Sono-transformation of tannic acid into biofunctional ellagic acid micro/nanocrystals with distinct morphologies

Article abstract of DOI:10.1039/c7gc03163g

A sustainable, reagent-less and one-pot ultrasonic methodology has been developed to transform amorphous tannic acid into regularly shaped crystalline ellagic acid particles. Multiple and consecutive reactions have been performed on tannic acid, in aqueous solution without the addition of any external reagent. The size, morphology and bio-activity of ellagic acid micro-nanocrystals can be finely tuned by choosing appropriate ultrasonic parameters.

Full text of DOI:10.1039/c7gc03163g

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Sono-transformation of tannic acid into
biofunctional ellagic acid micro/nanocrystals with
Cite this: DOI: 10.1039/c7gc03163g
distinct morphologies†
Received 20th October 2017,
Accepted 9th January 2018
a
a
a
DOI: 10.1039/c7gc03163g
Sukhvir Kaur Bhangu,
Ritu Singla, Enrico Colombo,
a
b,c
Muthupandian Ashokkumar * and Francesca Cavalieri
*
rsc.li/greenchem
A sustainable, reagent-less and one-pot ultrasonic methodology sation reactions and (iv) crystallisation of the product into
has been developed to transform amorphous tannic acid into regu- regularly shaped particles. To our knowledge, multiple and
larly shaped crystalline ellagic acid particles. Multiple and consecu- consecutive reactions induced by acoustic cavitation have
tive reactions have been performed on tannic acid, in aqueous never been reported before.
solution without the addition of any external reagent. The size,
As a proof of concept, we have developed a sustainable,
morphology and bio-activity of ellagic acid micro-nanocrystals can reagent-less and cost effective synthetic methodology to trans-
be finely tuned by choosing appropriate ultrasonic parameters.
form amorphous tannic acid into regularly shaped crystalline
ellagic acid particles, with tunable morphologies and function-
ality. The size and shape of ellagic acid micro-nanocrystals can
be finely tuned by choosing appropriate ultrasonic parameters
such as sonication time, power and frequency. Ellagic acid
powder is conventionally obtained either through the enzy-
matic hydrolysis oxidation of ellagitannins with tannase and
Acoustic cavitation in aqueous solutions results in the gene-
ration of a reactive green processing environment enabling a
number of reactions without the addition of any external
reagents. Extremely high local temperatures and pressures, oxi-
dizing and reducing agents and physical effects (microjets, tur-
bulence, shear forces, etc.) are generated by acoustic cavitation,
which may exert synergistic effects. High and low frequency
ultrasound has been employed in materials synthesis,
polymerization, degradation of organic and inorganic pollu-
tants, wastewater treatment, crystallisation, and microcapsule
7,8
9
decarboxylase or multistep organic synthesis without any
control over size and shape.
Conversely, in this work we demonstrate a remarkably
simple and unique green synthetic route to generate ellagic
acid micro-nanocrystals. In addition, our approach should be
readily extendable to other sono-responsive biomolecules.
The chemical functionalization and assembly of phenolic
biomolecules such as gallotannins, ellagitannins, catechins
and flavonoids have recently attracted much attention due to
their potential application in different fields including
1
–4
synthesis encapsulating food and cosmetic ingredients.
Recently, we have demonstrated a new concept that cavita-
tion bubbles generated by high frequency ultrasound in the
range of 0.35–1 MHz can act as catalytic binding sites for the
5,6
radical mediated C–C coupling of phenolic moieties. Simple
phenolic molecules such as tyrosine and phenol were coupled
by a C–C linkage to form oligomeric bio-functional phenolic
systems, which are not obtained with other radical sources
sensing, imaging, drug delivery, live-cell protection, and
10–13
catalysis.
The current approaches for the manipulation of
phenol moieties into the desired nanoarchitectures require the
(such as Fenton’s reaction).
formation of catechol–metal coordination complexes or oxi-
Herein, we show that cavitation bubbles are simple micro-
14
dative coupling reactions catalyzed by metals or enzymes.
reactors with reactive surfaces to perform one-pot multiple
reactions on complex organic molecules, namely (i) hydrolysis
of an ester linkage, (ii) C–C coupling reactions, (iii) conden-
The biomolecule obtained in this study, ellagic acid (EA), is of
particular interest in the biomaterials area because in vitro and
in vivo studies have shown that EA can significantly inhibit
cancer formation in the colon, oesophagus, liver, lung, tongue
and skin and the infection of human T-cell lymphotropic virus
a
School of Chemistry, The University of Melbourne, VIC 3010, Australia.
E-mail: masho@unimelb.edu.au
type-1-carrying MT-4 cells by human immunodeficiency virus
b
7,15
Department of Chemical and Biomolecular Engineering, The University of
(
HIV).
Melbourne, VIC 3010, Australia. E-mail: francesca.cavalieri@unimelb.edu.au
The direct sono-transformation of tannic acid into ellagic
c
Dipartimento di Scienze e Tecnologie Chimiche, Universita’ di Roma “TorVergata”,
acid micro-nanoparticles is a versatile method for the engin-
eering of ellagic acid-based biomaterials with tailored struc-
tural and functional properties.
via della ricerca scientifica 1, 00133 Rome, Italy
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7gc03163g
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Fig. 1 The effect of sonication time on the TAS particles obtained. (a) 2 h, (b) 3 h, (c) 4 h., and (d) 6 h; (e) average length of the particles as a function
of sonication time (scale bar = 10 µm). Samples were sonicated at 355 kHz and 5.5 W cm−3
.
Tannic acid is a biomass belonging to the category of hydro-
lysable tannins with a multiple number of gallic acid and
galloyl units attached to the central sugar core which is mostly
1
6,17
D-glucopyranose.
The aim of the study was to explore the
chemical transformation of tannic acid induced by ultrasound.
The sonication of an aqueous solution of tannic acid at 355
kHz and 5.5 W cm− was carried out at pH 7 for up to six
hours. Surprisingly, we discovered that the sonicated tannic
acid undertook a more complex modification pathway. We
observed the formation of nano-micrometer sized crystalline
particles hereafter referred to as TAS particles. The SEM
images of the TAS particles obtained after different sonication
times are shown in Fig. 1a–d. It can be seen that the size,
shape and morphology of the crystals depends on the soni-
cation time. Nanoparticles with a discoidal shape turned into
a flat ellipsoidal shape growing in size. The graph shown in
Fig. 1e illustrates that the average length of particles increased
from 650 nm to 7 µm when the sonication time was increased
from 2 h to 6 h.
3
We sought to investigate the chemical composition and
the mechanism of the formation of the obtained particles. In
order to gain insight into the composition of TAS particles
mass spectrometry, spectroscopy, chromatography and NMR Fig. 2 Schematic of the proposed mechanism for the ultrasonic trans-
analysis were conducted. The electrospray mass spectrometry formation of TA to ellagic acid. The core of cavitation bubbles generat-
ing high temperatures leading to the homolysis of the water molecule to
(
ESMS) and MALDI-TOF data (Fig. S1a, ESI†) acquired after
•
•
H and OH , step 1 showing the hydrolysis of tannic acid to gallic acid,
step 2 the oxidative coupling of gallic acid units at the bubble surface to
form a dimer and step 3 where the dimer spontaneously lactonized to
the dissolution of the TAS particles in Milli-Q water have
shown the presence of a major peak at m/z 301 that could be
assigned to ellagic acid (see Fig. 2 for the structure), form EA and its derivatives.
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accompanied by some small peaks ascribed to higher mole-
cular weight species (Fig. S1a and b, ESI†). The sono-trans-
formation of tannic into ellagic acid was further confirmed
1
13
by HPLC (Fig. S1, ESI†), absorption spectroscopy, H and
C
NMR spectroscopy (Fig. S2–S5, ESI† respectively) and PXRD
analyses.
In particular the X-ray diffraction patterns indicate that
commercial tannic acid is amorphous, which is consistent
1
8
with the literature, whereas the TAS particles are crystalline
Fig. S6, ESI†). The X-ray diffraction pattern of the TAS particles
(
1
9
was identical to the commercial ellagic acid, for example,
peaks at 2θ values of 17.9°, 21.4°, 24.8°, 26.7° and 28.5°, indi-
2
0
cating the crystalline nature of the particles. However, some
additional peaks were also observed. Overall our data suggest
that high frequency sonication provides a simple one-step
approach for the synthesis of crystalline ellagic acid particles
from tannic acid with different shapes and sizes. Fig. 2 shows
the possible mechanism involved in the formation of ellagic
acid from TA. It is well known that cavitation bubbles driven
by high frequency ultrasound can give rise to physical effects
such as shear forces, turbulence, microjets, etc. and chemical
effects such as the generation of radicals due to the homolysis
•
•
•
of water to H to OH (Fig. 2). The recombination of 2 OH
Fig. 3 SEM images of the TAS particles obtained using different TA con-
centrations and ultrasonic parameters: (a) TA 0.5 mM at 355 kHz and 5.5
1
,3
would lead to the formation of H
these effects can play a significant role in inducing different
chemical reactions. TA is composed of a multiple number of
galloyl units and shear stress and radicals generated by cavita- (f) SEM image of the TAS particles at 355 kHz and 20 W cm ; (g) the
2
O
2
.
The combination of all
W cm− , (b) TA 1 mM at 355 kHz and 5.5 W cm ; (c) TA 1 mM at 1 MHz
3
−3
−3
and 5.5 W cm ; (d) SEM image of the TAS particles at 355 kHz and 6.7
−3
−3
W cm , (e) SEM image of the TAS particles at 355 kHz and 12 W cm
,
−3
tion induces the hydrolysis of galloyl moieties (step 1) without graph provides the average length of the TAS particles at different con-
centrations of TA as a function of ultrasonic frequency and (h) average
length of the TAS particles as a function of ultrasonic power.
the use of any acid or base catalyst. Subsequently, two gallic
acid units undergo catalytic oxidative coupling through a C–C
linkage to form a dimer at the bubble−solution interface (step 2)
5,6
as observed in our previous studies.
The spontaneous
lactonization likely occurs in the bulk phase to form EA (step 3). bubble interface are necessary for the coupling of gallic acid
Ellagic acid can further react with gallic acid molecules on molecules.
the surface of cavitation bubbles to form derivatives by C–C
In order to gain further insight into the effect of ultrasonic
oxidative coupling mediated by OH radicals. Finally, the ultra- parameters on EA particle morphologies, further experiments
sound assisted assembly and crystallisation of ellagic acid were carried out. Fig. 3 shows the SEM images of the TAS par-
•
completes the series of reactions triggered by ultrasound.
ticles obtained as a function of different ultrasonic frequencies
Ultrasound assisted crystallisation has been reported in the (355 kHz and 1 MHz), initial concentrations of TA (0.5 mM
literature and it was suggested that the ultrasonic parameters and 1 mM) and ultrasonic powers (5.5, 6.7, 12, and 20 W
−
3
can be used to control size distribution, average crystal size cm ). Micro-nanoparticles with different morphologies were
2
1–23
and type of polymorph obtained.
Particularly, crystal size obtained including small and large discoidal shape particles,
can be controlled by two cavitation effects, the generation of flat ellipsoidal particles and brick-like particles. These images
shock waves by acoustic cavitation which increases the nuclea- suggest that the morphology of the TAS particles is affected
tion rate and microturbulence that can direct the subsequent significantly by the experimental parameters. When the initial
2
2
growth of the crystals. Interestingly, different crystal mor- concentration of TA was increased from 0.5 mM (Fig. 3a) to
phology was observed at different sonication times. After two 1 mM (Fig. 3b), the size of the particles increased from 3.7 to
hours of sonication, the EA concentration reaches the super- 7 µm at 355 kHz. Also, when the ultrasonic frequency was
saturation condition, forming submicron particles (Fig. 1a). switched from 355 kHz (Fig. 3b) to 1 MHz (Fig. 3c) at 1 mM
Subsequently the ultrasonically generated EA, with increasing concentration of TA, changes in the morphology, viz., size
sonication time, contributes to the particle growth up to (from 7 to 9.5 microns) as well as the aspect ratio, i.e., the
•
7
micrometers (Fig. 1d). It is worth noting that the use of OH
length to width ratio (from 1.8 to 1.1) of the crystals, were
radicals generated from hydrogen peroxide sources, such as observed.
Fenton’s reagents, didn’t result in the formation of fluorescent
Similarly, the effect of ultrasonic power was studied on the
and regularly shaped particles. This suggests that the simul- morphology and the size of the TAS crystals. Fig. 3d, e and f
taneous presence of OH radicals and a reactive cavitation represent the SEM images of the particles obtained when TA
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solution was sonicated for 5 hours at 6.7 W cm 3, 12 W cm−3
−
Similarly, ultrasonic power can be also used to tune the
and 20 W cm− , respectively. The results showed that when the size of the particles (Fig. 3d–f). The crystal size decreases
3
−
3
−3
power was increased from 5 W cm to 20 W cm , the crystal with increasing power due to the enhanced physical effects
size reduced from 7 microns to 200–350 nm (Fig. 3h). Fig. 3g and chemical effects. Peculiarly, increased shear forces, tur-
summarises how the average length of the TAS crystals bulence and shock waves as well as the number of radicals
changed with changes in the frequency and TA concentration. (required for the oxidative coupling reaction) can significantly
Also, the yield of EA produced varied from 40 to 70% when the improve the local supersaturation resulting in higher nuclea-
−
3
−3
21–23
power was increased from 5.5 W cm to 20 W cm
.
tion rates and smaller crystal sizes.
Hence, the decrease
Fig. 4 shows a schematic to explain how the morphology, in the crystal size is observed with the upsurge of ultrasonic
size and aspect ratio of the TAS particles are affected by power.
various parameters such as sonication time, concentration of
The aspect ratio of microparticles is dictated by the concen-
TA, ultrasonic power and frequency. As crystal size and size tration of ultrasonically produced EA. EA assembles into dis-
distribution are influenced by the population and growth rate coidal shaped particles at a higher frequency (Fig. 3c) when
•
of the nuclei, a rise in the number of nuclei formed (nuclea- the rate of generation of OH radicals is low and also at low
tion rate) would lead to a reduction in the amount of the concentrations of TA (Fig. 3a), consequently, decreasing the
solute available for the growth of individual crystals leading to concentration of EA. However, at a higher concentration of EA,
2
2,23
small sized crystals.
With an increase in the ultrasonic frequency from 355 kHz
microparticles preferentially form ellipsoidal crystals.
The optical properties of the TAS particles were investigated
•
to 1 MHz, a decrease in the number of OH radicals produced by fluorescence spectroscopy and microscopy. Fig. 5a rep-
and shear forces has been reported.
1
,3
•
OH radicals are resents the fluorescence emission spectra of the TAS particles
required for the oxidative coupling reaction; therefore, at a at different excitation wavelengths. It can be observed that λmax
higher frequency the amount of EA generated would decrease is around 440 nm when excited at 360 nm. The observed spec-
resulting in the decrease in the local supersaturation and troscopic properties were similar to those of pure EA (Fig. S7,
nucleation rate; thus an increase in the crystal size and aspect ESI†). The dependence of the emission wavelength on the exci-
ratio is observed (Fig. 3b and c).
tation wavelength indicates the presence of multiple other oli-
Fig. 5 (a) Fluorescence emission spectra of 1 mM TA solution sonicated
at 355 kHz and 5.5 W cm− recorded at different excitation wavelengths,
3
(b) fluorescence microscopy images of the TAS particles showing blue,
green and red emission. (c) DPPH radical scavenging activity of TA and
EA particles as a function of concentrations after 3 min incubation time,
(d) cytotoxicity of TA and TAS as a function of the concentration after
24 h incubation at 37 °C with MDA-MB-231 cells.
Fig. 4 Schematic illustration of the effect of sonication time, frequency,
power and concentration of TA on the morphology and size of the TAS
particles.
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gomeric species along with the formation of EA, in agreement
with previous results. The fluorescence of the TAS particles
Conclusions
was confirmed by fluorescence microscopy where blue, green We have demonstrated the unexpected ability of ultrasound to
and red emissions (Fig. 5b) were observed. perform multiple and consecutive reactions on tannin mole-
To demonstrate the functional properties of the TA and TAS cules. The product, i.e., ellagic acid assembles and crystallizes
particles, further studies were performed. The thermal stability into regularly shaped particles. The particle sizes and mor-
study of the TA and TAS particles was performed by thermo- phologies can be tuned by changing the ultrasonic parameters
gravimetric analysis. Fig. S8, ESI† shows that the TAS particles and concentration of tannic acid. We have demonstrated that
are also more thermostable compared to tannic acid. The the radical yield and shear forces can alter the size and mor-
ζ-potentials of the TAS particles were measured to be around phology of the particles. TAS particles possess optical pro-
−56 mV ± 11 mV in deionized water. This indicates that the perties in a wide range of wavelengths as well as anticancer
surface of the particles is negatively charged most likely due to and antioxidant properties. In particular, TAS nanoparticles
the presence of acidic phenoxy groups and the ζ-potential of showed significantly higher cytotoxicity and higher radical
the TAS particles is in the range for the colloidal stability of scavenging activity as compared to that of soluble TA and EA.
the particles. Hence, the TAS particles did not aggregate in In conclusion, this study highlights the importance of using a
aqueous solution.
powerful and green ultrasonic methodology to synthesize EA
The antioxidant properties of TAS and EA were studied particles starting from a biomass such as TA. The synthesis
and compared using a DPPH assay. Fig. 5c shows the percen- was performed without using any reagents or organic solvents
tage radical scavenging activity of the TAS and EA particles as with a fine control over particle size and morphology. We are
a function of concentration after 3 min incubation. Firstly, it currently investigating the potential use of TA nanoparticles as
was observed that the activity increased as a function of TAS drug delivery carriers. In addition, future efforts will be
and EA concentrations. Secondly, it was also observed that directed towards the utilization of this strategy to modify other
TAS nanoparticles had a better radical scavenging activity sono-reactive molecules to obtain biofunctional molecules.
than EA at all the concentrations. Polyphenolic molecules
have the capacity to prevent DNA and protein damage as they
can scavenge the OH radicals, peroxyl radicals and nitrogen Conflicts of interest
2
4
reactive species due to the high degree of hydroxylation.
These results confirm that the antioxidant properties of TA
are improved when converted to TAS particles. The anti-
oxidant properties can be exploited in various biomedical
applications to protect the cells and biomolecules against
free radicals.
There are no conflicts to declare.
Acknowledgements
This research was funded by the ARC Future Fellowship 2014
Finally, the cytotoxicity and anticarcinogenic activity of
the TA and TAS nanoparticles were assessed by an MTT via-
bility assay. MDA-MB-231 breast cancer cells were incubated
with TA and EA at different concentrations. Fig. 5d shows
that the TAS nanoparticles have higher anticancer activity as
compared to TA at all concentrations. The IC value of the
(
project number FT140100873, to FC). S. K. B. acknowledges
the University of Melbourne for the scholarships (MRS). We
thank Prof. Frank Caruso for helpful discussion.
5
0
Notes and references
−
1
TAS particles was between 20 and 10 µg mL , whereas TA
−
1
has a value >80 µg mL . The chemo-preventive properties of
different polyphenols have been determined both in vivo and
in vitro in the past. They exert such effects as they have the
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