NMR (CDCl 400 MHz): ı 1.35–1.40 (m, 2H, CH ), 1.62–1.73 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.35 (t, 2H, J = 7.2 Hz, CH2 ), 2.45–2.50 (m, 1H, CH2 ), 3.10–3.25 (m, 2H), 3.5–3.6 (m, 1H, CH), 5.02 (s, 2H, CH2 O), 6.8–6.95 (m, 3H, Ph). IR (cm−1 ): 3374, 2930, 2857, 1703, 1520, 1445. HR-MS (EI, m/z) [M+Na]+ calculated for C15 H20 O4 S2 Na 351.0701; Found: 351.0807. 2.3.2. Compound F: 2-(3,4-dihydroxyphenyl) ethyl 5-(1,2-dithiolan-3-yl) pentanoate 1 H NMR data was consistent with the previously reported data (15) and is as follows: 1 H NMR (CDCl 400 MHz): ı 1.35–1.45 (m, 2H, CH ), 1.60–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.2 Hz, CH2 ), 2.45–2.50 (m, 1H, CH2 ), 2.85 (t, 2H, J = 6.8 Hz, CH2 ), 3.15–3.25 (m, 2H), 3.53–3.60 (m, 1H, CH), 4.3 (t, 2H, J = 6.8 Hz, CH2 O), 5.3 (s, 1H, OH), 6.65–6.85 (m, 3H, Ph). IR (cm−1 ): IR (cm−1 ): 3400, 2945, 2870, 1701, 1530, 1465. HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O4 S2 Na 365.0857; Found: 365.0888. 2.4. Determination of antioxidant activity 2.4.1. DPPH radical assay The DPPH radical scavenging activities of the synthesized lipoic acid derivatives were measured according to a reported method with slight modification (Akowuah et al., 2006). DPPH is a commercially available stable free radical (dark purple color), which is reduced to DPPH (light yellow colored) by reacting with an antioxidant. A 0.1 mL aliquot of each prepared compound or reference antioxidants (1 or 2 mM) in ethanol: DMSO (95:5; v/v) were added to 2 mL of ethanolic DPPH solution (0.1 mM) and the volume was made up to 3 mL with the same solvent mixture. The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 60 min. The decrease in absorbance of the resulting solution was then measured spectrophotometrically at 517 nm against ethanol containing DMSO as blank and the control sample had all the reagents except test sample. BHT and ␣-tocopherol served as reference compounds. All measurements were made in triplicate and averaged. The ability to scavenge DPPH radical was calculated by the following equation: radical scavenging activity (FRSA) Free (%) = [(Ac − As )/(Ac )] × 100, where As is the absorbance of sample and Ac is the absorbance of control.
2.4.2. Inhibition of lipid oxidation Oxidation was induced in a fish oil emulsion, with or without the addition of antioxidant, and the level of oxidation was quantified by measuring the thiobarbuturic acid reactive substances (TBARS) using reported methods with slight modifications (Rupasinghe et al., 2009; Guzel et al., 2010). Fish oil (5 mg/mL) in a buffer solution (0.05 M TRIS–HCl, 0.15 M KCl, 1% Tween 20, pH 7.0) was sonicated at 30 s intervals for 2 min by placing the glass tubes in a sonicator to form an opalescent emulsion system. The sonicated stock emulsion was divided into 0.5 mL aliquots and the emulsion was maintained by shaking the tubes at 200 rpm. The reference and test antioxidants, 100 L of 0.2 mM FeCl3 were added to the emulsion system and incubated at 37 ◦ C for 30 min and the inhibition of lipid oxidation was determined by the TBARS assay. Briefly, the oxidized emulsion sample was mixed with 1 mL of TBA–TCA reagent (15% (w/v) TCA and 0.375 (w/v) TBA in 0.25 M HCl) and heated in a boiling water bath for 20 min. The samples were then cooled on ice and centrifuged for 10 min and the absorbance of the supernatant was recorded at 532 nm against a control which contained all reagents except the test compounds. BHT and ␣-tocopherol served as reference compounds and all measurements were made in triplicate and averaged. Inhibition of lipid oxidation was calculated using the formula: % inhibition = (1 − (As /Ac ) × 100, where As is the absorbance of sample and Ac is the absorbance of control. 2.5. Calculation of partition coefficient (log P values) The lipophilicity and the physico-chemical parameters and the theoretical drug likeness properties of the synthesized derivatives were determined using the Molinspiration software available online at http://www.molinspiration.com/cgi-bin/properties (MolinspirationCheminformatics, Bratislava, Slovak Republic). 3. Results and discussion 3.1. Synthesis and characterization The present work describes the chemo-enzymatic synthesis of ␣-lipoic acid esters of phenolic compounds (Fig. 1). To the best of our knowledge this is the first report on the enzymatic esterification of lipoic acid with phenolics for the synthesis of potential antioxidant molecules. The prepared molecules can have beneficial effects due to the modification in their structure causing effects on solubility and bioavailability. The synthetic routes employed are shown in Schemes 1 and 2. The phenolic compounds were enzymatically esterified with ␣-lipoic acid with the immobilized lipase B from C. antarctica (Novozym 435). This enzyme is known to be a very efficient catalyst for esterification of a wide range of alcohols. Primary alcohols are especially good substrates and the enzyme should thus be suitable for the esterification of the polyphenolic substrates used in the present study. Furthermore, this enzyme can preferably be used at quite low thermodynamic water activities, thus making it possible to achieve very high yields in esterification reactions (Petersson et al., 2007). The molar ratios taken for phenolic compound to ␣-lipoic acid was 1:1.2 and the biocatalyst used was 15% based on the total weight of substrates to ensure complete conversion of the phenolic substrates. The solubility of both the phenolics and lipoic acid was tested in solvents such as hexane, toluene, acetone and tertiary butanol for carrying out the enzymatic reaction. The solvent of choice was a mixture of 2-butanone and n-hexane. It was earlier reported that solvent mixtures composed of 2-butanone and hexane were useful for the esterification reactions using lipase (Sabally
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Fig. 1. Structures of the prepared novel esters of phenolics and ␣-lipoic acid.
et al., 2005). Solvent mixtures were chosen to get complete dissolution of the substrate mixture. The solubility increased with increasing concentration of 2-butanone and solvent ratios were chosen to be 2:1 for compounds C and D and 1:1 for compounds A and B. The reaction proceeded smoothly at ambient temperatures (25 ◦ C) until complete conversion of the phenol substrate resulting in the formation of expected products in satisfactory yields without any formation of by-products. No esterification of the phenolic hydroxyl groups was observed. The losses in the work up procedure could most probably be reduced by further optimization. The yields obtained demonstrate that the lipase from C. antarctica (Novozym 435) efficiently catalyzed the esterification reaction between the two non-natural substrates without any difficulty in the selected organic medium. In order to study the effect of additional phenolic groups, compounds E and F were prepared by aromatic hydroxylation of compounds A and C respectively with IBX followed by reduction with Na2 S2 O4 with good isolated yields (Scheme 2). Hydroxylation reactions can be conducted using enzymatic methods as well. However, these monooxygenase-catalyzed reactions are in most cases still not efficient enough to compete with chemical methods (Leak
et al., 2009). All the synthesized compounds were purified by silica gel column chromatography and characterized by TLC, IR, 1 H NMR and high resolution mass spectrometry. The reaction systems employed in the synthesis do not involve toxic reagents or catalysts and are therefore attractive steps in environmental friendly production of biologically active compounds. Previous studies have shown that Novozym 435 can be re-used several times, thus making the methodology even more attractive (Hagström et al., 2009). 3.2. Antioxidant activity The prepared phenolic esters of lipoic acid were tested for antioxidant activity employing two methods. One is the widely used DPPH radical scavenging assay and the other method measures the inhibition of the oxidation in a fish oil emulsion system. Antioxidants act by different mechanisms and scavenging of free radicals is one important mechanism. Though the DPPH radical is not identical to the radical species occurring in biological systems, it gives information on the general free radical scavenging capacity. It can be observed in Table 1 that all of the prepared
Scheme 1. Enzymatic synthesis of lipoic acid ester of 4-hydroxybenzyl alcohol (A).
Scheme 2. Synthetic route employed for compounds E and F.
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Table 1 Free radical scavenging activity, FRSA (%) of the novel esters and the reference antioxidants as measured by DPPH radical assay. Compound
FRSA (%) ± S.D.
86.37 91.11 18.75 44.54 15.19 83.95 77.25 90.92
% Inhibition ± S.D
Compound
1 mM BHT ␣-Tocopherol A B C D E F
Table 2 Antioxidant activity of the novel esters and reference antioxidants in fish oil emulsion system as measured by the TBARS assay.
2 mM ± ± ± ± ± ± ± ±
0.08 0.07 0.37 0.07 0.28 0.34 0.28 0.08
90.75 93.25 23.91 61.75 22.83 91.15 91.95 92.40
1 mM ± ± ± ± ± ± ± ±
0.27 0.07 0.47 0.2 0.8 0.13 0.27 0.08
novel derivatives were showing activity in the DPPH radical scavenging assay. It has been reported that the antioxidant activity of the phenolic compounds depends on their molecular structure, especially on their hydrogen-donating ability and subsequent stabilization of the formed phenoxy radical (Borges et al., 2000). Compounds A and C were showing least activity, and compound B was showing moderate activity whereas compounds D, E and F were showing very good scavenging activities. Infact D, E and F were showing activities close to that of the reference compounds. The free radical scavenging activity was in the following order: ␣tocopherol > F > BHT > D > E > B > A > C. It is interesting to note that the aromatic hydroxylation of A and C which resulted in compounds E and F greatly improved their activity compared to the precursors. This increase in activity can be explained on the basis of their structure as these derivatives posses two phenolic hydroxyls which are available as hydrogen donors to the DPPH radical. It is especially remarkable that compound C which was showing the least activity increased its activity dramatically when converted into compound F which was the most efficient radical scavenger among the esters synthesized. Methoxy groups (in compounds B and D) also increased the radical scavenging activity, but not as much as the hydroxyl groups. Compound D was considerably more effective than compound B, maybe due to its additional double bond. Among all the tested compounds, the hydroxytyrosol ester of lipoic acid, compound F exhibited the highest radical scavenging activity which was similar to ␣-tocopherol and better than the other commercial reference antioxidant, BHT in both the tested concentrations. Hydroxytyrosol derivatives have been reported to exhibit good antiproliferative effect on human colon cancer cells as well (Bernini et al., 2011). Most of the lipids in foods are present as emulsions and oxidation takes place at a faster rate at the oil water interface of emulsions than in bulk lipid systems. Therefore, we decided to test the efficiency of the prepared compounds as antioxidants in an emulsion system containing fish oil. Oxidation products were measured by the TBARS assay and the antioxidant activity of the test compounds were compared with those of the standard antioxidants, BHT and ␣-tocopherol. It can be observed in Table 2, that all of the prepared derivatives showed antioxidant activity in the tested system. The order of antioxidant efficacy in
BHT ␣-Tocopherol A B C D E F
54.50 57.28 39.20 48.14 50.13 56.29 23.11 57.08
2 mM ± ± ± ± ± ± ± ±
0.28 0.84 1.68 1.4 0.28 4.49 0.28 3.83
61.85 64.43 49.93 50.52 58.07 63.44 35.62 65.29
± ± ± ± ± ± ± ±
2.8 2.53 0.56 0.28 1.97 1.12 1.68 1.79
inhibition of lipid peroxidation decreased in the following order; ␣-tocopherol = F > BHT > D > C > B > A > E. The results are a bit more difficult to rationalize compared to those from the DPPH assay. Comparing compounds A, B and E, the methoxy group seems to be the most efficient substituent, while the extra hydroxyl group instead causes a decrease in antioxidant efficiency. On the other hand, when comparing compounds C and F, the hydroxyl group has a clear positive influence. Compound D is the second most efficient of the esters synthesized. This may be attributed to the presence of the cinnamic double bond in the molecule along with a methoxyl substituent at ortho position to hydroxyl, which is reported to increase the antioxidant activity efficiency (Scaccini et al., 1999). It is remarkable that the small structural difference between compounds E and F causes such a large difference in antioxidant activity in the emulsion assay. Among all the tested compounds, F was exhibiting higher activity than the other derivatives including BHT and it is interesting to note that it was showing equal activity as tocopherol at both the tested concentrations. This could be due to the difference in the partitioning of antioxidants between different phases of the emulsions due to their amphiphilic nature, in accordance with previous studies on similar systems (Jacobsen, 2010). Tocopherols are reported to be more active than their hydrophilic analogues due to their surface active nature and lipophilic character. Compound F had excellent antioxidant activity in both assays. Its antioxidative properties might be part of the reason of the interesting biological activity of this substance in other systems. In order to get indications concerning the potential applicability of the synthesized derivatives as drugs or prodrugs, theoretical calculations of molecular properties were carried out using the Molinspiration software and are presented in Table 3. The calculated log P values show that the synthesized derivatives had higher hydrophobicity than the parent compounds. The increased lipophilicity of the studied phenolics is an advantageous modification for an antioxidant to act at the water–lipid interface to prevent the initial reaction between aqueous radicals and lipid components (Borges et al., 2010). However, the log P value should not be too high. According to Lipinski’s rule-of-five, molecules should have log P values of ≤5 in order to be readily bioavailable (Lipinski et al., 1997). All the esters synthesized obey this rule (Table 3).
Table 3 Physico-chemical properties of the novel esters calculated theoretically. Properties
Compound A
Log P Mol wt n ON n OHNH
3.678 312.456 3 1
B 3.496 342.482 4 1
C 3.887 326.483 3 1
D 4.252 368.52 4 1
E 3.188 328.455 4 2
F 3.398 342.482 4 2
Log P, logarithm of the octanol-water partition coefficient; n ON, number of hydrogen acceptors (sum of O and N atoms); n OHNH, number of hydrogen bond donors (sum of OH and NH groups). All the values were calculated using Molinspiration program.
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Furthermore, Lipinski’s rule says that the molecular weight should be below 500, the number of hydrogen bond acceptors should not be more than 5 and the number of hydrogen bond donors should not be more than 5 (Lipinski et al., 1997). These numbers are the upper limits for drugs to be able to penetrate through biomembranes. Compounds A–F all have molecular weights below 500 and only 3–4 hydrogen bond acceptors. Furthermore, the number of hydrogen bond donors, hydroxyl and methoxyl groups are only 1–2 (Table 3). These values suggest that the molecules can have good absorption and permeation and oral bioavailability (Lipinski et al., 1997). Therefore, the prepared derivatives could find applications as pro drugs as the cleavage of the molecules will result in release of compounds which are both beneficial for human health. In conclusion, an efficient chemo-enzymatic route for the synthesis of novel esters of natural phenolics and ␣-lipoic acid is described. The data reported in the present study suggests that the ␣-lipoic acid–phenolic derivatives are effective antioxidants which can inhibit lipid oxidation and scavenge radicals. The synthetic protocol is simple, efficient and proceeded smoothly at ambient temperatures with excellent isolated yields of all the products. References Adlercreutz, P., Mbatia, B., Kaki, S.S., Mattiasson, B., Mulaa, F., 2011. Enzymatic synthesis of lipophilic rutin and vanillyl esters from fish by products. J. Agric. Food Chem. 59, 7021–7027. Adlercreutz, P., 2000. Biocatalysis in non-conventional media. In: Straathof, A.J.J., Adlercreutz, P. (Eds.), Applied Biocatalysis. , 2nd ed. Harwood Academic Publishers, Amsterdam, pp. 295–316. Akowuah, G.A., Zhari, I., Norhayati, I., Mariam, A., 2006. HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata. J. Food Comp. Anal. 19, 118–126. Bernini, R., Crisante, F., Merendino, N., Molinari, R., Soldatelli, M.C., Velotti, F., 2011. Synthesis of a novel ester of hydroxytyrosol and alpha-lipoic acid exhibiting an antiproliferative effect on human colon cancer HT-29 cells. Eur. J. Med. Chem. 46, 439–446. Bernini, R., Mincione, E., Barontini, M., Crisante, F., 2008. Convenient synthesis of hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol. J. Agric. Food Chem. 56, 8897–8904. Borges, F., Silva, F.A.M., Guimaraes, C., Lima, J.L.F.C., Matos, C., Reis, S., 2000. Phenolic acids and derivatives: studies on the relationship among structure, radical scavenging activity, and physicochemical parameters. J. Agric. Food Chem. 48, 2122–2126. Borges, F., Roleira, F.M.F., Siquet, C., Orru, E., Garrido, E.M., Garrido, J., Milhazes, N., Podda, G., Paiva-Martins, F., Reis, S., Carvalho, R.A., da Silva, E.J.T., 2010. Lipophilic phenolic antioxidants: correlation between antioxidant profile, partition coefficients and redox properties. Bioorg. Med. Chem. 18, 5816–5825. Chou, T.C., Lai, Y.S., Shih, C.Y., Huang, Y.F., 2010. Antiplatelet activity of alpha-lipoic acid. J. Agric. Food Chem. 58, 8596–8603. Figueroa-Espinoza, M.C., Villeneuve, P., 2005. Phenolic acids enzymatic lipophilization. J. Agric. Food Chem. 53, 2779–2787. Guzel, O., Karali, N., Ozsoy, N., Ozbey, S., Salman, A., 2010. Synthesis of new spiroindolinones incorporating a benzothiazole moiety as antioxidant agents. Eur. J. Med. Chem. 45, 1068–1077.
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Hagen, T.M., Smith, A.R., Shenvi, S.V., Widlansky, M., Suh, J.H., 2004. Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr. Med. Chem. 11, 1135–1146. Hagström, A., Nordblad, M., Adlercreutz, P., 2009. Biocatalytic polyester acrylation – process optimization and enzyme stability. Biotechnol. Bioeng. 102, 693–699. Igglessi-Markopoulou, O., Melagraki, G., Afantitis, A., Detsi, A., Koufaki, M., Kontogiorgis, C., Hadjipavlou-Litina, D.J., 2009. Synthesis and evaluation of the antioxidant and anti-inflammatory activity of novel coumarin-3aminoamides and their alpha-lipoic acid adducts. Eur. J. Med. Chem. 44, 3020–3026. Jacobsen, C., 2010. Enrichment of foods with omega-3 fatty acids: a multidisciplinary challenge. Ann. N. Y. Acad. Sci. 1190, 141–150. Kermasha, S., Sabally, K., Karboune, S., St-Louis, R., 2006. Lipase-catalyzed transesterification of dihydrocaffeic acid with flaxseed oil for the synthesis of phenolic lipids. J. Biotechnol. 127, 167–176. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241–246. Lawrence Lowell, J., Eisenberg, R.L., 2007. Novel esters of lipoic acid. US Patent No. 2007/0055070 A1. Lazos, E.S., Lafka, T.I., Lazou, A.E., Sinanoglou, V.J., 2011. Phenolic and antioxidant potential of olive oil mill wastes. Food Chem. 125, 92–98. Leak, D.J., Sheldon, R.A., John, M.W., Adlercreutz, P., 2009. Biocatalysts for selective introduction of oxygen. Biocatal. Biotransform. 27, 1–26. Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 1997. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliver. Rev. 23, 3–25. Nagendran Balasundram, K.S., Samman, S., 2006. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem. 99, 191–203. Packer, L., Witt, E.H., Tritschler, H.J., 1995. Alpha-lipoic acid as a biological antioxidant. Free Radic. Biol. Med. 19, 227–250. Petersson, A.E.V., Adlercreutz, P., Bo, M., 2007. A water activity control system for enzymatic reactions in organic media. Biotechnol. Bioeng. 97, 235–241. Quideau, S., Ralph, J., 1992. Facile large-scale synthesis of coniferyl, sinapyl, and para-coumaryl alcohol. J. Agric. Food Chem. 40, 1108–1110. Rupasinghe, H.P.V., Huber, G.M., Shahidi, F., 2009. Inhibition of oxidation of omega3 polyunsaturated fatty acids and fish oil by quercetin glycosides. Food Chem. 117, 290–295. Sabally, K., Karboune, S., Yebaoh, F.K., Kermasha, S., 2005. Enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol in organic solvent media. Biocatal. Biotransform. 23, 37–44. Scaccini, C., Natella, F., Nardini, M., Di Felice, M., 1999. Benzoic and cinnamic acid derivatives as antioxidants: structure–activity relation. J. Agric. Food Chem. 47, 1453–1459. Shahidi, F., Wanasundara, U.N., 1998. Antioxidant and pro-oxidant activity of green tea extracts in marine oils. Food Chem. 63, 335–342. Shahidi, F., Zhong, Y., 2010. Novel antioxidants in food quality preservation and health promotion. Eur. J. Lipid Sci. Technol. 112, 930–940. Villeneuve, P., 2007. Lipases in lipophilization reactions. Biotechnol. Adv. 25, 515–536. Viskupicova, J., Danihelova, M., Ondrejovic, M., Liptaj, T., Sturdik, E., 2010. Lipophilic rutin derivatives for antioxidant protection of oil-based foods. Food Chem. 123, 45–50. Zhang, W.J., Bird, K.E., McMillen, T.S., LeBoeuf, R.C., Hagen, T.M., Frei, B., 2008. Dietary alpha-lipoic acid supplementation inhibits atherosclerotic lesion development in apolipoprotein E-deficient and apolipoprotein E/low-density lipoprotein receptor-deficient mice. Circulation 117, 421–428. Zhu, L.M., Zheng, Y., Wu, X.M., Branford-White, C., Ning, X., Quan, J., 2009. Enzymatic synthesis and characterization of novel feruloylated lipids in selected organic media. J. Mol. Catal. B: Enzym. 58, 65–71.
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and ␣-lipoic acid Shiva Shanker Kaki, Carl Grey, Patrick Adlercreutz ∗ Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 16 August 2011 Received in revised form 17 October 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: ␣-Lipoic acid Phenolics Antioxidant Synthesis Lipase
a b s t r a c t Chemo-enzymatic synthesis of six esters of natural phenolics and ␣-lipoic acid was carried to produce novel compounds with potential bioactivity. The synthetic route was mild, simple, and efficient with satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay and inhibition of lipid oxidation in fish oil emulsion system. Among the prepared derivatives, three compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alphatocopherol in the DPPH radical scavenging assay, where as in fish oil emulsion system, two derivatives showed activity, which was similar to the reference antioxidants. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Phenolic compounds, one of the most widely occurring groups of phytochemicals, are of considerable interest as they are reported to exhibit anti-allergenic, anti-artherogenic, anti-inflammatory, anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects (Nagendran and Samman, 2006; Lazos et al., 2011). In recent years there has been a growing interest to produce molecules by lipophilization of phenolic compounds with fatty acids and the product molecules thus produced are reported to have improved function as antioxidants for food and pharma applications (Figueroa-Espinoza and Villeneuve, 2005). The molecules thus produced can have the beneficial effects of both the parent compounds and also help in the delivery and uptake of the compounds. Also, synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertbutylhydroquinone (TBHQ) have been used widely as antioxidants in foods, but concerns over the safety of their use have led towards interests in natural antioxidants (Shahidi and Wanasundara, 1998). This has led to an increased interest in research related to modification of the natural phenolic compounds in order to produce molecules with a wide range of applications in food and pharma industries. The examples include the combination of different fatty acids and phenolics such as ferulic acid, dihydrocaffeic acid,
∗ Corresponding author. E-mail address: [email protected] (P. Adlercreutz). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.11.012
rutin, and vanillyl alcohol (Kermasha et al., 2006; Zhu et al., 2009; Viskupicova et al., 2010; Adlercreutz et al., 2011). Another important molecule which shows antioxidant and anticancer properties is lipoic acid. It has been reported that lipoic acid is used to treat various diseases such as atherosclerosis, thrombosis and diabetes. It is used as a dietary supplement (Hagen et al., 2004; Zhang et al., 2008). Recently it was reported that lipoic acid can inhibit the formation of reactive oxygen species and cyclooxygenase-1 activity which are stimulated by arachidonic acid and it may also exhibit a therapeutic benefit in treatment of platelet hyperaggregation-based diseases (Chou et al., 2010). A number of experimental and clinical studies have been carried out which showed that ␣-lipoic acid is potentially useful as a therapeutic agent in conditions related to diabetes, ischemia-reperfusion injury, heavy-metal poisoning, radiation damage, neurodegeneration and HIV infection (Packer et al., 1995). It has been reported that molecular combinations obtained by joining two biologically active molecules can result in novel compounds with enhanced biologically activities (IgglessiMarkopoulou et al., 2009; Shahidi and Zhong, 2010). Therefore, the combination of these two natural biologically active molecules into a same structure could result in the formation of novel hybrid molecules with significant biological activity. The aim of the present study was to synthesize novel esters involving phenolics and ␣-lipoic acid. Six esters of lipoic acid with natural phenolics (A–F) were chemo-enzymatically prepared. Two of those are already known compounds (C and F) and F was reported to exhibit antiproliferative effect on human colon cancer HT-29 cells superior to the compounds from which it was derived (Bernini
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et al., 2011). The earlier chemical synthesis reported involved about four to five steps and therefore, we thought it would be more advantageous to combine the functional phenolics enzymatically with ␣-lipoic acid. The enzyme catalyzed route would be more favourable compared to chemical synthesis for such biologically active compounds because of the mild reaction conditions and the selectivity of enzymes, which lead to high product yield and purity. The use of organic media for enzymatic reactions makes it possible to carry out reversed hydrolytic reaction, such as ester synthesis, very efficiently (Adlercreutz, 2000; Klibanov, 2001). Lipases are particularly useful because they are able to convert many non-natural substrates. There are several reports on modification of natural phenolics to produce new products with multifunctional properties for use in food, pharma and cosmetic industries (Villeneuve, 2007).
2.2.1. Compound A ((4-hydroxyphenyl) methyl 5-(1,2-dithiolan-3-yl) pentanoate) 4-Hydroxybenzyl alcohol (1 mmol; 124.2 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2butanone:hexane; 2:1 (v/v)). The reaction was carried out as described above and compound A was obtained as oily liquid in 68% yield (212 mg). 1 H NMR (CDCl 400 MHz): ı 1.35–1.55 (m, 2H, CH ), 1.65–1.75 3 2 (m, 4H, 2 × CH2 ), 1.9 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.2 Hz, COCH2 ), 2.5 (m, 1H, CH2 ), 3.10–3.25 (m, 2H), 3.6 (m, 1H, CH), 5.0 (s, 2H, CH2 O), 5.1 (s, 1H, OH), 6.8–6.85 (d, 2H, J = 6.8 Hz, Ph), 7.25–7.3 (2H, J = 6.8 Hz, Ph). IR (cm−1 ): 3460, 2994, 2911, 1724, 1435, 1406. HR-MS (EI, m/z) [M+Na]+ calculated for C15 H20 O3 S2 Na 335.0752; Found: 335.0997.
2. Materials and methods
2.2.2. Compound B ((4-hydroxy-3-methoxy-phenyl) methyl 5-(1,2-dithiolan-3-yl) pentanoate) Vanillyl alcohol (1 mmol; 154 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2-butanone:hexane; 1:1 (v/v)). The reaction was carried out as described above and compound B was obtained as oily liquid in 64% yield (220 mg). 1 H NMR (CDCl 400 MHz): ı 1.45–1.5 (m, 2H, CH ), 1.65–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.6 Hz, CH2 ), 2.5 (m, 1H, CH2 ), 3.05–3.15 (m, 2H), 3.55–3.6 (m, 1H, CH), 3.9 (s, 3H, OCH3 ), 5.05 (s, 2H, CH2 O), 5.65 (s, 1H, OH), 6.85–6.9 (m, 3H, Ph). IR (cm−1 ): 3427, 2929, 2854, 1724, 1515, 1452. HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O4 S2 Na 365.0857; Found: 365.0741.
2.1. Materials and enzymes ␣-Lipoic acid, vanillyl alcohol, 4-hydroxybenzyl alcohol, 4hydroxyphenylethyl alcohol, ferulic acid, acetyl chloride, IBX, diisobutyl aluminium hydride and sodium dithionite were purchased from Sigma (Sigma–Aldrich Sweden AB, Stockholm, Sweden). Immobilized lipase B from Candida antarctica (Novozym 435) was a generous gift from Novozymes A/S (Bagsvaerd, Denmark). Tuna fish oil was supplied by LYSI, Reykjavik, Iceland. All other solvents and reagents were of highest grade of purity available and were purchased from VWR International Sweden (Stockholm, Sweden). Coniferyl alcohol was synthesized from ferulic acid employing a reported protocol in which ferulic acid was converted to ethyl ferulate and consequently reduced to coniferyl alcohol using DIBAL (Quideau and Ralph, 1992). The spectral data was similar to the published data and the pure isolated compound was used for the enzymatic reaction. The structures of the purified products were determined by 1 H NMR using 400 MHz NMR (Bruker, UltraShield Plus 400, Germany). Mass spectral analysis was carried out on hybrid QStar Pulsar quadrupole time-of-flight mass spectrometer (PE Sciex Instruments, Toronto, Canada), equipped with a nano electrospray ionization (ESI) source. IR spectra were recorded on a Perkin-Elmer spectrometer. Antioxidant assays were performed using PerkinElmer Lambda Bio+ spectrophotometer. Enzymatic reactions were carried out on a thermo shaker (MKR 13, HLC BioTech, Bovenden, Germany).
2.2. Synthesis of compounds A–D as depicted in Scheme 1
2.2.3. Compound C (2-(4-hydroxyphenyl) ethyl 5-(1,2-dithiolan-3-yl) pentanoate) 4-Hydroxyphenyl ethanol (1 mmol; 138.2 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2butanone:hexane; 1:1 (v/v)). The reaction was carried out as described above and compound C was obtained as oily liquid in 80% yield (260 mg). 1 H NMR data was consistent with the previously reported data (15) and is as follows: 1 H NMR (CDCl 400 MHz): ı 1.35–1.50 (m, 2H, CH ), 1.60–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.28 (t, 2H, J = 7.6 Hz, CH2 ), 2.45–2.50 (m, 1H, CH2 ), 2.9 (t, 2H, J = 6.8 Hz, CH2 ), 3.10–3.25 (m, 2H), 3.50–3.60 (m, 1H, CH), 4.25 (t, 2H, J = 6.8 Hz, CH2 O), 4.90 (s, 1H, OH), 6.75–6.80 (d, 2H, J = 6.4 Hz, Ph), 7.10 (2H, J = 6.4 Hz, Ph). IR (cm−1 ): 3362, 2929, 2858, 1726, 1515, 1436. HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O3 S2 Na 349.0908; Found: 349.0958.
Enzymatic synthesis was carried out as follows. To a mixture of phenolic compound (1 mmol) and lipoic acid (1.2 mmol) in mixtures of 2-butanone: hexane (v/v, 6 mL) was added lipase from C. antarctica (15% based on total substrate weight). l-Cysteine (0.5%) was added to inhibit the polymerization of lipoic acid (Lawrence Lowell and Eisenberg, 2007). The reaction mixture was shaken at 25 ◦ C for 15 h. At the end of the reaction, enzyme was filtered and to the reaction mixture ethyl acetate (10 mL) was added and washed with 5% sodium bicarbonate. The aqueous phase was extracted with ethyl acetate (2 × 10 mL) and the combined organic phases were washed with water and finally with saturated sodium chloride solution and dried over anhydrous sodium sulphate. The crude product was purified on a silica gel column chromatography with ethyl acetate:hexane (2:1, v/v) to obtain the pure ester compound as an oily liquid.
2.2.4. Compound D ([(E)-3-(4-hydroxy-3-methoxy-phenyl) allyl] 5-(1,2-dithiolan-3-yl) pentanoate) Coniferyl alcohol (1 mmol; 180 mg) and lipoic acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2-butanone:hexane; 2:1 (v/v)). The reaction was carried out as described above and compound D was obtained as oily liquid in 70% yield (258 mg). 1 H NMR (CDCl 400 MHz): ı 1.45–1.55 (m, 2H, CH ), 1.60–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.35 (t, 2H, J = 7.6 Hz, CH2 ), 2.5 (m, 1H, CH2 ), 3.15–3.25 (m, 2H), 3.55–3.65 (m, 1H, CH), 3.9 (s, 3H, OCH3 ), 4.7 (d, 2H, J = 6.8 Hz, CH2 O), 5.6 (s, 1H, OH), 6.15–6.2 (m, 1H, CH), 6.6 (d, 1H, J = 16 Hz, CH), 6.85–6.95 (m, 3H, Ph). IR (cm−1 ): 3433, 2932, 2855, 1724, 1594, 1450. HR-MS (EI, m/z) [M+Na]+ calculated for C18 H24 O4 S2 Na 391.1014; Found: 391.1169.
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2.3. Synthesis of compounds E and F as depicted in Scheme 2 The compounds E and F were prepared by aromatic hydroxylation of compounds A and C respectively following a reported protocol with slight modification (Bernini et al., 2008). Briefly, to the substrate (0.5 mmol) in methanol (5 mL) at 0 ◦ C was added IBX (0.6 mmol; 374 mg of 45% IBX) and the mixture was magnetically stirred until the end of the reaction time (3 h). Then, water (5 mL) and sodium dithionite (1.2 mmol; 209 mg) were added and the mixture was further stirred for 10 min at room temperature. Solvent was evaporated and the residue was solubilized in ethyl acetate (15 mL) and treated with saturated sodium bicarbonate (15 mL). The aqueous phase was extracted with ethyl acetate (2 × 15 mL) and the combined organic phases were washed with sodium chloride solution and dried over anhydrous sodium sulphate. The crude reaction product was purified by silica gel chromatography with ethyl acetate: hexane (2:1, v/v) to obtain compounds E and F as oily liquids in 65% (106 mg) and 68% (120 mg) yields respectively. 2.3.1. Compound E: (3,4-dihydroxyphenyl) methyl 5-(1,2-dithiolan-3-yl) pentanoate 1 H NMR (CDCl 400 MHz): ı 1.35–1.40 (m, 2H, CH ), 1.62–1.73 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.35 (t, 2H, J = 7.2 Hz, CH2 ), 2.45–2.50 (m, 1H, CH2 ), 3.10–3.25 (m, 2H), 3.5–3.6 (m, 1H, CH), 5.02 (s, 2H, CH2 O), 6.8–6.95 (m, 3H, Ph). IR (cm−1 ): 3374, 2930, 2857, 1703, 1520, 1445. HR-MS (EI, m/z) [M+Na]+ calculated for C15 H20 O4 S2 Na 351.0701; Found: 351.0807. 2.3.2. Compound F: 2-(3,4-dihydroxyphenyl) ethyl 5-(1,2-dithiolan-3-yl) pentanoate 1 H NMR data was consistent with the previously reported data (15) and is as follows: 1 H NMR (CDCl 400 MHz): ı 1.35–1.45 (m, 2H, CH ), 1.60–1.75 3 2 (m, 4H, 2 × CH2 ), 1.85–1.95 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.2 Hz, CH2 ), 2.45–2.50 (m, 1H, CH2 ), 2.85 (t, 2H, J = 6.8 Hz, CH2 ), 3.15–3.25 (m, 2H), 3.53–3.60 (m, 1H, CH), 4.3 (t, 2H, J = 6.8 Hz, CH2 O), 5.3 (s, 1H, OH), 6.65–6.85 (m, 3H, Ph). IR (cm−1 ): IR (cm−1 ): 3400, 2945, 2870, 1701, 1530, 1465. HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O4 S2 Na 365.0857; Found: 365.0888. 2.4. Determination of antioxidant activity 2.4.1. DPPH radical assay The DPPH radical scavenging activities of the synthesized lipoic acid derivatives were measured according to a reported method with slight modification (Akowuah et al., 2006). DPPH is a commercially available stable free radical (dark purple color), which is reduced to DPPH (light yellow colored) by reacting with an antioxidant. A 0.1 mL aliquot of each prepared compound or reference antioxidants (1 or 2 mM) in ethanol: DMSO (95:5; v/v) were added to 2 mL of ethanolic DPPH solution (0.1 mM) and the volume was made up to 3 mL with the same solvent mixture. The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 60 min. The decrease in absorbance of the resulting solution was then measured spectrophotometrically at 517 nm against ethanol containing DMSO as blank and the control sample had all the reagents except test sample. BHT and ␣-tocopherol served as reference compounds. All measurements were made in triplicate and averaged. The ability to scavenge DPPH radical was calculated by the following equation: radical scavenging activity (FRSA) Free (%) = [(Ac − As )/(Ac )] × 100, where As is the absorbance of sample and Ac is the absorbance of control.
2.4.2. Inhibition of lipid oxidation Oxidation was induced in a fish oil emulsion, with or without the addition of antioxidant, and the level of oxidation was quantified by measuring the thiobarbuturic acid reactive substances (TBARS) using reported methods with slight modifications (Rupasinghe et al., 2009; Guzel et al., 2010). Fish oil (5 mg/mL) in a buffer solution (0.05 M TRIS–HCl, 0.15 M KCl, 1% Tween 20, pH 7.0) was sonicated at 30 s intervals for 2 min by placing the glass tubes in a sonicator to form an opalescent emulsion system. The sonicated stock emulsion was divided into 0.5 mL aliquots and the emulsion was maintained by shaking the tubes at 200 rpm. The reference and test antioxidants, 100 L of 0.2 mM FeCl3 were added to the emulsion system and incubated at 37 ◦ C for 30 min and the inhibition of lipid oxidation was determined by the TBARS assay. Briefly, the oxidized emulsion sample was mixed with 1 mL of TBA–TCA reagent (15% (w/v) TCA and 0.375 (w/v) TBA in 0.25 M HCl) and heated in a boiling water bath for 20 min. The samples were then cooled on ice and centrifuged for 10 min and the absorbance of the supernatant was recorded at 532 nm against a control which contained all reagents except the test compounds. BHT and ␣-tocopherol served as reference compounds and all measurements were made in triplicate and averaged. Inhibition of lipid oxidation was calculated using the formula: % inhibition = (1 − (As /Ac ) × 100, where As is the absorbance of sample and Ac is the absorbance of control. 2.5. Calculation of partition coefficient (log P values) The lipophilicity and the physico-chemical parameters and the theoretical drug likeness properties of the synthesized derivatives were determined using the Molinspiration software available online at http://www.molinspiration.com/cgi-bin/properties (MolinspirationCheminformatics, Bratislava, Slovak Republic). 3. Results and discussion 3.1. Synthesis and characterization The present work describes the chemo-enzymatic synthesis of ␣-lipoic acid esters of phenolic compounds (Fig. 1). To the best of our knowledge this is the first report on the enzymatic esterification of lipoic acid with phenolics for the synthesis of potential antioxidant molecules. The prepared molecules can have beneficial effects due to the modification in their structure causing effects on solubility and bioavailability. The synthetic routes employed are shown in Schemes 1 and 2. The phenolic compounds were enzymatically esterified with ␣-lipoic acid with the immobilized lipase B from C. antarctica (Novozym 435). This enzyme is known to be a very efficient catalyst for esterification of a wide range of alcohols. Primary alcohols are especially good substrates and the enzyme should thus be suitable for the esterification of the polyphenolic substrates used in the present study. Furthermore, this enzyme can preferably be used at quite low thermodynamic water activities, thus making it possible to achieve very high yields in esterification reactions (Petersson et al., 2007). The molar ratios taken for phenolic compound to ␣-lipoic acid was 1:1.2 and the biocatalyst used was 15% based on the total weight of substrates to ensure complete conversion of the phenolic substrates. The solubility of both the phenolics and lipoic acid was tested in solvents such as hexane, toluene, acetone and tertiary butanol for carrying out the enzymatic reaction. The solvent of choice was a mixture of 2-butanone and n-hexane. It was earlier reported that solvent mixtures composed of 2-butanone and hexane were useful for the esterification reactions using lipase (Sabally
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Fig. 1. Structures of the prepared novel esters of phenolics and ␣-lipoic acid.
et al., 2005). Solvent mixtures were chosen to get complete dissolution of the substrate mixture. The solubility increased with increasing concentration of 2-butanone and solvent ratios were chosen to be 2:1 for compounds C and D and 1:1 for compounds A and B. The reaction proceeded smoothly at ambient temperatures (25 ◦ C) until complete conversion of the phenol substrate resulting in the formation of expected products in satisfactory yields without any formation of by-products. No esterification of the phenolic hydroxyl groups was observed. The losses in the work up procedure could most probably be reduced by further optimization. The yields obtained demonstrate that the lipase from C. antarctica (Novozym 435) efficiently catalyzed the esterification reaction between the two non-natural substrates without any difficulty in the selected organic medium. In order to study the effect of additional phenolic groups, compounds E and F were prepared by aromatic hydroxylation of compounds A and C respectively with IBX followed by reduction with Na2 S2 O4 with good isolated yields (Scheme 2). Hydroxylation reactions can be conducted using enzymatic methods as well. However, these monooxygenase-catalyzed reactions are in most cases still not efficient enough to compete with chemical methods (Leak
et al., 2009). All the synthesized compounds were purified by silica gel column chromatography and characterized by TLC, IR, 1 H NMR and high resolution mass spectrometry. The reaction systems employed in the synthesis do not involve toxic reagents or catalysts and are therefore attractive steps in environmental friendly production of biologically active compounds. Previous studies have shown that Novozym 435 can be re-used several times, thus making the methodology even more attractive (Hagström et al., 2009). 3.2. Antioxidant activity The prepared phenolic esters of lipoic acid were tested for antioxidant activity employing two methods. One is the widely used DPPH radical scavenging assay and the other method measures the inhibition of the oxidation in a fish oil emulsion system. Antioxidants act by different mechanisms and scavenging of free radicals is one important mechanism. Though the DPPH radical is not identical to the radical species occurring in biological systems, it gives information on the general free radical scavenging capacity. It can be observed in Table 1 that all of the prepared
Scheme 1. Enzymatic synthesis of lipoic acid ester of 4-hydroxybenzyl alcohol (A).
Scheme 2. Synthetic route employed for compounds E and F.
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Table 1 Free radical scavenging activity, FRSA (%) of the novel esters and the reference antioxidants as measured by DPPH radical assay. Compound
FRSA (%) ± S.D.
86.37 91.11 18.75 44.54 15.19 83.95 77.25 90.92
% Inhibition ± S.D
Compound
1 mM BHT ␣-Tocopherol A B C D E F
Table 2 Antioxidant activity of the novel esters and reference antioxidants in fish oil emulsion system as measured by the TBARS assay.
2 mM ± ± ± ± ± ± ± ±
0.08 0.07 0.37 0.07 0.28 0.34 0.28 0.08
90.75 93.25 23.91 61.75 22.83 91.15 91.95 92.40
1 mM ± ± ± ± ± ± ± ±
0.27 0.07 0.47 0.2 0.8 0.13 0.27 0.08
novel derivatives were showing activity in the DPPH radical scavenging assay. It has been reported that the antioxidant activity of the phenolic compounds depends on their molecular structure, especially on their hydrogen-donating ability and subsequent stabilization of the formed phenoxy radical (Borges et al., 2000). Compounds A and C were showing least activity, and compound B was showing moderate activity whereas compounds D, E and F were showing very good scavenging activities. Infact D, E and F were showing activities close to that of the reference compounds. The free radical scavenging activity was in the following order: ␣tocopherol > F > BHT > D > E > B > A > C. It is interesting to note that the aromatic hydroxylation of A and C which resulted in compounds E and F greatly improved their activity compared to the precursors. This increase in activity can be explained on the basis of their structure as these derivatives posses two phenolic hydroxyls which are available as hydrogen donors to the DPPH radical. It is especially remarkable that compound C which was showing the least activity increased its activity dramatically when converted into compound F which was the most efficient radical scavenger among the esters synthesized. Methoxy groups (in compounds B and D) also increased the radical scavenging activity, but not as much as the hydroxyl groups. Compound D was considerably more effective than compound B, maybe due to its additional double bond. Among all the tested compounds, the hydroxytyrosol ester of lipoic acid, compound F exhibited the highest radical scavenging activity which was similar to ␣-tocopherol and better than the other commercial reference antioxidant, BHT in both the tested concentrations. Hydroxytyrosol derivatives have been reported to exhibit good antiproliferative effect on human colon cancer cells as well (Bernini et al., 2011). Most of the lipids in foods are present as emulsions and oxidation takes place at a faster rate at the oil water interface of emulsions than in bulk lipid systems. Therefore, we decided to test the efficiency of the prepared compounds as antioxidants in an emulsion system containing fish oil. Oxidation products were measured by the TBARS assay and the antioxidant activity of the test compounds were compared with those of the standard antioxidants, BHT and ␣-tocopherol. It can be observed in Table 2, that all of the prepared derivatives showed antioxidant activity in the tested system. The order of antioxidant efficacy in
BHT ␣-Tocopherol A B C D E F
54.50 57.28 39.20 48.14 50.13 56.29 23.11 57.08
2 mM ± ± ± ± ± ± ± ±
0.28 0.84 1.68 1.4 0.28 4.49 0.28 3.83
61.85 64.43 49.93 50.52 58.07 63.44 35.62 65.29
± ± ± ± ± ± ± ±
2.8 2.53 0.56 0.28 1.97 1.12 1.68 1.79
inhibition of lipid peroxidation decreased in the following order; ␣-tocopherol = F > BHT > D > C > B > A > E. The results are a bit more difficult to rationalize compared to those from the DPPH assay. Comparing compounds A, B and E, the methoxy group seems to be the most efficient substituent, while the extra hydroxyl group instead causes a decrease in antioxidant efficiency. On the other hand, when comparing compounds C and F, the hydroxyl group has a clear positive influence. Compound D is the second most efficient of the esters synthesized. This may be attributed to the presence of the cinnamic double bond in the molecule along with a methoxyl substituent at ortho position to hydroxyl, which is reported to increase the antioxidant activity efficiency (Scaccini et al., 1999). It is remarkable that the small structural difference between compounds E and F causes such a large difference in antioxidant activity in the emulsion assay. Among all the tested compounds, F was exhibiting higher activity than the other derivatives including BHT and it is interesting to note that it was showing equal activity as tocopherol at both the tested concentrations. This could be due to the difference in the partitioning of antioxidants between different phases of the emulsions due to their amphiphilic nature, in accordance with previous studies on similar systems (Jacobsen, 2010). Tocopherols are reported to be more active than their hydrophilic analogues due to their surface active nature and lipophilic character. Compound F had excellent antioxidant activity in both assays. Its antioxidative properties might be part of the reason of the interesting biological activity of this substance in other systems. In order to get indications concerning the potential applicability of the synthesized derivatives as drugs or prodrugs, theoretical calculations of molecular properties were carried out using the Molinspiration software and are presented in Table 3. The calculated log P values show that the synthesized derivatives had higher hydrophobicity than the parent compounds. The increased lipophilicity of the studied phenolics is an advantageous modification for an antioxidant to act at the water–lipid interface to prevent the initial reaction between aqueous radicals and lipid components (Borges et al., 2010). However, the log P value should not be too high. According to Lipinski’s rule-of-five, molecules should have log P values of ≤5 in order to be readily bioavailable (Lipinski et al., 1997). All the esters synthesized obey this rule (Table 3).
Table 3 Physico-chemical properties of the novel esters calculated theoretically. Properties
Compound A
Log P Mol wt n ON n OHNH
3.678 312.456 3 1
B 3.496 342.482 4 1
C 3.887 326.483 3 1
D 4.252 368.52 4 1
E 3.188 328.455 4 2
F 3.398 342.482 4 2
Log P, logarithm of the octanol-water partition coefficient; n ON, number of hydrogen acceptors (sum of O and N atoms); n OHNH, number of hydrogen bond donors (sum of OH and NH groups). All the values were calculated using Molinspiration program.
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Furthermore, Lipinski’s rule says that the molecular weight should be below 500, the number of hydrogen bond acceptors should not be more than 5 and the number of hydrogen bond donors should not be more than 5 (Lipinski et al., 1997). These numbers are the upper limits for drugs to be able to penetrate through biomembranes. Compounds A–F all have molecular weights below 500 and only 3–4 hydrogen bond acceptors. Furthermore, the number of hydrogen bond donors, hydroxyl and methoxyl groups are only 1–2 (Table 3). These values suggest that the molecules can have good absorption and permeation and oral bioavailability (Lipinski et al., 1997). Therefore, the prepared derivatives could find applications as pro drugs as the cleavage of the molecules will result in release of compounds which are both beneficial for human health. In conclusion, an efficient chemo-enzymatic route for the synthesis of novel esters of natural phenolics and ␣-lipoic acid is described. The data reported in the present study suggests that the ␣-lipoic acid–phenolic derivatives are effective antioxidants which can inhibit lipid oxidation and scavenge radicals. The synthetic protocol is simple, efficient and proceeded smoothly at ambient temperatures with excellent isolated yields of all the products. References Adlercreutz, P., Mbatia, B., Kaki, S.S., Mattiasson, B., Mulaa, F., 2011. Enzymatic synthesis of lipophilic rutin and vanillyl esters from fish by products. J. Agric. Food Chem. 59, 7021–7027. Adlercreutz, P., 2000. Biocatalysis in non-conventional media. In: Straathof, A.J.J., Adlercreutz, P. (Eds.), Applied Biocatalysis. , 2nd ed. Harwood Academic Publishers, Amsterdam, pp. 295–316. Akowuah, G.A., Zhari, I., Norhayati, I., Mariam, A., 2006. HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata. J. Food Comp. Anal. 19, 118–126. Bernini, R., Crisante, F., Merendino, N., Molinari, R., Soldatelli, M.C., Velotti, F., 2011. Synthesis of a novel ester of hydroxytyrosol and alpha-lipoic acid exhibiting an antiproliferative effect on human colon cancer HT-29 cells. Eur. J. Med. Chem. 46, 439–446. Bernini, R., Mincione, E., Barontini, M., Crisante, F., 2008. Convenient synthesis of hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol. J. Agric. Food Chem. 56, 8897–8904. Borges, F., Silva, F.A.M., Guimaraes, C., Lima, J.L.F.C., Matos, C., Reis, S., 2000. Phenolic acids and derivatives: studies on the relationship among structure, radical scavenging activity, and physicochemical parameters. J. Agric. Food Chem. 48, 2122–2126. Borges, F., Roleira, F.M.F., Siquet, C., Orru, E., Garrido, E.M., Garrido, J., Milhazes, N., Podda, G., Paiva-Martins, F., Reis, S., Carvalho, R.A., da Silva, E.J.T., 2010. Lipophilic phenolic antioxidants: correlation between antioxidant profile, partition coefficients and redox properties. Bioorg. Med. Chem. 18, 5816–5825. Chou, T.C., Lai, Y.S., Shih, C.Y., Huang, Y.F., 2010. Antiplatelet activity of alpha-lipoic acid. J. Agric. Food Chem. 58, 8596–8603. Figueroa-Espinoza, M.C., Villeneuve, P., 2005. Phenolic acids enzymatic lipophilization. J. Agric. Food Chem. 53, 2779–2787. Guzel, O., Karali, N., Ozsoy, N., Ozbey, S., Salman, A., 2010. Synthesis of new spiroindolinones incorporating a benzothiazole moiety as antioxidant agents. Eur. J. Med. Chem. 45, 1068–1077.
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