FARMACIA, 2023, Vol.

71, 4
https://doi.org/10.31925/farmacia.2023.4.11 ORIGINAL ARTICLE

CATECHIN-ZINC-COMPLEX: SYNTHESIS, CHARACTERIZATION

AND BIOLOGICAL ACTIVITY ASSESSMENT

IONUȚ IULIAN LUNGU 1#, DENISA MARIN-BATÎR 2#, ALINA PANAINTE 1, CORNELIA
MIRCEA 1, CRISTINA TUCHILUȘ 1, ALINA ȘTEFANACHE 1, FLORIN ADRIAN SZASZ 3,
DANIEL GRIGORIE 4, SILVIA ROBU 3, OANA CIOANCĂ 1*, MONICA HĂNCIANU 1
1“Grigore T. Popa” University of Medicine and Pharmacy, Faculty of Pharmacy, Iași, Romania
2“Dunărea de Jos” University, Faculty of Medicine and Pharmacy, Galați, Romania
3University of Medicine and Pharmacy Oradea, Faculty of Medicine, Oradea, Romania
4“Carol Davila” University of Medicine and Pharmacy, Faculty of Medicine, Bucharest, Romania

*corresponding author: oana.cioanca@umfiasi.ro

#Authors with equal contribution.

Manuscript received: March 2023

Abstract
Flavonoids have been the subject of research due to their abundance and versatility, which have been attributed to therapeutic
benefits. Flavonoids have the potential to form metal complexes, which have been the focus of research due to their potential
bioactivity and pharmacological effects. They often form complexes with a metal to ligand (M:L) molar ratio of 1:2 or 1:1,
which have strong stability constants. The effectiveness with which flavonoids attach to metal ions is strongly connected with
the type and state of transition metal ions. In this paper we displayed some of the methods of synthesizing flavonoid-metal
complex and the biological activity assessment. The antioxidant effects were evaluated against free radicals (iron chelation and
hydroxyl radical tests) and the inhibition of lipoxygenase. On the other hand, the antidiabetic potential was investigated by
using alpha-amylase and alpha-glucosidase assays. The infrared, UV spectrophotometry and the scanning electronic microscopy
results indicated the formation of small acicular crystals with specific absorption bands confirming the obtaining of the
catechin-zinc complex (Cat-Zn complex). The antioxidant assays indicated changes in the potential of the newly formed
complex, with different intensity of action depending on the test, whereas the inhibition of alpha-amylase and alpha-glucosidase
showed better activity for the complex as compared to catechin and zinc acetate. In conclusion, maintaining or improving the
antioxidant or enzymatic inhibitory properties of catechin, after complexation with zinc ions, may represent a benefit for the
use of such compounds in preclinical studies, but further testing is necessary to fully understand the in vivo potential.

Rezumat
Flavonoidele au fost intens cercetate datorită abundenței și versatilității lor, și au fost atribuite o serie de beneficii terapeutice.
Flavonoidele au potențialul de a forma complecși cu ionii metalici, aceștia fiind scopul unor studii de evaluare a potențialului
biologic și farmacologic. Ele formează adesea complexe într-un raport molar metal la ligand (M:L) de 1:2 sau 1:1, care au
constante de stabilitate puternice. Eficacitatea cu care flavonoidele se atașează la ionii metalici este strâns legată de tipul și
starea ionilor de metal tranzițional. În acest studiu, am inclus câteva metode de sinteză a complexului flavonoid-metal și
evaluarea activității biologice. Efectele antioxidante au fost evaluate împotriva radicalilor liberi (teste de chelatare a fierului
și radicali hidroxil) și inhibarea lipoxigenazei. Pe de altă parte, potențialul antidiabetic a fost investigat prin utilizarea testelor
alfa-amilazei și alfa-glucozidazei. Rezultatele spectrofotometriei în infraroșu, UV și microscopiei electronice de scanare au
indicat formarea de mici cristale aciculare cu benzi de absorbție specifice confirmând obținerea complexului catechină-zinc
(Cat-Zn complex). Testele antioxidante au indicat modificări ale potențialului complexului nou format, cu intensitate diferită
de acțiune în funcție de test, în timp ce inhibarea alfa-amilazei și alfa-glucozidazei a arătat o activitate mai bună pentru
complex în comparație cu catechina și acetatul de zinc. În concluzie, menținerea sau îmbunătățirea proprietăților antioxidante
sau inhibitoare enzimatice ale catechinei, după complexarea cu ionii de zinc, poate reprezenta un beneficiu în cazul utilizării
acestor compuși în studii preclinice, dar sunt necesare teste suplimentare pentru a înțelege pe deplin potențialul in vivo.

Keywords: flavonoids, metal-ion-complex, catechin-zinc-complex, antioxidant, antidiabetic potential

Introduction in terms of their structure and distribution, and as a

result, grouped them into a variety of categories, such
Since the beginning of time, people have understood
as alkaloids, terpenoids, chromones, xanthones, phytides,
and studied the benefits of consuming different products
carotenoids and flavonoids. Flavonoids are also an
derived from plants, which allows them to take advantage
important and very studied category. Among these,
of the nutritional and medicinal benefits [3, 4, 11, 43].
flavonoids have been the subject of a significant amount
Furthermore, the research was focused on the phyto-
of research over the years due to the abundance and
chemical components that differ from one another
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versatility of flavonoids as well as their distinctive
properties, which have been attributed to a large
spectrum of therapeutic benefits [9, 24, 35, 37, 39,
42, 44].
Even now, the researchers try to acquire innovative
pharmacological properties and to fully understand
the mechanisms of action, this aim is being carried
out by the idea that a lot of phytochemicals are being
continuously uncovered in terms of therapeutics and
clinical implementation [8-11, 13, 18, 31].
Flavonoids are a group of compounds that are based
on the structure of 2-phenylchromen-4-one (2-phenyl-
1-benzopyran-4-one). The majority of flavonoids, if
not all of them, are coloured substances (the word
flavonoid comes from the Latin word flavus, which
means yellow). Research on flavonoids did not make
significant headway until the 1990s, when the number
of publications increased by approximately sixfold,
from 524 in 1990 to 3147 in 2017. This was the first
decade in which flavonoid research saw significant
progress. As a consequence of this, it is now well
established that flavonoids possess a wide and diverse
range of biological activities [10, 11, 13, 23, 28-30,
46]. Some examples of these activities include anti-
inflammatory [21, 24], antibacterial and antifungal [1,
47], neuroprotective [25, 37, 42, 44], cardioprotective
[13], antioxidant [15, 18, 26, 29] and anticancer [18,
20, 26, 27, 31] properties.
In recent years, the ability of flavonoids to form
metal complexes generated intense attention in both
scientific and industrial communities due to the potential
of an enhanced bioactivity and different pharmacological
[8, 15, 17, 31, 38, 41, 43]. Metal chelation is one of
the many features of flavonoids, and it is one of the
most important one since it has such a significant
impact on the amplitude of the pharmacological actions
that flavonoids may accomplish modulating the bio-
availability of various minerals and ions [1, 5, 11, 12,
17, 20, 22, 30, 37, 39, 42, 45].
Catechins are natural polyphenolic compounds –
flavanols, belonging to the flavonoid family [8, 10].
They are found in abundant concentrations in a variety
of fruits, vegetables and plant-based beverages. The
name catechin is derived from Cutch tree (Acacia
catechu L.f.). High concentrations of catechin can
be found in fresh tea leaves, rock-rose leaves, broad
beans, red wine, black grapes, strawberries and apricots.
Apples, blackberries, broad beans, cherries, black
grapes, pears, raspberries and chocolate are rich in
epicatechin. The main dietary source of catechins is
green tea [2, 10, 12, 23-26, 35, 37].
Due to the presence of carbonyl and hydroxyl functional
groups, available groups can be chelated by metal
ions, especially using various salts of transition metals,
which have a role as a biological constituent in the
body [9, 17, 20, 25, 32, 37, 42, 43, 45]. These
complexes are considered to be responsible for the
manifestation of the antioxidant and antiradical action.
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Studies have shown that these metal complexes with
flavonoids determine the obtaining of a wider palette
of properties and potentiate those characteristic of
the initial compounds [8, 17, 22, 31, 38, 42-44].
(-)-epicatechin (+)-epicatechin
gallocatechin epigallocatechin
Figure 1.
Chemical structures of the most common catechins
(Structures obtained using PubChem,
https://pubchem.ncbi.nlm.nih.gov/)
Starting from the fact that multiple food supplements
contain high levels of flavonoid derivatives or plant
extracts rich in catechins along with minerals and
vitamins, we wanted to investigate the possible changes
that occur when such components are taken together.
Thus, our aim was to obtain and characterize a catechin-
zinc complex (Cat-Zn complex), and to assess its in
vitro potential in various tests.
Materials and Methods
Starting materials
Catechin (Cat, 98%), zinc acetate (Zn2+, 98%), sodium
hydroxide (NaOH, 99%) were purchased from Sigma-
Aldrich (Germany). Ethanol (EtOH, HPLC grade),
Methanol (MeOH, ≥ 99.8%), dimethylsulfoxide (DMSO,
≥ 99.5%, p.a.), acetone (≥ 99.5%, p.a.), chloroform
(dried, ≥ 99.8% CHCl3, ≤ 50 ppm H2O) were
purchased from Carl Roth (Germany). All reagents
were used as received without any further purification.
Instrumentation
Structural characterization of the newly formed complex
was carried out by FTIR (Fourier-transform infrared
spectroscopy). The FTIR spectra were registered on
an FT-IR Bruker Vertex 70 Spectrophotometer in the
reflexional mode, by ATR technique. Analysis was
done at a scan rate of 40/s in the range 4000 cm-1 -
380 cm-1. The determination was performed with
approx. 1 mg of sample.
The UV-vis spectra were recorded on a Shimadzu
UV-1280 spectrophotometer using very diluted solution
samples (approx. 10-5 M) in methanol [2, 6, 17, 18,
32, 34, 41].
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The morphology of the complex evaluated as powder
was observed using by Environmental Scanning Electron
Microscope type Quanta 200-FEI (SEM) at a resolution
2, 5 and 20 μm and obtained crystals were photographed
with the incorporated Cannon camera [1, 2, 30, 31,
45, 47].
Synthesis
The synthesis protocol was followed by previous
mentions by the literature with some modifications
[32, 40, 41, 45, 47]. Briefly, for the catechin-zinc
complex the catechin and the zinc salt was taken
using a 1:1 molar ratio. Catechin was dissolved in
methanol under continuous stirring, then the zinc salt
was gradually added over 30 minutes. The covered
flask was maintained for 3 hours at 40ºC (on a hot
plate magnetic stirrer). The reaction was stopped and
allowed to cool to room temperature. After removing
the solvent by filtration, a precipitate of yellowish-
white colour that was washed several times with
acetone and dried in an oven (40ºC). The obtained
precipitate was dried to constant mass and used for
further studies.
In vitro biological activity evaluation
The investigation of the potential of the newly obtained
complex consisted in the in vitro antioxidant and
antidiabetic activity assessment.
Antioxidant potential
The antioxidant activity was established by the following
assays.
Iron (II) chelation test. Ferrozine can quantitatively
form complexes with ferrous iron yielding a pink
colour. However, in the presence of chelating agents,
there is disruption of the formation of the complex
which leads to a decrease in the colour intensity. The
ferrous ion was monitored by measuring the formation
of a pink ferrous ion-ferrozine complex at 562 nm
[12, 15, 41, 42]. The used method was similar with
the one described by Burlec et al., 2022 [13].
Hydroxyl radical test. The hydroxyl radical, formed
in the reaction between the ferrous ion and hydrogen
peroxide, will hydroxylate the salicylic acid with the
formation of a pink-purple compound with maximum
absorbance at 562 nm [27]. Briefly, over 0.225 mL of
sample solution in dimethyl sulfoxide (DMSO) were
added 0.750 mL of 1.5 mM iron (II) sulphate solution,
0.9 mL of 20 mM sodium salicylate solution and
0.525 mL of 6 mM hydrogen peroxide solution. The
mixture was kept for 30 minutes at 37ºC, and after
cooling to room temperature, the absorbance of the
sample (control) was read at 562 nm compared to
the control sample (control) in which the ferrous
sulphate solution was replaced by bi-distilled water.
The positive control was processed under the same
conditions as the samples, but DMSO was used instead
of the sample solution.
Inhibition of lipoxygenase (15-LOX) activity. The
activity of 15-LOX was determined following the
formation of reaction products at 234 nm [14]. All
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reactions were performed at a final volume of 2 mL
and stirred using a magnetic bar at room temperature.
The reaction medium used contained 0.1 M HEPES
buffer (pH 7.4). The reaction was carried out by adding
the inhibitor (complex) in methanol to the cuvette
with the substrate buffer, and finally the enzyme was
added. All steps were similar to the already described
method [23, 26].
Antidiabetic effect
The antidiabetic activity was established by the
following assays.
Alpha-amylase assay. Alpha-amylase catalyses the
hydrolysis of starch with the release of glucose which
reacts with dinitro-salicylic acid and forms a yellow-
orange coloured compound with maximum absorbance
at 540 nm. In the presence of enzyme inhibitors, enzyme
activity is blocked or reduced with a reduction in the
absorbance of the solution at 540 nm [3]. 0.4 mL
sample solution in DMSO was mixed with 0.08 mL 2
IU/mL enzyme solution, 0.2 mL 0.5% starch and 0.16
mL 20 mM phosphate buffer pH 6.7 and maintained
the solution for 10 minutes at 37ºC. After 10 minutes,
0.32 mL of dinitro-salicylic reagent solution was added,
and the reaction mixture was maintained for 15 minutes
at 100ºC. The solution was cooled, and the absorbance
of the sample was read against the sample control in
which no enzyme was added. The positive control
was obtained in a similar manner using DMSO [26].
Alpha-glucosidase assay. Alpha-glucosidase catalyses
the hydrolysis of pNFG to p-nitrophenylphosphate, a
yellow compound with maximum absorbance at 405
nm. In the presence of inhibitors, the enzyme activity
decreases or is blocked with the reduction of absorbance
of the solution at 405 nm [4]. The methodology was
similar to the one described by Iancu et al., 2020 and
consisted in the mixing of 0.1 mL sample (DMSO)
with 0.25 mL enzyme solution 1 IU/mL and the
solution was maintained for 5 minutes at 37ºC. After
5 minutes, 0.25 mL of 3 mM pNPG solution was
added and maintained for 10 minutes at 37ºC. The
reaction mixture was brought to room temperature,
1 mL of 0.2 M Na2CO3 solution was added, and the
absorbance of the sample (control) was read at 405 nm
against the blank of the sample (control). The positive
control included DMSO instead of the sample [26].
For the samples in which an enzyme inhibition capacity
of over 50% was obtained, the IC50 value expressed
in µg sample/mL final solution was calculated. IC50
was calculated taking into account the first lower
value and, respectively, the first higher value of 50%,
obtaining, by linear interpolation, the concentration
of the antioxidant agent solution that corresponds to
an activity of 50%.
Statistical analysis
All determinations were performed in triplicate, the
results being expressed as the mean of 3 determinations ±
standard deviation. For correct correlations and statistical
 FARMACIA, 2023, Vol. 71, 4
significance, t-student analysis was performed (p <
0.001).
Results and Discussion
Synthesis and Complexation yield
In this study, the most important assessed parameters
were the molar ratio of the reactants, the solvents used
to reach the highest yield, adjustment of reaction
temperature and the pH of the solution to encourage
the formation of the precipitate, and to increase the
reaction yield [6, 17, 30]. Also, stirring time and
speed were adjusted to favour intimate contact between
the reactants (catechin and zinc salt) and the reaction
Sample
Water Ethanol
Cat-Zn-complex 35.00 44.00
pH 5 5.5
1 N NaOH - -
Boric acid - -
Cat-Zn-complex
Phosphate buffer - -
1 N HCl 10.11
FT-IR analysis
The infrared spectrum of free catechin showed the
ν(C=O) band shifted from 1657 cm −1 to 1598 cm−1
upon binding with Zn (II) ion. This was due to the
formation of the coordination bond between the carbonyl
oxygen and the metal ions. A new ring was formed
in the complex which accentuated the conjugative
effect, thus the characteristic IR absorption band
ν(C=C) moved from 1619 cm−1 to 1509 cm−1. The
vibrations at 634 cm −1 observed in the complex implied
the presence of the O-Zn bond, which was not present
in free catechin. The FT-IR spectra of free catechin
and the newly formed complex is shown in Figure 2.
Figure 2.
Comparative FT-IR spectra of free catechin and the
Cat-Zn complex
UV-VIS spectra
For the catechin-zinc complex formed, the UV-VIS
absorption spectra were recorded when a shift of the
absorption maximum of the complex compared to the
medium. In Table I are displayed the reaction yields
obtained changing these parameters. Although we tried
to modulate the reaction medium by adjusting the pH,
the most interesting results were obtained for the
system where the pH was adjusted at pH = 8.5 using
a 1 N NaOH solution.
Scientific data indicates that the best parameters should
be adjusted to the methodology, solvents, lab equipment
and the complexation process is also dependent on
the metal salts, organic and inorganic derivatives
allowing the formation of certain compounds in higher
amounts [6, 17, 30, 31, 34, 38, 39].
Table I
Yields obtained changing the pH and solvent
Complexation yield, %
Methanol 96% Ethanol Hydroalcoholic mix (1:1)
59.20 48.10 37.90
Complexation yield, %
7 7.5 8 8.5 9 10 11
55.20 56.35 59.82 60.20 61.80 60.80 59.10
34.80 35.20 36.25 37.25 38.35 37.02 36.85
28.22 28.30 29.71 30.80 31.05 30.51 30.35
12.01 - - - - - - -
absorption maximum of catechin can be observed,
which confirms the formation of the complex (Figure 3).
The results of the UV-VIS spectra provided significant
information on the formation mode of catechin-zinc
bonds. The Zn(II) ion induced a π → π∗ transition
and formed a condensed ring with catechin through
the 3-OH and 4-oxo groups. The 5-OH group was not
involved due to the lower protonic acidity and steric
hindrance in the complex.
Figure 3.
UV-VIS spectra of catechin and the newly formed
complex with zinc
Morphology
SEM images of zinc complex with catechin revealed a
fibrous morphology with acicular structure and uneven
fracture. We observed the formation of small aggregates
of crystals of 1 μm in size that show adhesion at the
level of some larger segments, which may indicate
centres of aggregation of different amounts of the
complex. Various aspects of the obtained images
are included in Figure 4.
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Figure 4.
SEM images of the Cat-Zn complex

Our method was similar to other described previously they can also present pro-oxidant effects through the
in the scientific literature. Flavonoids often function ability to reduce the ferric ion to ferrous ion which,
as hydrogen donors, which is one of the factors that through the Fenton reaction, generates hydroxyl radicals
leads to the development of metal coordination complexes with an important oxidizing effect [42, 43].
that have a high degree of stability. In most cases, Iron chelation assay
reflux condensation or synthesis at room temperature Chelation of ferrous ions also represents an indirect
is used to create metal ion complexes, however reflux anti-inflammatory mechanism considering the fact that
condensation is also an option. In order to improve inflammatory processes are accompanied by oxidative
flavonoid metal coordination, the phenolic hydroxyl phenomena in which ferrous ions are involved [24].
groups are often deprotonated prior to metal coordination Based on these observations, there is a risk that some
[41, 47]. Numerous research have revealed that plant compounds generate ferrous ions with a pro-
flavonoids most often form complexes with a metal oxidant effect, and thus it is important to evaluate the
to ligand (M:L) molar ratio of 1:2, which has strong capacity of extracts or plant compounds to chelate
stability constants [9, 12, 22, 30]. The hydroxyl groups ferrous ions. The obtained results showed that through
in the flavonoid structure and the keto group make up complexation the inhibitory activity of catechin decreases,
the chelating sites in the flavonoids. The literature whereas the activity of Zn2+ increases (Figure 5).
also suggests that the effectiveness with which flavonoids
attach to metal ions is strongly connected with the
type and state of transition metal ions [2, 5-8, 17,
18]. Such changes are noted for our complex in the
FTIR and UV spectra. Therefore, all the obtained data
from FTIR, UV spectrophotometry and SEM analysis
indicated the formation of the Cat-Zn complex. Taking
into account all the results, the best conditions to
obtain such a complex were given by methanol as
solvent in a pH of 8.5, using NaOH (1 N) solution
for optimum reaction environment.
Biologic Potential Assessment
Figure 5.
Flavonoids are a class of polyphenolic compounds
Iron (II) chelation activity of the Cat-Zn complex
that are naturally found in plants and are involved in
a wide variety of biological and chemical processes.
Catechin, through its hydroxyl groups, has the ability
They have a C6-C3-C6 ring system, and they can
to chelate ferrous ions, a capacity that is lower compared
either be esterified or glycosylated in their natural
to compounds that contain carbonyl groups in the
form. Such compounds of vegetal origin, exhibit anti-
structure, such as quercetin.
oxidant action by neutralizing free radicals or other
pro-oxidant compounds in living systems. In particular,
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The complexation of catechin with zinc ions causes
an important reduction of its chelating capacity for
ferrous ions; so, if we compare the IC50 value (1653.79
for Cat-Zn-complex versus 290.88 for catechin), a
decrease in the chelating capacity of more than 5.5
times is observed. The zinc ion prevents the formation
of the coloured Fe2+-ferrozine complex, a phenomenon
that can be explained by the fact that the zinc ion is
also a bivalent cation.
Maintaining the chelating capacity of catechin even
after complexation with zinc ions has antioxidant
benefits through the catechin itself, but also through
the zinc ions that enter the body can be used for the
synthesis of superoxide dismutase, an enzyme with a
role in the decomposition of the superoxide radical
anion. Catechin also has the ability to cross the blood-
brain barrier and thus can reduce neuroinflammatory
phenomena [25, 27, 37].
Hydroxyl radical scavenger test
Hydroxyl radicals are extremely aggressive prooxidant
compounds because they induce oxidation reactions
in the chain and can affect cellular and subcellular
biological structures. Hydroxyl radicals also modify
functional groups in the structure of proteins by changing
their spatial structure and affecting biological function
or inducing pathological phenomena such as athero-
sclerosis, neurological disorders and cancer [33].
Similar to the lipoxygenase inhibition test and in this
hydroxyl radical scavenger test, the Cat-Zn complex
showed a much-reduced action compared to catechin
(over 7-fold reduction), a phenomenon that could be
determined by the partial blocking of the hydroxyl
groups in the structure catechins (Figure 6). Hydroxyl
groups, especially those of the phenolic type, are
proton donors and can neutralize free radicals. On the
other hand, the complexation has a better impact on
the zinc ions scavenger activity, which increased
significantly (p < 0.001).
Figure 6.
Hydroxyl radical scavenger activity of the Cat-Zn
complex
The reduced capacity of the Cat-Zn complex to
neutralize the hydroxyl radical is an expected phenomenon
considering the reduced effect of this complex, of
chelating the ferrous ion that is involved in the synthesis
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of the hydroxyl radical, in this in vitro test. Catechin,
compared to other compounds from the class of catechins
(epigallocatechin and epigallocatechin gallate), has a
lower antioxidant effect and to evaluate the influence
of complexation with zinc, it is necessary to evaluate
the complexes formed with these catechins [10].
Lipoxygenase inhibition assay
Lipoxygenases are enzymes that catalyse the oxidation
of unsaturated lipid compounds, mainly unsaturated
fatty acids to lipid peroxides that can have physiological
effects (as signal molecules), but also numerous
pathological implications, given that most of the time
their synthesis capacity exceeds the neutralization
capacity of the endogenous antioxidant systems [19].
Lipid peroxides formed under the action of these
enzymes will generate oxidative stress and inflammatory
phenomena, including neuroinflammation and cancer
[8, 10, 14, 19, 20, 21].
Our results in this assay highlighted the fact that by
complexing catechin with zinc ions, the inhibitory
effect is greatly reduced both by comparison with
catechin, but also by comparison with zinc ions.
The t-student values (p < 0.001) indicated highly
statistically significant results for catechin versus
Cat-Zn-complex, and in regards to Zn versus Cat-
Zn-complex.
Figure 7.
Lipoxygenase inhibition of the Cat-Zn complex
Studies in which catechin and zinc were used showed
also that the antioxidant effects of catechin are better
maintained when synthesizing nanoparticles with zinc
oxide, compared to the catechin-zinc ion complex [8].
Antidiabetic potential of the Cat-Zn-complex
The digestion of food starch involves the participation
of pancreatic alpha-amylase and alpha-glucosidase
which will transform the starch into glucose molecules
that are absorbed in the intestine, pass into the blood
and cause postprandial hyperglycaemia. Its value is
dependent on the available starch for digestion, the
activity of the enzymes involved in digestion and the
absorption capacity of the intestinal systems. The
increase in blood sugar, above the physiological levels,
determines the accentuation of the phenomenon of
oxidative stress and the uncontrolled glycosylation of
proteins with the worsening of symptoms in diabetes
and the risk of organic complications [28, 36].
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Inhibition of alpha-amylase
The Cat-Zn-complex analysed in the present study
has a better alpha-amylase inhibition capacity, compared
to zinc or catechin (Figure 8).
Figure 8.
Alpha-amylase inhibition potential for the Cat-Zn
complex
Inhibiting or reducing the activity of alpha-amylase
and alpha-glucosidase is one of the therapeutic options
in diabetes because the intestinal digestion of carbo-
hydrates is reduced with the reduction of available
glucose for absorption. Through this mechanism,
postprandial hyperglycaemia and, indirectly, its negative
consequences are reduced. Classical inhibitors, such
as acarbose, present the risk of adverse digestive
reactions and thus, compounds of vegetable origin
or metal ions with a moderate inhibitory effect, may
present an advantage [46].
Zinc ions reduce or block the activity of the enzyme
by changing its secondary structure, with an increase
in the frequency of areas with a beta-folded or linear
structure at the expense of those with an alpha-helix
structure. Changing the secondary structure will affect
the structure of the active centre of the enzyme and
reduce the interaction with the substrate [32].
Flavonoids modify the activity of the enzyme by
creating hydrophobic interactions and hydrogen bonds
with the amino acids in the enzyme structure and thus
modify its spatial structure, affecting the correct enzyme-
substrate interaction [29]. Spectroscopy and in silico
studies have shown that flavanols and catechin can
also interact with starch, blocking its access to the
active centre of the enzyme [29, 32, 40, 41, 49].
Matowane et al. showed a 2.6-fold increase in the
alpha-amylase and alpha-glucosidase inhibition capacity
of the caffeic acid-zinc acetate complex compared to
caffeic acid and a two-fold increase in the anti-
oxidant effect of the complex compared to caffeic
acid. The same study highlighted the ability of the
complex to increase the action of insulin on the cellular
uptake of glucose through GLUT-4 transporters [38].
The inhibition capacity of alpha-amylase, determined
in the present study (IC50 = 147.33 ± 5.19, p < 0.01 –
very significant), is close to that highlighted by Yilmazer-
Musa in the study on salivary alpha-amylase, for which
the IC50 value was 160 μg/mL.
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Inhibition of alpha-glucosidase
Similar to the alpha-amylase inhibition test, the Cat-
Zn-complex also reduced alpha-glucosidase activity,
but with a much higher efficiency (Figure 9). The
complex is approximately four times more active
compared to the zinc sulphate that was used in the
preparation of the complex. Compared to catechin,
the complex presented a slightly increased inhibitory
efficiency (p < 0.001, extremely significant).
Figure 9.
Alpha-glucosidase inhibition potential for the Cat-
Zn complex
Catechin reduces the activity of alpha-glucosidase by
making hydrogen bonds with the functional groups
of the amino acids in the enzyme structure [16].
Numerous studies highlight the ability of catechin
and other polyphenols of vegetable origin to inhibit
or reduce the activity of alpha-amylase or alpha-
glucosidase, with sometimes much greater efficiency
compared to acarbose, which is used as a pharmaceutical
substance to reduce the digestion of carbohydrates
[26, 28, 29, 32, 40, 48, 49].
The effect of zinc inhibiting alpha-amylase or alpha-
glucosidase is also maintained when using it in the
form of nanoparticles, observing similar to those
obtained in the present study, a more intense action on
alpha-glucosidase, compared to alpha-amylase [40].
All in all, the structure of the newly formed complex
is determined by the structure of flavonoid and metallic
ion that is involved which may impact the complex's
biological interactions, which may be unique from the
flavonoids that it was generated from [18, 22, 30-32,
42]. For example, transition metal ion complexes of
quercetin and genistein dramatically modify the chemical
characteristics of their particular parent flavonoids
[34, 43, 45, 47]. There are also many synthetic
substances that are derived from flavonoids that try
to simulate the biological effects of the natural ones,
or to achieve even greater effects by modulating the
pharmacological activity to target different tissues and
sustain normal function of affected organs [30, 42, 45].
Conclusions
The complex obtained by using zinc acetate and
catechin was obtained with a good yield by using
 FARMACIA, 2023, Vol. 71, 4
methanol as solvent, and NaOH as coregulator. All
modern techniques indicated the formation of the
complex. Moreover, the antioxidant capacity varies
depending on the assay, and the potential of the
obtained complex is different from the initial reagents
(zinc, catechin). Considering the importance of zinc
for maintaining insulin stability and the beneficial
effects of the complex to reduce carbohydrate digestion,
we can appreciate the fact that such complexes could
be useful in the therapy of patients with diabetes,
and their in vivo toxicological and pharmacokinetic
evaluation is necessary. Further research is needed to
establish the true potential of catechin-zin complexes
found often in food supplements.
Conflict of interest
The authors declare no conflict of interest.
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