Anda di halaman 1dari 6

Journal of Biotechnology 157 (2012) 344349

Contents lists available at SciVerse ScienceDirect

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 a b s t r a c t

Article history: Chemo-enzymatic synthesis of six esters of natural phenolics and -lipoic acid was carried to produce
Received 16 August 2011 novel compounds with potential bioactivity. The synthetic route was mild, simple, and efcient with
Received in revised form 17 October 2011 satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared
Accepted 16 November 2011
derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay
Available online 25 November 2011
and inhibition of lipid oxidation in sh oil emulsion system. Among the prepared derivatives, three
compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alpha-
Keywords:
tocopherol in the DPPH radical scavenging assay, where as in sh oil emulsion system, two derivatives
-Lipoic acid
Phenolics
showed activity, which was similar to the reference antioxidants.
Antioxidant 2011 Elsevier B.V. All rights reserved.
Synthesis
Lipase

1. Introduction rutin, and vanillyl alcohol (Kermasha et al., 2006; Zhu et al., 2009;
Viskupicova et al., 2010; Adlercreutz et al., 2011).
Phenolic compounds, one of the most widely occurring groups Another important molecule which shows antioxidant and anti-
of phytochemicals, are of considerable interest as they are reported cancer properties is lipoic acid. It has been reported that lipoic acid
to exhibit anti-allergenic, anti-artherogenic, anti-inammatory, is used to treat various diseases such as atherosclerosis, throm-
anti-microbial, antioxidant, anti-thrombotic, cardioprotective and bosis and diabetes. It is used as a dietary supplement (Hagen
vasodilatory effects (Nagendran and Samman, 2006; Lazos et al., et al., 2004; Zhang et al., 2008). Recently it was reported that
2011). In recent years there has been a growing interest to pro- lipoic acid can inhibit the formation of reactive oxygen species
duce molecules by lipophilization of phenolic compounds with and cyclooxygenase-1 activity which are stimulated by arachidonic
fatty acids and the product molecules thus produced are reported to acid and it may also exhibit a therapeutic benet in treatment
have improved function as antioxidants for food and pharma appli- of platelet hyperaggregation-based diseases (Chou et al., 2010). A
cations (Figueroa-Espinoza and Villeneuve, 2005). The molecules number of experimental and clinical studies have been carried out
thus produced can have the benecial effects of both the par- which showed that -lipoic acid is potentially useful as a therapeu-
ent compounds and also help in the delivery and uptake of tic agent in conditions related to diabetes, ischemia-reperfusion
the compounds. Also, synthetic antioxidants such as butylated injury, heavy-metal poisoning, radiation damage, neurodegenera-
hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert- tion and HIV infection (Packer et al., 1995).
butylhydroquinone (TBHQ) have been used widely as antioxidants It has been reported that molecular combinations obtained
in foods, but concerns over the safety of their use have led towards by joining two biologically active molecules can result in
interests in natural antioxidants (Shahidi and Wanasundara, 1998). novel compounds with enhanced biologically activities (Igglessi-
This has led to an increased interest in research related to mod- Markopoulou et al., 2009; Shahidi and Zhong, 2010). Therefore,
ication of the natural phenolic compounds in order to produce the combination of these two natural biologically active molecules
molecules with a wide range of applications in food and pharma into a same structure could result in the formation of novel hybrid
industries. The examples include the combination of different molecules with signicant biological activity.
fatty acids and phenolics such as ferulic acid, dihydrocaffeic acid, The aim of the present study was to synthesize novel esters
involving phenolics and -lipoic acid. Six esters of lipoic acid with
natural phenolics (AF) were chemo-enzymatically prepared. Two
of those are already known compounds (C and F) and F was reported
Corresponding author. to exhibit antiproliferative effect on human colon cancer HT-29
E-mail address: Patrick.Adlercreutz@biotek.lu.se (P. Adlercreutz). cells superior to the compounds from which it was derived (Bernini

0168-1656/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2011.11.012
S.S. Kaki et al. / Journal of Biotechnology 157 (2012) 344349 345

et al., 2011). The earlier chemical synthesis reported involved about 2.2.1. Compound A ((4-hydroxyphenyl) methyl
four to ve steps and therefore, we thought it would be more 5-(1,2-dithiolan-3-yl) pentanoate)
advantageous to combine the functional phenolics enzymatically 4-Hydroxybenzyl alcohol (1 mmol; 124.2 mg) and lipoic
with -lipoic acid. The enzyme catalyzed route would be more acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2-
favourable compared to chemical synthesis for such biologically butanone:hexane; 2:1 (v/v)). The reaction was carried out as
active compounds because of the mild reaction conditions and the described above and compound A was obtained as oily liquid in
selectivity of enzymes, which lead to high product yield and purity. 68% yield (212 mg).
The use of organic media for enzymatic reactions makes it possible 1 H NMR (CDCl 400 MHz): 1.351.55 (m, 2H, CH ), 1.651.75
3 2
to carry out reversed hydrolytic reaction, such as ester synthe- (m, 4H, 2 CH2 ), 1.9 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.2 Hz, COCH2 ),
sis, very efciently (Adlercreutz, 2000; Klibanov, 2001). Lipases 2.5 (m, 1H, CH2 ), 3.103.25 (m, 2H), 3.6 (m, 1H, CH), 5.0 (s, 2H,
are particularly useful because they are able to convert many CH2 O), 5.1 (s, 1H, OH), 6.86.85 (d, 2H, J = 6.8 Hz, Ph), 7.257.3 (2H,
non-natural substrates. There are several reports on modication J = 6.8 Hz, Ph).
of natural phenolics to produce new products with multifunc- IR (cm1 ): 3460, 2994, 2911, 1724, 1435, 1406.
tional properties for use in food, pharma and cosmetic industries HR-MS (EI, m/z) [M+Na]+ calculated for C15 H20 O3 S2 Na
(Villeneuve, 2007). 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)
2.1. Materials and enzymes 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
-Lipoic acid, vanillyl alcohol, 4-hydroxybenzyl alcohol, 4- (v/v)). The reaction was carried out as described above and com-
hydroxyphenylethyl alcohol, ferulic acid, acetyl chloride, IBX, pound B was obtained as oily liquid in 64% yield (220 mg).
1 H NMR (CDCl 400 MHz): 1.451.5 (m, 2H, CH ), 1.651.75
diisobutyl aluminium hydride and sodium dithionite were pur- 3 2
chased from Sigma (SigmaAldrich Sweden AB, Stockholm, (m, 4H, 2 CH2 ), 1.851.95 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.6 Hz, CH2 ),
Sweden). Immobilized lipase B from Candida antarctica (Novozym 2.5 (m, 1H, CH2 ), 3.053.15 (m, 2H), 3.553.6 (m, 1H, CH), 3.9 (s, 3H,
435) was a generous gift from Novozymes A/S (Bagsvaerd, OCH3 ), 5.05 (s, 2H, CH2 O), 5.65 (s, 1H, OH), 6.856.9 (m, 3H, Ph).
Denmark). Tuna sh oil was supplied by LYSI, Reykjavik, Iceland. IR (cm1 ): 3427, 2929, 2854, 1724, 1515, 1452.
All other solvents and reagents were of highest grade of purity HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O4 S2 Na
available and were purchased from VWR International Sweden 365.0857; Found: 365.0741.
(Stockholm, Sweden). Coniferyl alcohol was synthesized from fer-
ulic acid employing a reported protocol in which ferulic acid was
converted to ethyl ferulate and consequently reduced to coniferyl 2.2.3. Compound C (2-(4-hydroxyphenyl) ethyl
alcohol using DIBAL (Quideau and Ralph, 1992). The spectral data 5-(1,2-dithiolan-3-yl) pentanoate)
was similar to the published data and the pure isolated compound 4-Hydroxyphenyl ethanol (1 mmol; 138.2 mg) and lipoic
was used for the enzymatic reaction. acid (1.2 mmol, 248 mg) were solubilized 6 mL of solvent (2-
The structures of the puried products were determined by 1 H butanone:hexane; 1:1 (v/v)). The reaction was carried out as
NMR using 400 MHz NMR (Bruker, UltraShield Plus 400, Germany). described above and compound C was obtained as oily liquid in
Mass spectral analysis was carried out on hybrid QStar Pulsar 80% yield (260 mg).
quadrupole time-of-ight mass spectrometer (PE Sciex Instru- 1 H NMR data was consistent with the previously reported data
ments, Toronto, Canada), equipped with a nano electrospray (15) and is as follows:
ionization (ESI) source. IR spectra were recorded on a Perkin-Elmer 1 H NMR (CDCl 400 MHz): 1.351.50 (m, 2H, CH ), 1.601.75
3 2
spectrometer. Antioxidant assays were performed using Perkin- (m, 4H, 2 CH2 ), 1.851.95 (m, 1H, CH2 ), 2.28 (t, 2H, J = 7.6 Hz, CH2 ),
Elmer Lambda Bio+ spectrophotometer. Enzymatic reactions were 2.452.50 (m, 1H, CH2 ), 2.9 (t, 2H, J = 6.8 Hz, CH2 ), 3.103.25 (m, 2H),
carried out on a thermo shaker (MKR 13, HLC BioTech, Bovenden, 3.503.60 (m, 1H, CH), 4.25 (t, 2H, J = 6.8 Hz, CH2 O), 4.90 (s, 1H, OH),
Germany). 6.756.80 (d, 2H, J = 6.4 Hz, Ph), 7.10 (2H, J = 6.4 Hz, Ph).
IR (cm1 ): 3362, 2929, 2858, 1726, 1515, 1436.
HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O3 S2 Na
2.2. Synthesis of compounds AD as depicted in Scheme 1 349.0908; 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 mix- 2.2.4. Compound D ([(E)-3-(4-hydroxy-3-methoxy-phenyl) allyl]
tures of 2-butanone: hexane (v/v, 6 mL) was added lipase from C. 5-(1,2-dithiolan-3-yl) pentanoate)
antarctica (15% based on total substrate weight). l-Cysteine (0.5%) Coniferyl alcohol (1 mmol; 180 mg) and lipoic acid (1.2 mmol,
was added to inhibit the polymerization of lipoic acid (Lawrence 248 mg) were solubilized 6 mL of solvent (2-butanone:hexane; 2:1
Lowell and Eisenberg, 2007). The reaction mixture was shaken at (v/v)). The reaction was carried out as described above and com-
25 C for 15 h. At the end of the reaction, enzyme was ltered and to pound D was obtained as oily liquid in 70% yield (258 mg).
the reaction mixture ethyl acetate (10 mL) was added and washed 1 H NMR (CDCl 400 MHz): 1.451.55 (m, 2H, CH ), 1.601.75
3 2
with 5% sodium bicarbonate. The aqueous phase was extracted (m, 4H, 2 CH2 ), 1.851.95 (m, 1H, CH2 ), 2.35 (t, 2H, J = 7.6 Hz, CH2 ),
with ethyl acetate (2 10 mL) and the combined organic phases 2.5 (m, 1H, CH2 ), 3.153.25 (m, 2H), 3.553.65 (m, 1H, CH), 3.9 (s,
were washed with water and nally with saturated sodium chlo- 3H, OCH3 ), 4.7 (d, 2H, J = 6.8 Hz, CH2 O), 5.6 (s, 1H, OH), 6.156.2 (m,
ride solution and dried over anhydrous sodium sulphate. The crude 1H, CH), 6.6 (d, 1H, J = 16 Hz, CH), 6.856.95 (m, 3H, Ph).
product was puried on a silica gel column chromatography with IR (cm1 ): 3433, 2932, 2855, 1724, 1594, 1450.
ethyl acetate:hexane (2:1, v/v) to obtain the pure ester compound HR-MS (EI, m/z) [M+Na]+ calculated for C18 H24 O4 S2 Na
as an oily liquid. 391.1014; Found: 391.1169.
346 S.S. Kaki et al. / Journal of Biotechnology 157 (2012) 344349

2.3. Synthesis of compounds E and F as depicted in Scheme 2 2.4.2. Inhibition of lipid oxidation
Oxidation was induced in a sh oil emulsion, with or without the
The compounds E and F were prepared by aromatic hydrox- addition of antioxidant, and the level of oxidation was quantied
ylation of compounds A and C respectively following a reported by measuring the thiobarbuturic acid reactive substances (TBARS)
protocol with slight modication (Bernini et al., 2008). Briey, to using reported methods with slight modications (Rupasinghe
the substrate (0.5 mmol) in methanol (5 mL) at 0 C was added IBX et al., 2009; Guzel et al., 2010). Fish oil (5 mg/mL) in a buffer solution
(0.6 mmol; 374 mg of 45% IBX) and the mixture was magnetically (0.05 M TRISHCl, 0.15 M KCl, 1% Tween 20, pH 7.0) was sonicated
stirred until the end of the reaction time (3 h). Then, water (5 mL) at 30 s intervals for 2 min by placing the glass tubes in a sonicator to
and sodium dithionite (1.2 mmol; 209 mg) were added and the mix- form an opalescent emulsion system. The sonicated stock emulsion
ture was further stirred for 10 min at room temperature. Solvent was divided into 0.5 mL aliquots and the emulsion was maintained
was evaporated and the residue was solubilized in ethyl acetate by shaking the tubes at 200 rpm. The reference and test antioxi-
(15 mL) and treated with saturated sodium bicarbonate (15 mL). dants, 100 L of 0.2 mM FeCl3 were added to the emulsion system
The aqueous phase was extracted with ethyl acetate (2 15 mL) and incubated at 37 C for 30 min and the inhibition of lipid oxi-
and the combined organic phases were washed with sodium chlo- dation was determined by the TBARS assay. Briey, the oxidized
ride solution and dried over anhydrous sodium sulphate. The crude emulsion sample was mixed with 1 mL of TBATCA reagent (15%
reaction product was puried by silica gel chromatography with (w/v) TCA and 0.375 (w/v) TBA in 0.25 M HCl) and heated in a boil-
ethyl acetate: hexane (2:1, v/v) to obtain compounds E and F as ing water bath for 20 min. The samples were then cooled on ice and
oily liquids in 65% (106 mg) and 68% (120 mg) yields respectively. centrifuged for 10 min and the absorbance of the supernatant was
recorded at 532 nm against a control which contained all reagents
2.3.1. Compound E: (3,4-dihydroxyphenyl) methyl except the test compounds. BHT and -tocopherol served as ref-
5-(1,2-dithiolan-3-yl) pentanoate erence compounds and all measurements were made in triplicate
1 H NMR (CDCl 400 MHz): 1.351.40 (m, 2H, CH ), 1.621.73 and averaged. Inhibition of lipid oxidation was calculated using the
3 2
(m, 4H, 2 CH2 ), 1.851.95 (m, 1H, CH2 ), 2.35 (t, 2H, J = 7.2 Hz, CH2 ), formula:
2.452.50 (m, 1H, CH2 ), 3.103.25 (m, 2H), 3.53.6 (m, 1H, CH), 5.02 % inhibition = (1 (As /Ac ) 100, where As is the absorbance of
(s, 2H, CH2 O), 6.86.95 (m, 3H, Ph). sample and Ac is the absorbance of control.
IR (cm1 ): 3374, 2930, 2857, 1703, 1520, 1445.
HR-MS (EI, m/z) [M+Na]+ calculated for C15 H20 O4 S2 Na 2.5. Calculation of partition coefcient (log P values)
351.0701; Found: 351.0807.
The lipophilicity and the physico-chemical parameters and the
2.3.2. Compound F: 2-(3,4-dihydroxyphenyl) ethyl theoretical drug likeness properties of the synthesized derivatives
5-(1,2-dithiolan-3-yl) pentanoate were determined using the Molinspiration software avail-
1 H NMR data was consistent with the previously reported data able online at http://www.molinspiration.com/cgi-bin/properties
(15) and is as follows: (MolinspirationCheminformatics, Bratislava, Slovak Republic).
1 H NMR (CDCl 400 MHz): 1.351.45 (m, 2H, CH ), 1.601.75
3 2
(m, 4H, 2 CH2 ), 1.851.95 (m, 1H, CH2 ), 2.3 (t, 2H, J = 7.2 Hz, CH2 ),
3. Results and discussion
2.452.50 (m, 1H, CH2 ), 2.85 (t, 2H, J = 6.8 Hz, CH2 ), 3.153.25 (m,
2H), 3.533.60 (m, 1H, CH), 4.3 (t, 2H, J = 6.8 Hz, CH2 O), 5.3 (s, 1H,
3.1. Synthesis and characterization
OH), 6.656.85 (m, 3H, Ph).
IR (cm1 ): IR (cm1 ): 3400, 2945, 2870, 1701, 1530, 1465.
The present work describes the chemo-enzymatic synthesis of
HR-MS (EI, m/z) [M+Na]+ calculated for C16 H22 O4 S2 Na
-lipoic acid esters of phenolic compounds (Fig. 1). To the best of
365.0857; Found: 365.0888.
our knowledge this is the rst report on the enzymatic esterication
of lipoic acid with phenolics for the synthesis of potential antioxi-
2.4. Determination of antioxidant activity dant molecules. The prepared molecules can have benecial effects
due to the modication in their structure causing effects on solu-
2.4.1. DPPH radical assay bility and bioavailability. The synthetic routes employed are shown
The DPPH radical scavenging activities of the synthesized lipoic in Schemes 1 and 2.
acid derivatives were measured according to a reported method The phenolic compounds were enzymatically esteried with
with slight modication (Akowuah et al., 2006). DPPH is a com- -lipoic acid with the immobilized lipase B from C. antarctica
mercially available stable free radical (dark purple color), which is (Novozym 435). This enzyme is known to be a very efcient cat-
reduced to DPPH (light yellow colored) by reacting with an antiox- alyst for esterication of a wide range of alcohols. Primary alcohols
idant. A 0.1 mL aliquot of each prepared compound or reference are especially good substrates and the enzyme should thus be suit-
antioxidants (1 or 2 mM) in ethanol: DMSO (95:5; v/v) were added able for the esterication of the polyphenolic substrates used in the
to 2 mL of ethanolic DPPH solution (0.1 mM) and the volume was present study. Furthermore, this enzyme can preferably be used at
made up to 3 mL with the same solvent mixture. The mixture quite low thermodynamic water activities, thus making it possible
was shaken vigorously and allowed to stand in the dark at room to achieve very high yields in esterication reactions (Petersson
temperature for 60 min. The decrease in absorbance of the result- et al., 2007).
ing solution was then measured spectrophotometrically at 517 nm The molar ratios taken for phenolic compound to -lipoic acid
against ethanol containing DMSO as blank and the control sam- was 1:1.2 and the biocatalyst used was 15% based on the total
ple had all the reagents except test sample. BHT and -tocopherol weight of substrates to ensure complete conversion of the phe-
served as reference compounds. All measurements were made in nolic substrates. The solubility of both the phenolics and lipoic acid
triplicate and averaged. The ability to scavenge DPPH radical was was tested in solvents such as hexane, toluene, acetone and ter-
calculated by the following equation: tiary butanol for carrying out the enzymatic reaction. The solvent
Free radical scavenging activity (FRSA) of choice was a mixture of 2-butanone and n-hexane. It was earlier
(%) = [(Ac As )/(Ac )] 100, where As is the absorbance of sample reported that solvent mixtures composed of 2-butanone and hex-
and Ac is the absorbance of control. ane were useful for the esterication reactions using lipase (Sabally
S.S. Kaki et al. / Journal of Biotechnology 157 (2012) 344349 347

Fig. 1. Structures of the prepared novel esters of phenolics and -lipoic acid.

et al., 2005). Solvent mixtures were chosen to get complete dis- et al., 2009). All the synthesized compounds were puried by silica
solution of the substrate mixture. The solubility increased with gel column chromatography and characterized by TLC, IR, 1 H NMR
increasing concentration of 2-butanone and solvent ratios were and high resolution mass spectrometry.
chosen to be 2:1 for compounds C and D and 1:1 for compounds A The reaction systems employed in the synthesis do not involve
and B. The reaction proceeded smoothly at ambient temperatures toxic reagents or catalysts and are therefore attractive steps in envi-
(25 C) until complete conversion of the phenol substrate resulting ronmental friendly production of biologically active compounds.
in the formation of expected products in satisfactory yields with- Previous studies have shown that Novozym 435 can be re-used
out any formation of by-products. No esterication of the phenolic several times, thus making the methodology even more attractive
hydroxyl groups was observed. The losses in the work up procedure (Hagstrm et al., 2009).
could most probably be reduced by further optimization. The yields
obtained demonstrate that the lipase from C. antarctica (Novozym 3.2. Antioxidant activity
435) efciently catalyzed the esterication reaction between the
two non-natural substrates without any difculty in the selected The prepared phenolic esters of lipoic acid were tested for
organic medium. antioxidant activity employing two methods. One is the widely
In order to study the effect of additional phenolic groups, com- used DPPH radical scavenging assay and the other method mea-
pounds E and F were prepared by aromatic hydroxylation of sures the inhibition of the oxidation in a sh oil emulsion system.
compounds A and C respectively with IBX followed by reduction Antioxidants act by different mechanisms and scavenging of free
with Na2 S2 O4 with good isolated yields (Scheme 2). Hydroxylation radicals is one important mechanism. Though the DPPH radical
reactions can be conducted using enzymatic methods as well. How- is not identical to the radical species occurring in biological sys-
ever, these monooxygenase-catalyzed reactions are in most cases tems, it gives information on the general free radical scavenging
still not efcient enough to compete with chemical methods (Leak 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.


348 S.S. Kaki et al. / Journal of Biotechnology 157 (2012) 344349

Table 1 Table 2
Free radical scavenging activity, FRSA (%) of the novel esters and the reference Antioxidant activity of the novel esters and reference antioxidants in sh oil emul-
antioxidants as measured by DPPH radical assay. sion system as measured by the TBARS assay.

Compound FRSA (%) S.D. Compound % Inhibition S.D

1 mM 2 mM 1 mM 2 mM

BHT 86.37 0.08 90.75 0.27 BHT 54.50 0.28 61.85 2.8
-Tocopherol 91.11 0.07 93.25 0.07 -Tocopherol 57.28 0.84 64.43 2.53
A 18.75 0.37 23.91 0.47 A 39.20 1.68 49.93 0.56
B 44.54 0.07 61.75 0.2 B 48.14 1.4 50.52 0.28
C 15.19 0.28 22.83 0.8 C 50.13 0.28 58.07 1.97
D 83.95 0.34 91.15 0.13 D 56.29 4.49 63.44 1.12
E 77.25 0.28 91.95 0.27 E 23.11 0.28 35.62 1.68
F 90.92 0.08 92.40 0.08 F 57.08 3.83 65.29 1.79

novel derivatives were showing activity in the DPPH radical scav- inhibition of lipid peroxidation decreased in the following order;
enging assay. It has been reported that the antioxidant activity -tocopherol = F > BHT > D > C > B > A > E. The results are a bit more
of the phenolic compounds depends on their molecular struc- difcult to rationalize compared to those from the DPPH assay.
ture, especially on their hydrogen-donating ability and subsequent Comparing compounds A, B and E, the methoxy group seems to
stabilization of the formed phenoxy radical (Borges et al., 2000). be the most efcient substituent, while the extra hydroxyl group
Compounds A and C were showing least activity, and compound instead causes a decrease in antioxidant efciency. On the other
B was showing moderate activity whereas compounds D, E and F hand, when comparing compounds C and F, the hydroxyl group has
were showing very good scavenging activities. Infact D, E and F a clear positive inuence. Compound D is the second most efcient
were showing activities close to that of the reference compounds. of the esters synthesized. This may be attributed to the presence of
The free radical scavenging activity was in the following order: - the cinnamic double bond in the molecule along with a methoxyl
tocopherol > F > BHT > D > E > B > A > C. It is interesting to note that substituent at ortho position to hydroxyl, which is reported to
the aromatic hydroxylation of A and C which resulted in compounds increase the antioxidant activity efciency (Scaccini et al., 1999).
E and F greatly improved their activity compared to the precur- It is remarkable that the small structural difference between
sors. This increase in activity can be explained on the basis of their compounds E and F causes such a large difference in antioxidant
structure as these derivatives posses two phenolic hydroxyls which activity in the emulsion assay. Among all the tested compounds, F
are available as hydrogen donors to the DPPH radical. It is espe- was exhibiting higher activity than the other derivatives including
cially remarkable that compound C which was showing the least BHT and it is interesting to note that it was showing equal activity as
activity increased its activity dramatically when converted into tocopherol at both the tested concentrations. This could be due to
compound F which was the most efcient radical scavenger among the difference in the partitioning of antioxidants between different
the esters synthesized. Methoxy groups (in compounds B and D) phases of the emulsions due to their amphiphilic nature, in accor-
also increased the radical scavenging activity, but not as much as dance with previous studies on similar systems (Jacobsen, 2010).
the hydroxyl groups. Compound D was considerably more effec- Tocopherols are reported to be more active than their hydrophilic
tive than compound B, maybe due to its additional double bond. analogues due to their surface active nature and lipophilic charac-
Among all the tested compounds, the hydroxytyrosol ester of lipoic ter.
acid, compound F exhibited the highest radical scavenging activ- Compound F had excellent antioxidant activity in both assays.
ity which was similar to -tocopherol and better than the other Its antioxidative properties might be part of the reason of the inter-
commercial reference antioxidant, BHT in both the tested concen- esting biological activity of this substance in other systems.
trations. Hydroxytyrosol derivatives have been reported to exhibit In order to get indications concerning the potential applicabil-
good antiproliferative effect on human colon cancer cells as well ity of the synthesized derivatives as drugs or prodrugs, theoretical
(Bernini et al., 2011). calculations of molecular properties were carried out using the
Most of the lipids in foods are present as emulsions and oxi- Molinspiration software and are presented in Table 3. The cal-
dation takes place at a faster rate at the oil water interface of culated log P values show that the synthesized derivatives had
emulsions than in bulk lipid systems. Therefore, we decided to higher hydrophobicity than the parent compounds. The increased
test the efciency of the prepared compounds as antioxidants lipophilicity of the studied phenolics is an advantageous modica-
in an emulsion system containing sh oil. Oxidation products tion for an antioxidant to act at the waterlipid interface to prevent
were measured by the TBARS assay and the antioxidant activity the initial reaction between aqueous radicals and lipid compo-
of the test compounds were compared with those of the stan- nents (Borges et al., 2010). However, the log P value should not
dard antioxidants, BHT and -tocopherol. It can be observed in be too high. According to Lipinskis rule-of-ve, molecules should
Table 2, that all of the prepared derivatives showed antioxidant have log P values of 5 in order to be readily bioavailable (Lipinski
activity in the tested system. The order of antioxidant efcacy in 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 B C D E F

Log P 3.678 3.496 3.887 4.252 3.188 3.398


Mol wt 312.456 342.482 326.483 368.52 328.455 342.482
n ON 3 4 3 4 4 4
n OHNH 1 1 1 1 2 2

Log P, logarithm of the octanol-water partition coefcient; 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.
S.S. Kaki et al. / Journal of Biotechnology 157 (2012) 344349 349

Furthermore, Lipinskis rule says that the molecular weight should Hagen, T.M., Smith, A.R., Shenvi, S.V., Widlansky, M., Suh, J.H., 2004. Lipoic acid as
be below 500, the number of hydrogen bond acceptors should not a potential therapy for chronic diseases associated with oxidative stress. Curr.
Med. Chem. 11, 11351146.
be more than 5 and the number of hydrogen bond donors should Hagstrm, A., Nordblad, M., Adlercreutz, P., 2009. Biocatalytic polyester acryla-
not be more than 5 (Lipinski et al., 1997). These numbers are the tion process optimization and enzyme stability. Biotechnol. Bioeng. 102,
upper limits for drugs to be able to penetrate through biomem- 693699.
Igglessi-Markopoulou, O., Melagraki, G., Afantitis, A., Detsi, A., Koufaki, M.,
branes. Compounds AF all have molecular weights below 500 and Kontogiorgis, C., Hadjipavlou-Litina, D.J., 2009. Synthesis and evaluation
only 34 hydrogen bond acceptors. Furthermore, the number of of the antioxidant and anti-inammatory activity of novel coumarin-3-
hydrogen bond donors, hydroxyl and methoxyl groups are only 12 aminoamides and their alpha-lipoic acid adducts. Eur. J. Med. Chem. 44,
30203026.
(Table 3). These values suggest that the molecules can have good
Jacobsen, C., 2010. Enrichment of foods with omega-3 fatty acids: a multidisciplinary
absorption and permeation and oral bioavailability (Lipinski et al., challenge. Ann. N. Y. Acad. Sci. 1190, 141150.
1997). Therefore, the prepared derivatives could nd applications Kermasha, S., Sabally, K., Karboune, S., St-Louis, R., 2006. Lipase-catalyzed transes-
terication of dihydrocaffeic acid with axseed oil for the synthesis of phenolic
as pro drugs as the cleavage of the molecules will result in release
lipids. J. Biotechnol. 127, 167176.
of compounds which are both benecial for human health. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature
In conclusion, an efcient chemo-enzymatic route for the syn- 409, 241246.
thesis of novel esters of natural phenolics and -lipoic acid is Lawrence Lowell, J., Eisenberg, R.L., 2007. Novel esters of lipoic acid. US Patent No.
2007/0055070 A1.
described. The data reported in the present study suggests that the Lazos, E.S., Lafka, T.I., Lazou, A.E., Sinanoglou, V.J., 2011. Phenolic and antioxidant
-lipoic acidphenolic derivatives are effective antioxidants which potential of olive oil mill wastes. Food Chem. 125, 9298.
can inhibit lipid oxidation and scavenge radicals. The synthetic Leak, D.J., Sheldon, R.A., John, M.W., Adlercreutz, P., 2009. Biocatalysts for selective
introduction of oxygen. Biocatal. Biotransform. 27, 126.
protocol is simple, efcient and proceeded smoothly at ambient Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 1997. Experimental and com-
temperatures with excellent isolated yields of all the products. putational approaches to estimate solubility and permeability in drug discovery
and development settings. Adv. Drug Deliver. Rev. 23, 325.
Nagendran Balasundram, K.S., Samman, S., 2006. Phenolic compounds in plants and
References agri-industrial by-products: antioxidant activity, occurrence, and potential uses.
Food Chem. 99, 191203.
Adlercreutz, P., Mbatia, B., Kaki, S.S., Mattiasson, B., Mulaa, F., 2011. Enzymatic syn- Packer, L., Witt, E.H., Tritschler, H.J., 1995. Alpha-lipoic acid as a biological antioxi-
thesis of lipophilic rutin and vanillyl esters from sh by products. J. Agric. Food dant. Free Radic. Biol. Med. 19, 227250.
Chem. 59, 70217027. Petersson, A.E.V., Adlercreutz, P., Bo, M., 2007. A water activity control system for
Adlercreutz, P., 2000. Biocatalysis in non-conventional media. In: Straathof, A.J.J., enzymatic reactions in organic media. Biotechnol. Bioeng. 97, 235241.
Adlercreutz, P. (Eds.), Applied Biocatalysis. , 2nd ed. Harwood Academic Pub- Quideau, S., Ralph, J., 1992. Facile large-scale synthesis of coniferyl, sinapyl, and
lishers, Amsterdam, pp. 295316. para-coumaryl alcohol. J. Agric. Food Chem. 40, 11081110.
Akowuah, G.A., Zhari, I., Norhayati, I., Mariam, A., 2006. HPLC and HPTLC densitomet- Rupasinghe, H.P.V., Huber, G.M., Shahidi, F., 2009. Inhibition of oxidation of omega-
ric determination of andrographolides and antioxidant potential of Andrographis 3 polyunsaturated fatty acids and sh oil by quercetin glycosides. Food Chem.
paniculata. J. Food Comp. Anal. 19, 118126. 117, 290295.
Bernini, R., Crisante, F., Merendino, N., Molinari, R., Soldatelli, M.C., Velotti, F., 2011. Sabally, K., Karboune, S., Yebaoh, F.K., Kermasha, S., 2005. Enzymatic esterication
Synthesis of a novel ester of hydroxytyrosol and alpha-lipoic acid exhibiting an of dihydrocaffeic acid with linoleyl alcohol in organic solvent media. Biocatal.
antiproliferative effect on human colon cancer HT-29 cells. Eur. J. Med. Chem. Biotransform. 23, 3744.
46, 439446. Scaccini, C., Natella, F., Nardini, M., Di Felice, M., 1999. Benzoic and cinnamic acid
Bernini, R., Mincione, E., Barontini, M., Crisante, F., 2008. Convenient synthesis of derivatives as antioxidants: structureactivity relation. J. Agric. Food Chem. 47,
hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alco- 14531459.
hol. J. Agric. Food Chem. 56, 88978904. Shahidi, F., Wanasundara, U.N., 1998. Antioxidant and pro-oxidant activity of green
Borges, F., Silva, F.A.M., Guimaraes, C., Lima, J.L.F.C., Matos, C., Reis, S., 2000. Pheno- tea extracts in marine oils. Food Chem. 63, 335342.
lic acids and derivatives: studies on the relationship among structure, radical Shahidi, F., Zhong, Y., 2010. Novel antioxidants in food quality preservation and
scavenging activity, and physicochemical parameters. J. Agric. Food Chem. 48, health promotion. Eur. J. Lipid Sci. Technol. 112, 930940.
21222126. Villeneuve, P., 2007. Lipases in lipophilization reactions. Biotechnol. Adv. 25,
Borges, F., Roleira, F.M.F., Siquet, C., Orru, E., Garrido, E.M., Garrido, J., Milhazes, N., 515536.
Podda, G., Paiva-Martins, F., Reis, S., Carvalho, R.A., da Silva, E.J.T., 2010. Lipophilic Viskupicova, J., Danihelova, M., Ondrejovic, M., Liptaj, T., Sturdik, E., 2010. Lipophilic
phenolic antioxidants: correlation between antioxidant prole, partition coef- rutin derivatives for antioxidant protection of oil-based foods. Food Chem. 123,
cients and redox properties. Bioorg. Med. Chem. 18, 58165825. 4550.
Chou, T.C., Lai, Y.S., Shih, C.Y., Huang, Y.F., 2010. Antiplatelet activity of alpha-lipoic Zhang, W.J., Bird, K.E., McMillen, T.S., LeBoeuf, R.C., Hagen, T.M., Frei, B., 2008. Dietary
acid. J. Agric. Food Chem. 58, 85968603. alpha-lipoic acid supplementation inhibits atherosclerotic lesion development
Figueroa-Espinoza, M.C., Villeneuve, P., 2005. Phenolic acids enzymatic lipophiliza- in apolipoprotein E-decient and apolipoprotein E/low-density lipoprotein
tion. J. Agric. Food Chem. 53, 27792787. receptor-decient mice. Circulation 117, 421428.
Guzel, O., Karali, N., Ozsoy, N., Ozbey, S., Salman, A., 2010. Synthesis of new spiroin- Zhu, L.M., Zheng, Y., Wu, X.M., Branford-White, C., Ning, X., Quan, J., 2009. Enzymatic
dolinones incorporating a benzothiazole moiety as antioxidant agents. Eur. J. synthesis and characterization of novel feruloylated lipids in selected organic
Med. Chem. 45, 10681077. media. J. Mol. Catal. B: Enzym. 58, 6571.

Anda mungkin juga menyukai