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Formulation of Nanoencapsulated Catechin with Chitosan as Encapsulation

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Article · December 2014

DOI: 10.1016/j.proche.2014.05.028

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Procedia Chemistry 9 (2014) 235 – 241

International Conference and Workshop on Chemical Engineering UNPAR 2013, ICCE UNPAR
2013

Formulation of Nanoencapsulated Catechin with Chitosan as

Encapsulation Material
Sari Intan Kailakua*, Ira Mulyawantia, Andi Nur Alamsyaha
a
Indonesian Center for Agricultural Postharvest Research and Development, Jl. Tentara Pelajar no. 12, Bogor 16114, Indonesia
* Corresponding author. Sari Intan Kailaku Tel.: +62251- 8321762. fax: +62251-8350920 .E-mail address: sari.kaylaku@gmail.com.

Abstract

Gambier is one of Indonesian superior estate crop. Among several chemical compounds in gambier, catechin from polyphenols
group has the greatest benefit for health. However, catechin has a limitedness of stability that causes a low bioavailability
property. One way to maintain and improve the stability of catechin is the application of nanotechnology. The objective of this
study is to obtain the best formulation in producing a solution of catechin nanoparticles with particle droplet size <300 nm as an
antioxidant material. Observations were conducted to the formulations of the concentration of the solution of chitosan (0.2 and
0.4%), sodium tripolyphosphate (Na-TPP) (0.1 and 0.2%), catechin extract (0.3, 0.4, 0.5 and 0.6%) and the ratio of chitosan
solution with catechin solution in Na-TPP ( 5 : 1 and 7 : 1) in order to determine one that yields the best solution. Observations
were carried on the stability and characteristics of catechin nanoparticle solution including particle size, particle surface
topography and internal structure. Catechin nanoparticles with best particle size was found in formula with 0.2% chitosan
solution, 0.4% catechin solution, 0.1% Na-TPP solution and ratio of chitosan solution with withcatechin in Na-TPP solution 7 : 1,
that is 137.6 nm. Physically, the best formulation of catechin nanoparticleshas good particle surface topography, internal particle
structure and emulsion stability.
© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
© 2014 The Authors. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Selection and
Peer-review peer-review
under under
responsibility responsibility
of the Organizing of the Organizing
Committee of ICCECommittee of ICCE UNPAR 2013.
UNPAR 2013

Keywords: nanoencapsulation; catechin; chitosan

1. Introduction

Gambier (Uncaria gambier Roxb.) is one of Indonesian export commodity that mostly prfoduced in Aceh, West
Sumatera, South Sumatera, Riau and North Sumatera1. Among the various chemical compounds in gambier,

* Corresponding author. Sari Intan Kailaku Tel.: +62251- 8321762. fax: +62251-8350920 .E-mail address: sari.kaylaku@gmail.com.

1876-6196 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Peer-review under responsibility of the Organizing Committee of ICCE UNPAR 2013
doi:10.1016/j.proche.2014.05.028
236 Sari Intan Kailaku et al. / Procedia Chemistry 9 (2014) 235 – 241

catechin (poliphenol group) is the largest in amount and has the most important benefit for health. A number of
biological activities of catechin had been reported. Catechin is known to have an even better antioxidant properties
than -tocopherol, hydroxyanisolebutylatedor hydroxytoluenebutylated2.However, catechin has at least one
limitedness, that is instability that leads to a decrease of its bioavailability properties.

Nanoencapsulated compounds have been developedinitially for the design of drug delivery systems. The
application of nanoencapsulation technology can increase the stability of catechin, thus increase its solubility and
bioavailability3. Encapsulation of catechinsin carbohydrate nanoparticles, liposomes orother pre-formed
materials,represents a solutionto increase the antioxidant’s efficacy of therapy12According to National
Nanotechnology Initiave4, nanotechnology is known as a science that learns the characterization and manipulation
of biological and microbiological materials with a size of less than 100 nanometer, including the unique
phenomenon and a novel functional properties that will emerge.

Nanoparticle coloidal system using a non-toxic biopolymer material is expected to be able to protect functional
properties of certain bioactive compound like catechin.Mixtures of chitosan and sodium tripolyphosphate (Na-TPP)
as carrier for catechin should be protect, stabilize, or control the release of the core. Chitosan as hydrophilic
polymer can easily cross-link with counter poly anions like Na-TPP to control the release of the drug13. In this case,
composition of chitosan and Na-TPP is the key parameter in controlling the properties and the structur of the system.
The objective of this study is to obtain the best formulation in producing a solution of nanoencapsulated catechin
with particle droplet size <300 nm.

2. Method

Catechin used in the research was extracted from gambier leaves by Laboratorium of Medical and Pharmacy
Technology – Science and Technology Research Center, Serpong, Indonesia. Chitosan (DD 85), sodium
tripolyphosphate (Na-TPP), glacial acetic acid from Sigma Aldrich.

Solution of chitosan was made with a consentration of 0.2 and 0.4%. It was obtained by adding chitosan to a
solution of glacial acetic acid with 1:1 consentration ratio, then was stirred for 24 hours and filtered with Whatman
no. 1 filter paper. Solution of Na-TPP was obtained by adding Na-TPP to distilled water with a concentration of 0.1-
0.2%, stirred for 2 hours. This solution of Na-TPP will be used as solvent for catechin. Catechin solution was made
with concentrations of 0.3, 0.4, 0.5, dan 0.6%.

Nanoencapsulation process was carried by mixing Na-TPP solution containing catechin to chitosan solution by
stirring using magnetic stirrer. The catechin solution was shed using syringe. Stirring was done at room temperature
(280C) and was continued for 15 minutes after all catechin solution was added to chitosan solution in order to
improve the reaction.

Observations were conducted to the formulations of the concentration of the solution of chitosan (0.2 and 0.4%),
sodium tripolyphosphate (Na-TPP) (0.1 and 0.2%), catechin extract (0.3, 0.4, 0.5 and 0.6%) and the ratio of chitosan
solution with catechin solution in Na-TPP (5 : 1 and 7 : 1).
 Sari Intan Kailaku et al. / Procedia Chemistry 9 (2014) 235 – 241 237

Na-TPP solution stirrer

and catechin
extract

Chitosan solution

Figure 1. Scheme of nanoencapsulation of catechin extract.

Experiment design used was completely randomized factorial design with two repetition. All formulas were
analyzed for its particle size using Particle Size Analyzer/PSA. A product with smallest particle size was then
selected as the best nanoencapsulation formula. Selected nanoencapsulated catechin product were characterized for
their physicochemical and functional properties, including particle structure using Scanning Electron
Microscope/SEM, the internal structure of the nanoparticle using Transmission Electron Microscope/TEM, emulsion
stability using centrifuge, catechin content using pyrolisis gas chromatography, and antioxidant capacity using
DPPH.

3. Results and Discussion

The results of particle size analysis showed that particle size is highly influenced by the concentration of chitosan
solustion concentration. Products with chitosan solution concentration of 0.2% had smaller particle sizes compared
to those of products with chitosan solution concentration of 0.4% (Fig. 2). Moreover, analysis results also showed
the trend of increasing particle size with the increasing concentration of catechin solution, both with chitosan
solution concentration of 0.2% and 0.4%. TEM images of chitosan nanoparticles and catechin loaded chitosan
nanoparticles indicated that loading of catechin in chitosan nanoparticles increased the particle size13.

The higher concentration of Na-TPP (0.2%) with the concentration of chitosan of 0.2% produced a smaller particle
size of nanoencapsulated catechin compared to concentration of Na-TPP 0.1%. However, on products with chitosan
concentration of 0.4%, higher Na-TPP concentration resulted a bigger particle size, which is concurrent to the
finding of Dhudani et al. (2010). The increased concentration of dissolved solids and concentration of chitosan and
Na-TPP generated agglomeration and a bigger particle size.

Nanoencapsulated catechin product with chitosan solution concentration of 0.2%, catechin concentration of 0.4%,
Na-TPP concentration of 0.1% and ratio of chitosan and Na-TPP of 7 : 1 was selected as the best formula because of
its smallest particle size, 137,6 nm. SEM showed that catechin was comnpletely coated in the matrix of Na-TPP and
chitosan, with the distribution of similar size and shape in the solution, that is a firm round. The appearance of the
particle surface was smooth, non-porous and no agglomeration (Fig 3).
238 Sari Intan Kailaku et al. / Procedia Chemistry 9 (2014) 235 – 241

The TEM image showed that catechin compounds trapped in the matrix of the coating material. Catechin
compounds trapped in the matrix indicated by the dark color. The bright part of the image showed the area of
chitosan, and it appeared that there are bonds between the chitosan and Na-TPP, which contained catechin
compounds (Fig. 4).

Figure 2. Particle sizes of catechin nanoencapsulations. a1= catechin concentration 0,3%;a2 = catechin concentration 0,4%;a3 = catechin
concentration 0,5%;a4 = catechin concentration 0,6%;b1 = Na-TPP concentration 0,1%; b2 = Na-TPP concentration 0,2%; c1 =
chitosan : Na-TPP ratio 5 : 1; c2 = chitosan : Na-TPP ratio 7 : 1. Red bars = chitosan concentration 0.4%, blue bars = chitosan
concentration 0.2%.

(a) (b)

Figure3. Nanoencapsulated catechin imaging using Scanning Electron Microscopy (SEM). (a) 1,000 times and (b) 5,000 times
magnification.
 Sari Intan Kailaku et al. / Procedia Chemistry 9 (2014) 235 – 241 239

(a) (b)

Figure 4. Nanoencapsulated catechin imaging using Transmission Electron Microscopy (TEM). (a) 20,000 times and (b) 40,000
times (right) magnification.

Emulsion stability analysis was done to catechin solution and nanoencapsulated catechin. Separation using
centrifugation speed of 10,000 rpm for 25 minutes showed good stability with the absence of phase separation
(Fig.5). Chitosan is a cationic biopolymer composed of 2-amino-2-deoxy-D glucopyranose linked by -1,4 bonds.
Polymers consist of cationic polymers and anionic polymers. Chitosan is a positively charged polymer that can be
crosslinked with an anionic polymer which is negatively charged such as alginate, carrageenan, and Na-TPP.
Chitosan can easily bind with poly anions like TPP3. The use of multiple coating Alginate-Chitosan can reduce the
porosity and stability of the resulting emulsion capsules8. It also occured in the use of multiple coating system
Chitosan-NaTPP in the experiment that can improve emulsion stability in solution of nanoencapsulated catechin.

Figure 5. Appearance of chitosan solution (left) and nanoencapsulated catechin (right) after emulsion stability analysis.

The main components of gambier are catechins (catechin acid) and catechutannatacid. Gambier also contains a
little quercetine (yellow dye). Catechinis a phenolic compounds that inhibits and entraps free radicals.
Phenolic/phenol compounds are known to be the major phytochemicals responsible for the antioxidant activity of
240 Sari Intan Kailaku et al. / Procedia Chemistry 9 (2014) 235 – 241

plant material9. Several factors that affect the antioxidant activity are antioxidant concentration, pH, temperature,
processing and storage. Antioxidant activity increased with the increasing concentrations of catechin in solution. The
epimerization process or the structure changes of catechin from cis to trans also showed the instability ofcatechin.
Epimerization occurs at high temperatures. Therefore, processing with high temperatures lowers antioxidant
activity10.

Results of DPPH analysis showed that the nanoemulsification process lowers the antioxidant activity of catechin.
The antioxidant activity of pure catechin extract (concentration of 100%) was 315,733.53 ppm while that of the
nanoencapsulated catechin with catechin solution concentration of 0.4% (best treatment) was 223.75+4 ppm.
Catechin content was also shown decreased from dehydrated catechin extract solution tonanoencapsulated catechin.
Catechin extract had higher levels of catechin(42%), while the catechin content of nanoencapsulated catechinwas
16.8%.

Incomplete nanoemulsification was likely causing the decrease of antioxidant activity and catechin content.
Untrapped catechin extract in the nanoemulsion can form a sediment, thus lower the catechin in the solution.

The stability of catechin is influenced by pH, it is stable at pH < 4 and unstable at pH > 6 11. A prolonged heating
and heating in base solution can cause changes from catechin to catechutannic acid, and therefore decrease the
antioxidant activity. The instability of antioxidant is also influenced by the concentration of oxygen. The higher
oxygen amount and the lower antioxidant concentration will increase oxidation of catechin. The nanoencapsulation
process of catechin expanded the surface area thus allowed more catechins in contact with oxygen and increased
oxidation and decreased the activity of antioxidant of catechin. The instability of antioxidant molecules will reduce
the functional value of food. Entrapment of antioxidant in a macromolecule is a favorable way to reduce oxidation
due to limited access of oxygen to the molecules.

4. Conclusion

Nanoencapsulatedcatechin with the best particle size obtained from the formula with chitosan solution
concentration of 0.2%, 0.4% catechin solution, a solution of 0.1% Na-TPP and ratio of chitosan and Na-TPP 7: 1,
with the particle size of 137.6 nm. Nanoencapsulatedcatechinhadgood particle surface topography, internal structure
of the particles and emulsion stability. Based on the analysis of catechin content, it was found that the
nanoencapsulatedcatechincontained 16.8% catechin and 223.75+4 ppm antioxidant activity.

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