Functionalities of conjugated compounds of γ-aminobutyric acid with salicylaldehyde or cinnamaldehyde

Article abstract of DOI:10.1016/j.foodchem.2015.06.077

Aldehydes or ketones can react with amino compounds to form Schiff base adducts, which have been widely studied and shown to exhibit antimicrobial, antioxidant or antiviral activity. Salicylaldehyde (SA) and cinnamaldehyde (CA) are components of plant ess

Full text of DOI:10.1016/j.foodchem.2015.06.077

Accepted Manuscript
Functionalities of conjugated compounds of γ –aminobutyric acid with salicy-
laldehye or cinnamaldehyde
Tai-Ti Liu, Yi-Wei Tseng, Tsung-Shi Yang
PII:
S0308-8146(15)00969-3
DOI:
http://dx.doi.org/10.1016/j.foodchem.2015.06.077
FOCH 17759
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 March 2015
30 May 2015
22 June 2015
Please cite this article as: Liu, T-T., Tseng, Y-W., Yang, T-S., Functionalities of conjugated compounds of γ –
aminobutyric acid with salicylaldehye or cinnamaldehyde, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.foodchem.2015.06.077
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Functionalities of conjugated compounds of γ–aminobutyric acid with salicylaldehye
or cinnamaldehyde
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Running title: Functionalities of conjugated compounds of γ–aminobutyric acid…
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Tai-Ti Liua$, Yi-Wei Tseng a, and Tsung-Shi Yangb *
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aDepartment of Food Science, Yuanpei University, No. 306 Yuanpei Street, Hsinchu 30015, Taiwan
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bDepartment of Cosmeceutics, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402,
Taiwan
*Author to whom correspondence should be addressed (e-mail: tsyang@mail.cmu.edu.tw)
Tel.: +886 4 22053366; fax: +886 4 22078083.
$Co- corresponding author: e-mail: taiti@mail.ypuu.edu.tw
Tel.: +886 3 5381183
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20 ABSTRACT
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Aldehydes or ketones can react with amino compounds to form Schiff base adducts, which have been
widely studied and shown to exhibit antimicrobial, antioxidant or antiviral activity. Salicylaldehye
(SA) and cinnamaldehyde (CA) are components of plant essential oils. γ-Aminobutyric acid (GA) is
an important substance in the mammalian central nervous system and responsible for many
bioactivities. This study aimed to synthesize functional Schiff base adducts using GA and SA or CA;
to study the antimicrobial activity, antioxidant activity and tyrosinase-inhibition activity of these
adducts (SA-GA and CA-GA) and their metal complexes__SA-GA-Cu (or Zn) and CA-GA-Cu (or
Zn); and to find their applications in food systems. SA-GA and CA-GA both exhibited good
antibacterial effects, and so did their Cu complexes. As for antioxidant activity, SA-GA and CA-GA
were superior to their metal complexes in most tests. Regarding inhibition of enzymatic browning of
mushrooms, both SA-GA-Cu and CA-GA-Cu could inhibit tyrosinase activity effectively.
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Keywords：Salicylaldehye, Cinnamaldehyde, γ-Aminobutyric acid, Schiff base, Antimicrobial
activity, Antioxidant activity, Tyrosinase, Mushroom browning.
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38 1. Introduction
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Most foods are susceptible to microbial spoilage, or biochemical or chemical deterioration.
Therefore, a variety of synthetic food additives are used to enhance safety and quality of food
products in order to ensure consumer acceptance. As many negative effects have been reported about
synthetic food additives (Soni, Carabin, & Burdock, 2005), consumers have grown more concerned
about the safety of synthetic food additives. Regarding food preservatives, plant essential oils have
been investigated and found to have the potential to be used as natural preservatives (Yang, Liou, Hu,
Peng, & Liu, 2013). However, essential oils (EOs) generally have strong characteristic odours and
are immiscible with water due to hydrophobicity, which may limit their applications in many food
systems.
Aldehydes or ketones can react with amino compounds or amino groups of food proteins to form
Schiff base adducts, such as those in the Maillard reaction, which is commonly encountered in food
cooking, processing or storage. Schiff base adducts have been widely studied in organic, inorganic
and medicinal chemistry and show many biological activities, such as antimicrobial, antioxidant and
antiviral activity (Kumar, Dhar, & Saxena, 2009). Although many Schiff base adducts have been
synthesized in the literature, few have found applications in food systems because most of the
compounds used for the synthesis are not edible or allowed in foods. A recent study used
cinnamaldehyde and various amino acids, excluding γ-aminobutyric acid (GA), to generate Schiff
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base adducts and investigated their antimicrobial activity in vitro and in food systems (Wei, Xiong,
Jiang, Zhang, & Ye, 2011). The Schiff base adducts can reduce the odour intensity and increase the
solubility of cinnamaldehyde in aqueous systems.
Cinnamaldehyde and salicylaldehye are components of plant EOs, and both are food flavourings
assessed by the Flavor and Extraction Manufacturers Association as generally regarded as safe
(GRAS) ingredients (Adams et al., 2004; Adams et al., 2005). In addition, Schiff base adducts can be
hydrolyzed into their original reactive components in acidic conditions (Cordes & Jencks, 1963).
Therefore, the safety of these adducts can be mainly determined by the reactive components after
digestion. For example, Schiff base adducts of cinnamaldehyde and amino acids have been
investigated and do not show any oral toxicity in animal tests (Wei etal., 2011).
GA is a chief inhibitory neurotransmitter in the mammalian central nervous system. It can
regulate neuronal excitability throughout the nervous system. In humans, GA is also directly
responsible for the regulation of muscle tone (Watanabe, Maemura, Kanbara, Tamayama, &
Hayasaki, 2002). In addition, GA shows relaxation and immunity enhancement effects in humans
(Abdou, Higashiguchi, Horie, Kim, Hatta, & Yokogoshi, 2006); it can also reduce the blood pressure
in animals and humans (Aoki, Furuya, Endo, & Fujimoto, 2003). As a functional ingredient, GA has
been formulated into a number of commercial health-oriented products for use as a dietary
supplement. Regarding foods, GA tea is commercially produced in Taiwan with an emphasis on the
aforementioned bioactivities (Wang, Tsai, Lin, & Ou, 2006).
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In this study, we aimed to synthesize functional Schiff base adducts using GA and salicylaldehye
or cinnamaldehyde; to study the antimicrobial activity, antioxidant activity and tyrosinase-inhibition
activity of these adducts and their metal complexes; and to find their applications in food systems.
2. Materials and methods
2.1. Chemicals
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γ-Aminobutyric acid (GA), salicylaldehye (SA), cinnamaldehyde (CA), 2,2ˊ-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), nicotinamide adenine
dinucleotide (NADH), phenazine methosulfate (PMS), nitrotetrazolium blue chloride (NBT),
tyrosinase (50 KU) and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from
Sigma-Aldrich Co. (St. Louis, MO, USA). Potassium peroxidisulfate and ferrous chloride were
acquired from Mallinckrodt-Baker, Inc. (Phillipsburg, NJ, USA). Sodium nitroprusside (SNP),
sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride, ferrozine were obtained from
Alfa Aesar Co. (Ward Hill, MA, USA). Tryptic soy broth (TSB) (Bacto™), nutrition broth (NB) and
agar were obtained from Becton Dickinson & Company (San Jose, CA, USA).
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2.2. Microbial strains
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Staphylococcus aureus (BCRC10780) and Escherichia coli O157:H7 (BCRC14824) were
acquired from Bioresource Collection and Research Center (BCRC) at the Food Industry Research
and Development Institute, Hsinchu, Taiwan.
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2.3. Preparation of Schiff base adducts
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The preparation of Schiff base adducts followed the methods of Wei, Xiong, Jiang, Zhang and
Ye (2011) with some modifications. The KOH (0.38 g) was dissolved in methanol (25 ml) and GA
(0.59 g) was added to the solution with stirring for complete dissolution. The above solution was
reacted at 50˚C for 2 h in a round-bottomed flask equipped with a condenser. The mixture was
cooled and filtered to obtain the GA-containing filtrate. The methanol solution of SA or CA was
prepared by dissolving 0.7 ml SA or 0.84 ml CA in 10 ml methanol placed in a round-bottomed flask.
Then the GA-containing filtrate was added dropwise to the methanol solution of SA or CA within 1 h
under N2 gas. The precipitate was formed during the reaction and it was collected after the solvent
was evaporated by the gas. Anhydrous ether was added to the precipitate to remove residual reactants
and the precipitate was dried under vacuum.
2.4. Preparation of metal complexes of the adducts
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Metal (II) nitrates (1 mmol) were dissolved in methanol (10 ml) and the solution was filtered
and added dropwise into a (15 ml) methanolic solution of SA-GA or CA-GA (1 mmol). The above
mixture was stirred for 2 h. The metal complex obtained was filtered off, washed with methanol and
ether; and then dried under vacuum (Nair & Joseyphus, 2008).
2.5. Infrared spectral analysis
The sample was mixed with KBr powder at a 1:100 ratio and dried overnight at 50˚C. The
sample-KBr pellet was prepared by mechanically pressing the powder mixture. The IR spectra were
recorded by a FT-IR spectrophotometer (Prestige 21, Shimazu, Tokyo, Japan) with a scan range of
400-4000 cm-1 using KBr as a reference.
2.6. Antimicrobial activity assay for bacteria in vitro
A stock solution (7500 µg/ml) was prepared with SA-GA or CA-GA and its metal ion complexes
(Cu2+ and Zn2+) dissolved in DMSO. The bacteria from stock cultures were inoculated in 5 ml broth
and incubated at 37°C for 24 h to increase their population. The enriched cultures were controlled
with dilution to optical density of 1 at 630 nm, which were further diluted with appropriate media for
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each microbe to 103- 104 colony forming units (CFU/ml) as an inoculum. The media for S. aureus, E.
coli O157 were TSB and NB, respectively.
A microbial suspension was mixed with an amount of the test solution (20:1, v/v) at a series of
concentrations, and then added to a 96-well microplate. The plate was capped and incubated for 48 h
at 37˚C. The minimal bactericidal concentration (MBC) was determined by visually judging the
microbial growth in the tubes with a series of broth dilutions. Then, the existence of viable cells was
verified by a plate count method. An inoculated growth medium without the test compound was
employed as a control. All of the experiments were performed in triplicate and the average values
were reported as MBC. The MBC is defined as the lowest concentration of the test solution that
results in no viable cells in a culture plate.
2.7. Antimicrobial activity assay in food
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White button mushrooms were used for evaluating the antimicrobial activity of the SA-GA-Cu
or CA-GA-Cu in a real food system. Each mushroom (50 1 g) was weighed and dipped into a
SA-GA-Cu or CA-GA-Cu solution at a concentration of 187 or 375 µg/ml for 10 s and held until no
water dripped from the surface. Then, the samples were placed into a sterilized plastic bag and
zipped, followed by storage in a chamber at room temperature for 6 h. At sampling time, the sample
(16 g) and 144 ml PBS (0.85％) were added into a sterilized container with blades and then
homogenized. The homogenate was used for antimicrobial assay. The homogenate was taken for a
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series dilution, 1 ml of which was poured onto plate count agar and incubated for 48 h at 37˚C. The
population of the microorganisms was determined by counting the number of colonies from the
plates with 25-250 colonies and multiplying by the dilution factor. The experiments were performed
in triplicate.
2.8. Analysis of antioxidant activity
2.8.1. Scavange of ABTS radical
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The ABTS radical assay followed the method of Debnath, Park, Nath, Samad, Park and Lim
(2011) with some modifications. ABTS was dissolved in water to make a solution of 7 mM. Then 10
ml of the ABTS solution was mixed with 2 ml potassium peroxidisulfate (14 mM) and 12 ml sodium
phosphate buffer (0.01M, pH7.4) to generate ABTS by allowing the mixture to stand in the dark at
room temperature for 12-16 h before use. For sample testing, the ABTS solution was diluted with the
sodium phosphate buffer to an absorbance of 1 at 734 nm. The test samples (0.1 ml), each at a
different concentration, were mixed with 1 ml ABTS solution and reacted for 10 min in dark. The
absorbance was recorded at 734 nm. Each sample was measured in triplicate and averaged. This
antioxidant effect is given as percentage ABTS scavenging activity that is calculated by the
following formula:
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Scavenging activity (%) = [(Ac–As)/Ac] × 100
where AC is the absorbance value of the control and AS is the absorbance value of the test samples.
2.8.2. Scavenging of nitrite oxide
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Test samples (0.3 ml) at various concentrations were mixed with 0.88 ml PBS, and sodium
nitroprusside (25 mM) in PBS, and then reacted for 2 h at room temperature. A volume of 0.5 ml of
the mixture was taken and added to 0.3 ml 5% H3PO4 solution containing 1% sulfanilamide, and 0.3
ml 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride solution (Marcocci, Maguire, Droylefaix,
& Packer, 1994). The absorbance of the reacted sample solution was measured at 570 nm. The
scavenging activity is determined according to the following equation:
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Scavenging activity (%) = [(Ac–As)/Ac] × 100
where AC is the absorbance value of the control and AS is the absorbance value of the test samples.
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2.8.3. Scavenging of superoxide anion
The stock solutions of PMS (30 µM), NADH (936 µM) and NBT (300 µM) were prepared with
0.1 M phosphate buffer (pH 7.4). The 50 µl test solution was added into a 96-well microplate
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followed by addition of 62.5 µl NBT and 62.5 µl NADH. The mixture was left standing for 2 min for
concentration balance. The reaction was started by adding 62.5 µl PMS to the mixture. The reaction
mixture was incubated at room temperature for 5 min and absorbance at 560 nm was recorded (Bi et
al., 2013). The scavenging activity of superoxide anion is expressed by the following formula:
Scavenging activity (%) = [(Ac–As)/Ac] × 100
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where AC is the absorbance value of the control and AS is the absorbance value of the test samples.
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2.8.4. Ferric ion chelating activity
In the present assay, 0.5 ml of test solution in a series of concentrations was mixed with 1.6 ml
of double de-ionized water, 0.1 ml FeCl2 (2 mM) and 0.1 ml of 5 mM aqueous ferrozine solution.
The mixture was shaken vigorously and left standing at room temperature for 10 min. The
absorbance was measured at 562 nm (Demirtas, Erenler, Elmastas, & Goktasoglu, 2013). EDTA was
used as a reference compound for comparison. The calculation was performed using the following
formula:
Chelating activity (%) = [(Ac–As)/Ac] × 100
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where AC is the absorbance value of the control and AS is the absorbance value of the test sample
solution.
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2.9. Inhibition of tyrosinase in vitro
The test sample was dissolved in DMSO and diluted to a series of concentrations. An aliquot of
14 µl mushroom tyrosinase (30 U, 40 nM) was pre-incubated with the test sample in 76 µl sodium
phosphate buffer (50 mM, pH 6.8) for 10 min at 25°C. Then the 100 µl L-DOPA (4 µM) was added
to the sample mixture, which was allowed to stand for 10 min for the formation of dopaquinone. The
absorbance was measured at 475 nm by a microplate reader (SpectraMax® M2, Molecular Devices).
The inhibition of the enzyme activity was calculated as follows:
Percent inhibition (%) = [(B − S)/B] × 100, where B and S were the absorbance for the blank and
samples, respectively (Kumar, Yang, Chu, Chang, & Wang, 2010). Ascorbic acid (5 µg/ml) was used
as a standard tyrosinase inhibitor for comparison.
2.10. Inhibition of browning of mushroom
White button mushrooms (Agaricus bisporus) with similar size (16 0.5 g; 4 0.2 cm, id) were
selected and separated into two groups (10 pieces in each group), one for the treatment and the other
as a control. The mushrooms were dipped into a SA-GA-Cu or CA-GA-Cu solution at a
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concentration of 187 or 375 µg/ml for 10 s and held until no water dripped from the surface. Each
mushroom was weighed before and after the soaking procedure in order to calculate the amount of
the active component absorbed by the mushroom. The samples in the control group were subjected to
the same procedures except for the double de-ionized water used as the test solution. Each sample in
both groups was put in a 100 ml beaker and stored at room temperature. The colour change of
samples was monitored at the time period of 0, 4 and 8 h with a colorimeter (CR-200, Minolta, Japan)
using a CIE-L*a*b* system for data expression.
2.11. Statistical analysis
Statistical analysis of variance (ANOVA) was conducted using Statistica for Windows (StatSoft,
Tulsa, OK, USA). Duncan’s multiple range test was used to compare the mean values of data for
significant difference at a 95% level.
237 3. Results and discussion
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3.1. Infrared spectra of Schiff base adduct
The IR spectral data of SA-GA or CA-GA and its metal complex are given in Table 1. The C=N
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bond can be observed at ν(C-N) 1627-1635 cm−1 due to the azomethine stretching vibration, which
indicates the successful synthesis of the Schiff base adduct (Guo et al., 2005). Carboxylate (COO−)
stretching occurs at two different spectral regions: an asymmetrical stretching band near 1650-1550
cm−1, and weaker symmetric stretching bands near 1400 cm−1 (Gnanasambandam & Proctor, 2000).
On complexation in this study, the asymmetric carboxylate stretching νasym(COO−) is shifted from
1610-1618 cm−1 to the 1543–1573 cm−1 range and the symmetric carboxylate stretching νsym (COO−)
is shifted from 1404-1408 cm−1 to 1384 cm−1, indicating the linkage between the metal ion and
carboxylate oxygen atom (Nair & Joseyphus, 2008). The spectra of the complexes show broad bands
in the 3388–3471 cm−1 range, which can be attributed to the stretching vibration of the OH group.
This indicates the presence of water molecules in the structure (Hassan, Nassar, Hussien, & Elkmash,
2012). In the complexes, the bands in the 565–586 and 439–470 cm−1 range can be attributed to
ν(M-N) and ν(M-O), respectively (Nair & Joseyphus, 2008).
3.2. Antimicrobial activity assay for bacteria in vitro
The antimicrobial activity of SA-GA or CA-GA and its metal complex against S. aureus and E.
coli O157：H7 is shown in Table 2. Regarding SA-GA and its metal complexes, these compounds
exhibited a better antimicrobial effect against S. aureus than against E. coli O157：H7. The SA-GA
could inhibit S. aureus but failed to inhibit E. coli O157：H7 at the test concentration of 375 µg/ml. In
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contrast, both SA-GA-Cu and SA-GA-Zn could eradicate E. coli O157：H7 at 375 µg/ml. This
indicates that the metal complex can enhance the antimicrobial activity of SA-GA. Chelation
considerably reduces the polarity of the metal ion because of partial sharing of its positive charge
with donor groups and possible electron delocalization over the whole chelate ring. Such a chelation
could enhance the lipophilic character of the central metal atom, which subsequently favours its
permeation through the lipid layer of the cell membrane (Ceyhan, Çelik, Uruş, Demirtaş, Elmastaş,
& Tümer, 2011). Moreover, SA-GA-Cu exhibited a greater antimicrobial effect than SA-GA-Zn
against S. aureus. The reason for the higher antimicrobial activity of Cu2+ complex relative to the
Zn2+ complex may be that Cu2+ can form a stronger Cu2+–ligand bond than Zn2+–ligand bond, which
increases the lipophilic character of a Cu2+ complex compared to a Zn2+ complex. In addition, Cu2+
has a higher redox activity than Zn2+, which may also account for the higher antimicrobial activity of
Cu2+ complexes (El-Sherif & Eldebss, 2011).
As with SA-GA, CA-GA also had a good antimicrobial activity against S. aureus, which was
stronger than that of SA-GA by giving lower MBC values at each corresponding concentration.
However, as for the antimicrobial effect against E. coli O157：H7, CA-GA could inactivate E. coli
O157：H7 at 375 µg/ml, but SA-GA could not. The metal complexes of CA-GA also enhanced the
antimicrobial activity of CA-GA against S. aureus. Noticeably, CA-GA-Cu showed greater
antimicrobial activity than CA-GA against E. coli O157：H7. In addition, CA-GA-Cu was superior to
SA-GA-Cu in suppressing E. coli O157：H7. From the results, it seems that Gram (+) bacteria such
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as S. aureus is more susceptible to the test compounds than Gram (-) bacteria, namely E. coli O157：
H7. The resistance of Gram (-) bacteria toward antibacterial substances is ascribed to the hydrophilic
surface of their outer membrane, which is rich in lipopolysaccharide molecules, forming a barrier to
the penetration of numerous antibiotic molecules, and is also relevant to the enzymes in the
periplasmic space, which can break down the molecules introduced from outside (Nikaido, 1994;
Gao, Van Belkum, & Stiles, 1999).
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Since SA-GA-Cu and CA-GA-Cu showed a better antimicrobial activity than the other test
compounds in the SA or CA adduct group, both were selected as a representative for each group to
further assay their antimicrobial effect in a real food system - mushroom. As the mushrooms were
only very briefly (10 s) dipped into the test solutions, the amount of SA-GA-Cu or CA-GA-Cu
absorbed into the mushrooms was much lower than that in the test solution, which is listed in Table 3.
The initial microbial population in the untreated mushrooms was approximately 102 CFU/g. Two
concentration levels, 5.8 and 11.6 µg/g, were used for the test. At the concentration of 5.8 µg/g,
SA-GA-Cu gave an 82% inhibition ratio of total microorganisms at 2 h; however, the ratio decreased
to 67% at 6 h. This decrease may be due to the resuscitation of surviving microorganisms after the
administration of the test compound. In contrast, CA-GA-Cu exhibited a 99% inhibition ratio at
either 2 or 6 h at the same concentration. Namely, CA-GA-Cu gave a better antimicrobial effect than
SA-GA-Cu at this concentration.
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At the concentration of 11.6 µg/g, SA-GA-Cu showed a 99% inhibition ratio of the total
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microorganisms at 2 h and the ratio slightly declined to 93% at 6 h. This result suggests that
SA-GA-Cu at this concentration can effectively inhibit >90% microorganisms during 6 h of storage.
Comparatively, at the same concentration CA-GA-Cu exhibited a marked antimicrobial effect, which
could be observed from the 44% inhibition ratio at the initial time of administrating the test
compound (Table 3). CA-GA-Cu could kill or destroy the microorganisms instantly and the damaged
organisms seemed to lose their ability to resume growth in the later incubation period. Therefore, the
inhibition ratio remains 100% from 2 h to 6 h of storage.
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3.3. Antioxidant activity
3.3.1. Inhibition of ABTS radical
Assays
of
2,2’-azino-bis-3-ethylbenzthiazoline-6-sulfonic
acid
(ABTS)
and
1,1-diphenyl-2-picrylhydrazyl (DPPH) are the most popular among various spectrophotometric
methods for measuring the antioxidant capacity of foods. ABTS assay is superior to DPPH assay
when applied to a variety of plant foods containing hydrophilic, lipophilic and high-pigmented
antioxidant compounds (Floegel, Kim, Chung, Koo, & Chun, 2011). Therefore, the ABTS assay was
used in this study. The inhibition of ABTS radical activity by the metal complexes of SA-GA or
CA-GA is shown in Table 4. Generally, SA-GA and its metal complexes had a stronger scavenging
effect than CA-GA and its metal complexes. Noticeably, SA-GA seemed comparable with ascorbic
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acid in this activity. On the other hand, SA-GA-Cu or SA-GA-Zn did not show any better scavenging
activity than SA-GA, which reveals that the metal complexes do not enhance the scavenging effect
of SA-GA but slightly reduce it.
With respect to CA-GA and its metal complexes, CA-GA had a lower IC50 value than that of
CA-GA-Cu or CA-GA-Zn, which means that the metal complexes cannot increase the antioxidant
activity of CA-GA but slightly decrease it. The effect of metal ions on CA-GA in scavenging ABTS
radicals is similar to its effect on SA-GA. In addition, the scavenging activity of CA-GA-Cu is
similar to that of CA-GA-Zn. This phenomenon is also applicable to the scavenging activity between
SA-GA-Cu and SA-GA-Zn.
3.3.2. Inhibition of superoxide anion activity
Inhibition of superoxide anion activity by SA-GA or CA-GA and its metal complexes are listed
in Table 4. Regarding SA-GA and its metal complexes, SA-GA exhibited an inhibitory effect with an
IC50 value of 764 µg/ml. In contrast, SA-GA-Cu and SA-GA-Zn gave IC50 values of 5.7 and >6000
µg/ml, respectively. This result reveals that the metal complexes do not show a consistent trend in
enhancing or reducing the scavenging effect of SA-GA, but manifests that this effect is dependent on
the species of the metal ion. In this case, Cu2+ enhances the scavenging effect of SA-GA, while Zn2+
decreases it. Similarly, with respect to CA-GA and its metal complexes, CA-GA-Cu showed the
strongest scavenging effect (IC50: 5.8 µg/ml) among the test compounds, which is comparable with
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that (IC50: 5.7 µg/g) of SA-GA-Cu. The IC50 value for CA-GA for its inhibitory effect was 1482
µg/ml, which is about twice the value for SA-GA. Meanwhile, as with SA-GA-Zn, CA-GA-Zn did
not express any scavenging effect with an IC50 value > 6000 µg/ml.
Superoxide dismutases (SODs) are a diverse group of metalloenzymes, and they can catalyze
dismutation of two negatively charged superoxide radicals to molecular oxygen and hydrogen
peroxide. The redox potential of SOD containing Cu2+ and Zn2+ (CuZnSOD) during catalysis has
been investigated, and the result indicates that copper is the redox partner of the superoxide radical,
whereas the oxidative state of Zn2+ does not change during the dismutation reaction (Konecny et al.,
1999). Several other studies also showed that Cu2+complex of Schiff base adducts can mimic the
function of CuZnSOD in dismutating a superoxide anion (Bian et al., 2015; Patel, Patel, Shukla, &
Singh, 2013). From these studies, Cu2+ seems to play a more important role than Zn2+ in scavenging
superoxide anion, which is also evidenced from the results of this research.
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3.3.3. Inhibition of nitric oxide
Nitric oxide (NO), one of the smallest known bioactive products of mammalian cells, has
multiple biological effects. However, a number of disease states are characterized by abnormally
high NO production and removing the excess NO could have beneficial effects. In the inhibition of
NO radical assay, SA-GA was better than SA-GA-Cu or SA-GA-Zn (Table 4). In contrast,
surprisingly CA-GA and its metal complexes did not show any scavenging effect. This may be
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attributed to the effect of structural difference. The SA has an OH group on the aromatic ring but CA
does not. From literature, a study that compares the reactivity of NO with the metal complexes of
Schiff base ligand derived from SA or pyridine-2-carboxaldehyde shows that the
pyridine-2-carboxaldehyde-derived ligands do not promote reactivity with NO, whereas, the
SA-derived ligands do. Therefore, it is concluded that the substituents on the aromatic moiety have a
dramatic effect on the reactivity (Coleman & Taylor, 1980).
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3.3.4. Ferrous ion chelating activity
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The assay is based on the principle that ferrozine can bind Fe2+ rather than Fe3+ into a complex
that absorbs strongly at 562 nm (Im, Lee, & Löffler, 2013). If the Fe2+ is chelated by the test
compound, the absorption signal will be reduced. SA-GA or CA-GA shows higher Fe2+ chelating
activity than its metal complexes (Table 4), which is expected because it is a common characteristic
of many synthesized Schiff bases in chelating metal ions (Monier, Ayad, & Abdel-Latif, 2012).
However, it is noted that the metal complexes of SA-GA or CA-GA also give certain Fe2+ chelating
activity even though the ability is lower than that of the metal-free adduct. Several possibilities are
hypothesized to explain this. First, Fe2+ may compete with the metal ions in the complexed adducts
because of relatively high concentration gradient, and partly replace Cu2+ or Zn2+ in the chelated
structure, which may decrease the amount of Fe2+ available in the solution for binding ferrozine.
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Second, because the Cu2+ can catalyze the autoxidation of Fe2+ to Fe3+ (Sayin, 1982), SA-GA-Cu or
CA-GA-Cu may also have this ability to convert Fe2+ into Fe3+, and thus reduce the amount of Fe2+
for the reaction with ferrozine. In contrast, Zn2+ does not alter the absorbance of the Fe2+–ferrozine
complex (Sayin, 1982); therefore, this may account for the fact that SA-GA-Cu or CA-GA-Cu has
greater Fe2+ chelating activity than SA-GA-Zn or CA-GA-Zn. Comparatively, SA-GA-Zn has a
lower IC50 value (207µg/ml) than that (4341µg/ml ) of CA-GA-Zn, which may be attributed to the
OH group present in the SA structure. The unpaired electrons on the OH group may provide a
chelating site for Fe2+, which is not structurally available in the CA.
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3.3.5. Inhibition of tyrosinase activity
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Tyrosinase is a copper dependent enzyme which catalyzes two different reactions using
molecular oxygen: the hydroxylation of monophenols to o-diphenols (monophenolase activity) and
the oxidation of the o-diphenols to o-quinones (diphenolase activity) (Chen & Kubo, 2002). With
respect to SA-GA and its metal complexes, SA-GA-Cu and SA-GA-Zn exhibited a better inhibitory
effect on tyrosinase than SA-GA (Table 5). Moreover, SA-GA-Cu is better than SA-GA-Zn at this
effect. Similarly, CA-GA-Cu and CA-GA-Zn also gave a stronger inhibitory effect on tyrosinase than
CA-GA. In addition, CA-GA-Cu is superior to CA-GA-Zn in exhibiting this effect. From these
results, the metal complexes can reinforce the inhibitory ability of SA-GA or CA-GA on tyrosinase,
especially the CA-GA. The reason may be that the Cu2+ complex of Schiff base adducts could be
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attracted to tyrosinase and the Cu2+ in the structure could interfere with the electron transferring
process at the active site of tyrosinase, which would lead to inactivation of tyrosinase activity. In one
study, which used synthetic membranes to immobilize tyrosinase, the researchers found that when
Cu2+ is chelated on the membrane surfaces, the tyrosinase absorption efficiency can increase due to
the effect of binding affinity between the enzyme and the chelated Cu2+ (Arıca & Bayramoǧlu,
2004).
As for the effect of Zn2+ on tyrosinase, it has been suggested in a study that Zn2+ can directly
form a ligand-binding with tyrosinase at a position near or inside the active site, which induces
conformational change that inactivates the enzyme (Han et al., 2007). Although the Zn2+ is chelated
in the Schiff base adducts, the active site might not be shielded or might be only partially affected by
the structure of adducts, depending on the position and orientation of where the Zn2+ is present in the
structure. Thus, the Zn2+ adducts may still retain certain inhibitory effects on the tyrosinase activity.
On the other hand, it is probable that the metal ions complexed with the adducts form a more stable
or special conformational structure that is favourable for binding tyrosinase and interferes with its
activity. The interactive effects of chelators on metal ions have been reported in literature. For
example, EDTA and several other metal-ion chelators have been shown to enhance or inhibit
iron-dependent lipid peroxidation depending on the types of chelator and the ratio of iron salt to the
chelator in the reaction mixture. Regarding the enhancive effect, EDTA can increase the ability of
complexed iron to undergo redox cycling and thus accelerate the lipid peroxidation (Gutteridge,
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1984).
3.3.6. Inhibition of mushroom browning
The white button mushroom is widely recognized for its nutritional, organoleptic and medicinal
properties, and is highly popular among consumers (Beelman, Royse, & Chikthimmah, 2003).
However, the mushrooms are perishable during storage with a shelf life of about 3–4 days. Their
commercial value is lost within a few days due to browning, water loss, senescence and microbial
attack (Gao, Feng, & Jiang, 2014). As SA-GA-Cu and CA-GA-Cu display good effects on the
inhibition of tyrosinase activity in vitro, both compounds were used for inhibition of mushroom
browning. The colour change of the untreated and treated mushrooms during storage was compared.
A colorimeter with a colour scale- CIE-L*a*b* system was used for recording the colour change. L*
(0-100) represents lightness from low to high; a* is red, and -a* is green; b* and -b* stand for yellow
and blue, respectively. Regarding the colour scale of the control sample, the L* value decreased
while the a* and b* values both increased as the storage time increased. Compared with the L*a*b*
values of the control sample at the initial time, the colour scale differences (∆E*) were 4.42 and 8.32
for the same sample measured at 4 and 8 h, respectively (Table 6). This indicates that the colour of
mushrooms turns darker and consists of more red and yellow components, which is consistent with
the browning effect observed visually. When the mushrooms were treated with SA-GA-Cu at 5.8
µg/g, the ∆E* values were reduced to 3.57 and 6.85 at 4 h and 8 h, respectively. In addition, the
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∆E* values were 2.23 and 4.67 at 4 h and 8 h, respectively, for the samples treated with CA-GA-Cu
at the same concentration (Table 6). The results show that SA-GA-Cu and CA-GA-Cu can
effectively inhibit the mushroom browning and that CA-GA-Cu gives a stronger effect than
SA-GA-Cu, which is consistent with the results obtained from the in vitro test of tyrosinase
inhibition.
Zn and Cu are essential trace elements for human health but over intake of them may cause health
risks. Regarding the safety of Zn and Cu intakes, the tolerable upper levels for Zn andCuare 40 mg/d
and 10 mg/d, respectively (Goldhaber, 2003). The effective antimicrobial or antibrowning
concentration of the metal complexes used in the mushrooms is ca 12 µg/g. This means that if 200 g
(approximately 12 pieces) of the mushrooms are consumed at a meal, the intake of these elements is
approximately 2.4 mg, which is lower than the tolerable upper levels of both elements. It is worth
noting that the calculation is based on the raw materials; in practice, the mushrooms must be washed
or soaked and boiled in water before consumption, which may lead to a marked loss of the added
compounds. Therefore, the actual intake of these elements is much lower than the above estimated
value. In addition, Zn can compete with Cu and reduce the Cu absorption in digestion (de Romaña,
Olivares, Uauy, & Araya, 2011); as a result, one may also use both of the synthesized compounds
together as a mixture and adjust to an appropriate ratio between them by taking advantage of the
characteristic of Zn that has a higher tolerable upper level. Accordingly, the safety concern can be
eliminated when using these adducts and their metal complexes.
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451 4. Conclusions
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The Schiff base adducts of GA and SA or CA were synthesized and their functionalities were
assessed. These adducts and their metal complexes can impart antibacterial, antioxidant, and/or
tyrosinase-inhibition activities. Regarding antibacterial activity, SA-GA and CA-GA both exhibit
good antibacterial effects, which can be further enhanced by forming a complex with Cu2+ or Zn2+.
As for antioxidant activity, SA-GA and CA-GA adducts are more effective than their metal
complexes in ABTS, nitric oxide, and ferric ion chelating analyses, whereas their Cu metal
complexes are superior to these adducts in superoxide anion analysis. With respect to inhibition of
enzymatic browning of mushrooms, both SA-GA-Cu and CA-GA-Cu can inhibit tyrosinase activity
effectively. The multiple functionalities of these adducts render them very suitable for preservation of
foods such as mushrooms in extension of shelf life.
463
464
465 Acknowledgment
466
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469
This research was supported by Ministry of Science and Technology,Taiwan, R.O.C. (project no.:
NSC 102-2221-E-264 -004).
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