IOP Conference Series: Earth and Environmental Science

PAPER • OPEN ACCESS You may also like

- In Vitro antibacterial activity of Mindi (Melia
The effect of centrifugation speed and Chitosan- azedarach Linn.) leaf extract with
nanoencapsulation technology
Sodium Tripolyphosphate ratio toward the N A S Masjid, R Martien, Zuprizal et al.

- Action of the anti-tumoral

nanoencapsulation of Sambiloto (Andrographis zinc(II)phthalocyanine in solution or
encapsulated into nanoparticles of poly--
paniculata) for the formulation of Hepatitis B drug caprolactone internalized by peritoneal
macrophages
Amanda Santos Franco da Silva Abe,
Eduardo Ricci-Júnior, Morgana Teixeira
To cite this article: Devi Permata Sari et al 2018 IOP Conf. Ser.: Earth Environ. Sci. 105 012112 Lima Castelo Branco et al.

- Influence of diverse natural biopolymers

on the physicochemical characteristics of
borage seed oil-peppermint oil loaded
View the article online for updates and enhancements. W/O/W nanoemulsions entrapped with
lycopene
Abdur Rehman, Qunyi Tong, Sameh A
Korma et al.

This content was downloaded from IP address 125.164.17.143 on 05/02/2023 at 14:30

2nd international Tropical Renewable Energy Conference (i-TREC) 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
105 (2018) 012112 doi:10.1088/1755-1315/105/1/012112

The effect of centrifugation speed and Chitosan-Sodium

Tripolyphosphate ratio toward the nanoencapsulation of
Sambiloto (Andrographis paniculata) for the formulation of
Hepatitis B drug

Devi Permata Sari1, Tania Surya Utami1, Rita Arbianti1 and Heri Hermansyah1
1
Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia,
Depok, West Java 16424, Indonesia

E-mail: [email protected]

Abstract. Hepatits B is a viral infection which attack the liver. One of the compound that
can overcome and inhibits Hepatitis B is Andrographolide. The compound was derived from
Sambiloto plants (Andrographis paniculate).Andrographolide compound works by inhibiting
α-glucosidase which assists the secretion of Hepatitis B virus. The goal of this research is to
make nanoencapsulation of sambiloto leaf extracts that was encapsulated in chitosan and
STPP. The nanoencapsulation will increase the bioavailability of the body for the administered
Andrographolide. The size of the resulting particle at a variation of centrifugal speed of 8.000
RPM with the concentration ratio of chitosan : STPP equals to 0.2%:0.1% (g/mL), was
68.3nm. The loading capacity of the nanoparticles is 67.20% and the encapsulation efficiency
of the nanoparticles is 99.48%. The release profile has a cumulative release of 34.55% with
slow release in gastric pH conditions and followed by a burst release in intestine pH
conditions.

1. Introduction
One of the bad habits of society Indonesia is low in maintaining healthy. A less healthy causing the
incidence of various health problems, such as the emergence of a wide range of dangerous diseases
that can attack all of ages, from adult to Toddler-aged children.The cause of the occurrence of many
diseases is due to the inability of the body against viruses, bacteria, as well as hazardous substances
that are carcinogens. One of the dangerous diseases caused by virus is Hepatitis, which is comprised of
Hepatitis A, B, C, D and E. Hepatitis is a disease characterized by inflammation that occurs in the liver
organ [1]. Indonesia is a country with a high additional Hepatitis B is the second largest country in the
South East Asian Region (SEAR) after Myanmar [2]. There are about 2 billion people in the world
infected with the Hepatitis B virus and 240 million people of whom become people with Chronic
Hepatitis B [2] .
Wolrd Health Organization (WHO) states that the efforts that have been made to solve the
Hepatitis B sufferers is to use adrenokortikosteroid, corticosteroids, and lamivudin, but these things
have not been able to effectively solve the viral infections. Hepatitis B antivirals suchas drugs usage
lamivudin already available and in development stage, but the drugs have not been evaluated for the
treatment of acute hepatitis B [3].One of the compounds that can inhibit even the deadly hepatitis B

Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
2nd international Tropical Renewable Energy Conference (i-TREC) 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
105 (2018) 012112 doi:10.1088/1755-1315/105/1/012112

virus replication is andrographolide. The source of the andrographolide found in Sambiloto

(Andrographis paniculata). One of the andrographolide activity is can inhibits the α-glucosidase
enzyme [4][5] which is a constituent of glikohidrolase enzymes i.e. enzymes that contribute in the
formation of the viral envelope and is responsible for glikohidrolasi protein. The presence of inhibits
glikohidrolase enzyme, then virus replication is not perfect and the virus will die because of the viral
envelope formation will be blocked [6]
In this research, performed the extraction of sambiloto leaves with encapsulation by nano-sized
particles that will be used is Chitosan. Chitosan has a fragile mechanical properties, so it must be
stabilised by using sodium tripoliposphat (STPP) as a crosslinker, so when passing a very acidic pH,
Chitosan is still not degraded and active substances not apart before reaching the target [7].
In this research was conducted on variation towards the concentration ratio Chitosan and STPP,
because that affects the ratio of a particle may be nano-sized. Futhermore, it also conducted on
variation of the centrifugation speed, to known how the appropriate centrifugation speed so that the
particles that form can nano-sized. Then, controlled release by in vitro was done to know the
performance of the extract of sambiloto has in nanoencapsulation as the hepatitis B drug.

2. Methods
Variations on this research is ratio of concentrations chitosan-STPP and centrifugation
speed.Continued with particle distribution measurement by using particle size analyzer (PSA),
determination of morphology using SEM and FTIR, quantitative test of andrographolide by using
HPLC, and controlled drug release of nanoparticles in in vitro.

2.1. Extraction of Sambiloto and quantitative test of Andrographolide

Sambiloto leaves powder was dissolved in solvent ethanol 70%. Then extraction using sonicator for
50 minutes at frequency of 30 Hz and 25oC, continued by solvent evaporating using vacuum rotary
evaporator. Calculating of andrographolide in sambiloto extract by using HPLC.

2.2. Nanoencapsulation
The selected method in the nanoencapsulation is ionic gelation method. Chitosan 0.2 g was dissolved
in 100 mL acetic acid 1% (b/v), added 3 g of sambiloto extract, and dissolve 0.04 g 40 mL STPP into
akuades then added 200 μL Tween 80 0.1% (v/v).Solution of STPP then added into chitosan-extract
solution with flow speed of 0.75 ml/min accompanied with magnetic stirring for 2 hours. Then do size
diminution using sonikator with 130 Watts and frequency of 30 Hz for 50 minutes and centrifugation
for 15 minutes with a pause of 1 minute every 5 minutes. The last is drying using freeze dryer to a
powder.

2.3. Controlled drug release

Controlled drug release was done on a synthetic fluid medium, i.e. in series in a seven hours. Synthetic
fluid medium that is used in the two of kind of buffers that indicates the human digestive system, i.e.
the Simulated Gastric Fluid (SGF) pH 1.2 which indicate gastric conditions and Simulated Intestinal
Fluid (SIF) 7.4 pH which suggests intestinal conditions.

3. Result and discussion

3.1. Nanoencapsulation of Sambiloto extract

Method of extraction sonication produce yield 5.44% with active substance andrographolide 17.60%.
The concentration ratio of Chitosan: STPP (g/mL) are made in different variations, i.e. 0.1% : 0.1% ;
0.1% : 0.15% ; 0.15% : 0.1% and 0.2% : 0.1%. Continued with the variation of centrifugation speed,
i.e. 8,000 rpm, 10,000 rpm, 13,000 rpm and 15,000 rpm.

2
2nd international Tropical Renewable Energy Conference (i-TREC) 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
105 (2018) 012112 doi:10.1088/1755-1315/105/1/012112

3.2. Particle size distribution of Chitosan:STPPRatio

Particle Size Analizer (PSA) test beginning with the sample dispersion in the aquademin with the
addition of Dispersing Agent Coulter Type 1A. Here are the average particle size of the variation ratio
of Chitosan: STPP obtained from the PSA analysis.

Table 1. Result of particle size distribution of chitosan:STPP ratio.

Concentration Ratio of Z-Average Particle Size Distribution
chitosan : STPP (g/mL) (nm) (nm)
90.78 ± 12.15
0,1%:0,1% 1917 125.2 ± 17.38
232.7 ± 32.15
206.6 ±27.87
0,1%:0,15% 2569 131.9 ± 18.32
-
144.1 ± 17.03
0,15%:0,1% 1454 269.2 ± 47.53
62.68 ± 8.29
170.5 ± 24.99
0,2%:0,1% 1424 90.41 ± 12.91
1212 ± 153.2

Based on the table, that the higher concentration of chitosan than concentrations of STPP will earn
average particle size (z-average) getting smaller. It is caused due to an increased of STPP
concentration will enhance physical endurance of the particle, because of the increasing number of
STPP will make ionic-crosslinking reaction by the positive force of chitosan more stable forming
physically, but also increased nanoparticle diameter [8].Particle size distribution obtained from each
variation has a relatively small size and reach a size of nanometer, but z-average or average particle
size is still big even reach a size belongs to the micro.The Z-average size will only be comparable with
the size measured by other techniques if the sample is monomodal, spherical or near-spherical in
shape, monodisperse, and the sample is prepared in a suitable dispersant, as the Z-Average mean size
can be sensitive to even small changes in the sample, e.g. the presence of a small proportion of
aggregates[9].

3.3. Particle size distribution of Centrifugation Speed Variation

Based on the measurement of particle distribution, size obtained reach the nano is a variation of
centrifugation speed 8,000 rpm i.e. 68.3 nm and 1663.3 nm at centrifugation speed 15,000 rpm. It is
because at the high centrifugation speed will make particles collide and can cause clots flok. That is
corresponds to the Budiyono (1999)[10] who explains that fluid movement due to stirring will be
followed by microflok formed. Here are the average particle size of the centrifugation speed variation
obtained from the PSA analysis.

Table 2. Result of particle size distribution of centrifugation speed variation.

Centrifugation Speed Z-Average Particle Size Distribution
(rpm) (nm) (nm)
22.4 ± 3.2
8.000 68.3
86.3 ± 27.3
964.5 ± 161.8
10.000 1160.7
-
13.000 666.0 1.3 ± 0.3

3
2nd international Tropical Renewable Energy Conference (i-TREC) 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
105 (2018) 012112 doi:10.1088/1755-1315/105/1/012112

241.4 ± 78.1
15.000 1663.3
2780 ± 1619.4

According to Jong (2008)[11], the size of nanoparticles used as drug carriage has a range of <100
nm, so at the centrifugation speed 8,000 rpm can be said that the particles sucessfully formed with
nano size.

3.4. Morphology determination using SEM

Morphology of the particles observed with the Scanning Electron Microscope (SEM). The resulting
morphology is not smooth and has no pores on its surface, occurs the agglomeration, and the form is
not spherical.

Figure 1. SEM result of nanoparticle

3.5. Loading capacity and encapsulation efficiency

Loading capacity shows the number of drugs contained in nanoparticles formed in this paper, the drug
was active substance andrographolide in the crude extract of sambiloto. The results of the calculation
of loading capacity from each variation can be seen below:

(a) (b)
Figure 2. (a) Concentration ratio of chitosan:STPP (b) Variation of centrifugation speed

4
2nd international Tropical Renewable Energy Conference (i-TREC) 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
105 (2018) 012112 doi:10.1088/1755-1315/105/1/012112

In the figure (a) shows that with increasedof chitosan concentrations so the loading capacity and
encapsulation efficiency will also increase, while the addition of STPP concentration will decrease of
loading capacity and encapsulation efficiency. While in addition of the STPP concentrationwill
decrease loading capacity because the droplets rapidly solidification occurs when the process of ionic
gelation [12]. In figure (b) shows that increasing of the centrifugation speed will increase loading
capacity, because with the increasing of the centrifugation speed, then small-sized particles will also
precipitates (not only large-sized particles that precipitates), so the mass of nanoparticles after freeze
drying will be more.
Encapsulation efficiency obtained ranged from 99.47%-99.51%, this result is appropriate with
Chen et al. (2006)[13] who is state that good encapsulation efficiency is at least 80%, because it shows
the processes that do not eliminate existing active substances. The high value of the encapsulation
efficiency may also be evidenced by the results of morphology in FTIR in figure 2, because
andrographolide contained in the sambiloto extract have been encapsulation with chitosan, as
evidenced by the existence of andrographolide’s functional group on nanoparticle.

3.6. Controlled drug release of Nanoparticle

The results showed that the cumulative release is 34.55% at the 7 th hour. In figure 3 shows that at the
1st to 3rd hour which indicates gastric condition with the pH 1.2 longer than at pH 7.4 which indicates
intestinal conditions shows profile release to be faster.
In figure 3 shows that at the 1st to 3rd hour in which indicates gastric condition is slow release. This
is because in these conditions, the hydrogen bonds between molecules of Chitosan nanoparticles make
getting stronger which causes diffusion into the moleculebecomes more difficult, and then followed
the release of a drug that lasts longer [14]. The addition of surfactants such as tween 80 may increase the
endurance of the particles on the acid condition in gastric.
Then on the intestinal conditions pH 7.4 shows the release profiles tend to be faster.That is called
burst release i.e. mechanism of drug released actively, indicating the drug is at the surface of the
nanoparticles so that chitosan having deprotonation on the surface when mixed with synthetic fluid
medium [15].
35
Lambung (pH 1,2) Usus halus (pH 7,4)
30

25
% Rilis Kumulatif

20

15

10

0
0 1 2 3 4 5 6 7 8
Waktu (jam ke-)

Figure 3.Release profile of nanoparticle in series

At the Simulated Intestinal Fluid (SIF), the concentration of STPP contributed to the polymer chain
conformation more because deprotonation, so chitosan nanoparticle easy damaged [16]. Has been
previously reported also that on media with pH above 6 ionization of amine reduced drastically [17].
This triggered the deprotonation of the amine chitosan and chitosan particles causing crosslink with
STPP became unstable and start to degradation as reported on a similar research[18].

5
2nd international Tropical Renewable Energy Conference (i-TREC) 2017 IOP Publishing
IOP Conf. Series: Earth and Environmental Science1234567890
105 (2018) 012112 doi:10.1088/1755-1315/105/1/012112

4. Conclusion
Based on the results of the research, it can be concluded that the results of measurements of the
particles distribution have an average of 68.3 nm on a variation of the concentration ratio
chitosan:STPP 0.2%:0.1% (g/mL) with the centrifugation speed 8,000 rpm. The variation has loading
capacity and efficiency encapsulation each 67.20% and 99.48%, and cumulative release 34.55%.

5. References
[1] Misnadiarly A S 2008 Mengenal Penyakit Liver Mediakom Ed. XII (Jakarta: Badan Litbangkes)
[2] Kementrian Kesehatan Republik Indonesia 2014 Situasi dan Analisis Hepatitis. Pusat Data dan
Informasi
[3] World Health Organization 2002 Hepatitis B Departement of Communicable Diseases
Surveillance and Response
[4] Dai Gui-Fu et al. 2006 Studies on the novel α-glucosidase inhibitory activity and structure–
activity relationships for andrographolide analogues. Bioorganic & Medicinal Chemistry
Letters Vol 16
[5] Xu, Hai-Wei., Dai, Gui-Fu., Liu, Gai-Zhi., Wang, Jun-Feng., dan Liu, Hong-Min 2007
Synthesis of andrographolide derivatives: A new family of α-glucosidase inhibitors
Bioorganic & Medicinal Chemistry 15 4247-55
[6] Firdayani, et al. 2012 Pengembangan Kandidat Senyawa Obat Turunan Naftokuinon sebagai
Inhibitor Virus Hepatitis B (Jakarta: Pusat Teknologi Farmasi dan Medika BPPT)
[7] Aini, D et al. 2015 Uji Pelepasan Terkendali Ekstrak Daun Keji Beling (Strobilanthes crispus)
untuk Sediaan Obat Antihiperkolesterolemia. Prosiding Seminar Nasional Teknik Kimia
UNPAR: 101-104
[8] Ko J A, Park H J, Hwang S J, Park J B and Lee J S 2002 Preparation and characterization of
chitosan microparticles intended for controlled drug delivery Int. J. of Pharmaceutics 249
165-174
[9] Malvern Instruments Limited 2011 Dynamic Light Scattering Common Terms Defined. [online]
www.malvern.com/contact/
[10] Budiyono, et al. 1999 Pengaruh Kecepatan dan Waktu Pengadukan pada Proses Pengendapan
Limbah Radioaktif Cair yang Mengandung Aktinida dengan FeCl3 dan Al2(SO4)3 Prosiding
Pertemuan dan Presentasi Ilmiah P3TM-Batan Yogyakarta
[11] Jong W H 2008 Drug Delivery and Nanoparticles: Applications and Hazards. Int. J. of
Nanomedicine 133-149
[12] Mardliyati E, Muttaqien, Sjaikhurrizal E and Setyawati D R 2012 Sintesis Nanopartikel
Kitosan-Trypolyphosphate dengan Metode Gelasi Ionik: Pengaruh Konsentrasi dan Rasio
Volume terhadap Karakteristik Partikel Prosiding Pertemuan Ilmiah Ilmu Pengetahuan dan
Teknologi Bahan (Serpong: BPPT)
[13] Chen, et al. 2006 Food protein-based materials as nutraceutical delivery systems Trends in Food
Science & Technol. 17 272-283
[14] Dhanasingh, et al 2010 Chitosan/Casein Microparticles: Preparation, Characterization and Drug
Release Studies. World Academy of Science, Eng. and Technology 4 08-23
[15] Siegel R A and Rathbone M J 2012 Overview of Controlled Release Mechanisms. Advances in
Delivery Science and Technology
[16] Fauzia F 2014 Mikropartikel Kitosan Tertaut Silang Tripolifosfat untuk Pelepasan Terkendali
Senyawa Bioaktif Asetogenin dan Mangostin Fakultas Teknik Universitas Indonesia
[17] Yu C Y, Yin B C, Zhang W, Cheng S X, Zhang X Z and Zhuo R X 2009 Composite
microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH-
sensitive drug release property Colloids Surf B Biointerfaces 68 245-9
[18] Konecsni K, Low N H and Nickerson M T 2012 Chitosan-tripolyphosphate submicron particles
as the carrier of entrapped rutin Food Chem 134 1775-9