Jurnal Biodjati 5(2):214-222, November 2020
e-ISSN : 2541-4208
p-ISSN : 2548-1606
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TREATMENT OF PLGA NANOPARTICLES OINTMENT-ETHANOL
EXTRACT OF Archidendron pauciflorum IN THE WOUND HEALING IN
DIABETIC MICE
Desak Made Malini1*, Yasmi Purnamasari Kuntana2, Madihah3, Wildanul Furqon4,
Wawan Hermawan5
Received : August 08, 2020
Accepted : September 29, 2020
DOI: 10.15575/biodjati.v5i2.9256
1,2,3,4,5
Departement of Biology, Faculty
of Mathematics and Natural Sciences,
Universitas Padjadjaran. Jl. Bandung
-Sumedang Km 21, Jatinangor, Sumedang 45363, West Java, Indonesia.
Tel./Fax. +62-22-7796412
e-mail:
*1desak.made@unpad.ac.id
2
y.purnamasari@unpad.ac.id
3
madihah@unpad.ac.id
4
wildannurforqon@gmail.com
5
wawan. hermawan@unpad.ac.id
*Corresponding author
Abstract. Diabetic wounds lead to severe tissue damage and are difficult to cure. Jengkol (Archidendron pauciflorum) is a plant commonly
used by local Indonesian communities to treat diabetic wounds. The
efficiency of herbal medicine still has a deficiency of its ability to reach
the target organs, therefore nanotechnology is applied in the hope
that all drug concentrations can reach the target organs successfully.
This study aimed to evaluate the effectiveness of PLGA nanoparticle
ointment-ethanol extract jengkol fruit peel (EEJFP) to accelerate the
wound healing process in the skin of streptozotocin-induced diabetic
mice. The research method used was experimental with a completely
randomized design using six treatments and four replications. Diabetes was induced by intraperitoneal injection of streptozotocin 180 mg/
kg BW. Mice with a blood glucose level of ≥150 mg/dL were used for
diabetic mice models. The incision wound created at the dorsolateral
region of shaven skin at ±1 cm2 using sterile scissors. The treatments
given were vaseline for Control Negative (CN) and Control Positive
(CP), Betadine ointment (PB), 10% EEJFP ointment (P1), 5% PLGA
nanoparticle ointment-EEJFP (P2), and 2.5% PLGA nanoparticle
ointment-EEJFP (P3). The results showed that the administration
of PLGA nanoparticles ointment-EEJFP with a concentration of 5%
PLGA nanoparticle ointment-EEJFP (P2) resulted in the shortest
wound length on day 3, 7 and 14; narrower granulation tissue; a larger
number of blood capillaries; and denser collagen fibers (α <0.05)
compared to CP and PB treatments. The administration of PLGA
nanoparticle ointment-EEJFP with a concentration of 5% was the
most effective concentration in accelerating wound healing in the skin
of diabetic mice.
Keywords: diabetic wounds, jengkol, nanoparticles, ointment, PLGA
Citation
Malini, D. M., Kuntana, Y. P., Madihah, Furqon, W. & Hermawan, W. (2020). Treatment of PLGA
Nanoparticles Ointment-Ethanol Extract of Archidendron pauciflorum in the Wound Healing in
Diabetic Mice. Jurnal Biodjati, 5(2), 214-222.
Jurnal Biodjati 5(2):214-222, November 2020
http://journal.uinsgd.ac.id/index.php/biodjati
INTRODUCTION
Diabetes wound (diabetic ulcure) is a
further complication of the condition experienced by a person with diabetes mellitus,
characterized by an open wound on the epidermal layer to the dermis layer with blackish-red color, and foul-smelling. This occurs
because of the clogged blood vessel in the
palm or soles (Sudayo et al., 2009). The widely
used remedy for diabetic wounds is povidone
iodine (Betadine®). This drug can kill bacteria
(bactericides) and fungi (fungicides) at a
concentration of 0.1-1% but has several side
effects including irritating the wound during
the healing process (Rahmawati, 2014). Based
on this fact, it is necessary to cure a wound of
diabetes without any side effects that cause
more severe injury and safe.
One of the plants that have been used traditionally in several regions in Indonesia as a
cure for diabetes mellitus is jengkol fruit peel.
According to Malini et al. (2017), the people
of Karangwangi Village use have been using
jengkol fruit peel to treat diabetes wounds
because they are easy to obtain and have no
side effects. Jengkol fruit peel contains flavonoids, quinones, tannins, saponins, polyphenols, glycosides, and steroids. These compounds play a role in wound healing because
they are astringent, have antioxidant and antimicrobial properties, stimulates re-epithelialization and neovascularization, as well as triggers collagen growth (Francis et al., 2002).
The efficiency of herbal medicines derived from nature has a disadvantage, such as
its ability to reach the target organ as only a
small concentration can reach it, while most
of it is distributed to the other body parts.
Nanoparticles are a technology that aims to
make dosage forms in the range of 10 nm 1000 nm (Aloys et al., 2016). The smallest
capillaries in the body have a diameter of 5-6
Malini et al.
mm, therefore, natural medicines in nano size
can be distributed to all blood vessels in the
body (Mathur & Govind, 2013). Nano preparations must be encapsulated by polymers that
are biocompatible and biodegradable. Encapsulation polymers commonly used are PLGA
(polylactic glycolic acid) alginate, chitosan,
gelatin, and ethylcellulose. PLGA is the most
commonly used polymer in the encapsulation of nanoparticles and is an effective carrier in drug delivery to target organs. PLGA
polymers have been approved by the Food
and Drug Administration (FDA) for human
therapy (Danhier et al., 2012). According to
Chereddy et al. (2014), PLGA nanoparticles
are nano compatible polymer-carriers and act
as promoters of wound healing, stimulating
re-epithelialization, neovascularization, and
collagen fiber density.
Research on the effectiveness of PLGA
nanoparticle ointment-ethanol extract of
jengkol fruit peel to treat diabetic wounds
has not been widely reported. Therefore this
study aimed to test the effectiveness of PLGA
nanoparticle ointment-ethanol extract of jengkol fruit peel on the process of wound closure
and repair the histological damage of the skin
in mice (M. musculus) diabetes.
MATERIALS AND METHODS
The research was conducted at the
Laboratory of Structural and Animal Physiology, Universitas Padjadjaran from May to
July 2019.
Preparation of Ethanol Extract of Jengkol
Fruit Peel
Jengkol fruit peel was collected from
Karangwangi village, Cianjur District, West
Java Province, Indonesia. The samples were
identified in the Taxonomy laboratory in the
Biology Department, Faculty of Mathematics
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and Sciences, Universitas Padjadjaran. The
samples were air-dried to a constant weight
and blend to a coarse powder. The dried powder was soaked and macerated on ethanol 70%
(ratio 1:2) for 72 hours and every 24 hours the
macerate was collected. The macerate was
then evaporated using a rotary evaporator at
a temperature of 40-50°C, and freeze-dried to
obtain a paste extract (Khan et al., 2012).
Preparation of PLGA Nanoparticles Ethanol Extract of Jengkol Fruit Peel
The manufacture of PLGA nanoparticles was carried out by nanoprecipitation
techniques based on Fessi et al. (1989). Fifty
milligrams of PLGA and 10 mg of jengkol
fruit peel ethanol extract were weighed and
dissolved in 3 mL acetone, then added dropwise (0.5 mL/ min) to a solution containing 20
mL stabilizer (1% polyoxyethylene-polyoxypropylene in aquadest). The mixed solution
was stirred at 400 rpm using a stirrer at room
temperature until the organic solvent evaporates. Afterward, centrifugation was carried
out at 25,000 rpm and 4°C for 30 minutes. The
pellet was then suspended again in Milli-Q
water and washed three times. The suspension
contained solid particles roasted at a temperature of 30°C for 24 hours, after which they
were grounded until smooth to form a powder.
Preparation of PLGA Nanoparticles Ointment-Ethanol Extract of Jengkol Fruit
Peel and Ointment of Ethanol Extract of
Jengkol Fruit Peel
The PLGA-ethanol extract ointment for
jengkol fruit peel was made for 15 grams in
weight with two concentrations of 5% and
2.5%, while the ethanol extract ointment for
jengkol fruit peel was made for 15 grams in
weight with a concentration of 10%. Each
concentration of ointment was homogenized
with vaseline using a mortar that was heated
Jurnal Biodjati 5(2):214-222, November 2020
to a temperature of 75°C (Winarsih et al.,
2012). Ointments were stored in film bottles
and labeled.
Experimental Design
Twenty-four male Swiss-Webster mice
(8-12 weeks old, 30-40 g of weight) were
obtained from the Faculty of Animal Husbandry Universitas Padjadjaran. They were
housed in standard environmental conditions
and fed with piglet standard diets (CP-551,
PT. Charoen Pokphand) and water ad libitum.
The animals were acclimatized for seven days
before the experiment. The experiment used a
completely randomized design with six treatments and four replications.
Induction of Diabetes
The animals fasted for 4-6 hours, and
their baseline fasting blood glucose level
was measured using a glucometer, by collecting blood via tail cut before induction of
diabetes. Diabetes was induced by intraperitoneal injection of a freshly prepared solution
of STZ (Nacalai Tesque, Inc.) with a dose of
180 mg/kg BW in 10 mM citrate buffer solution pH 4.5 of five groups, while the negative
control mice were injected with the vehicle.
The mice were provided with 10% of sucrose
solution for three constitutive days to prevent
hypoglycemia after STZ induction. Four days
after administration of STZ, the mice fasted
and the blood was collected via tail cut
for measuring their fasting glucose levels.
The animals which have glucose level
more than 150 mg/dL were used for further
experiment and categorized as diabetic
mice (Wu & Huan, 2008; Furman, 2015).
The Procedure of the Wound Creation and
Treatment with the Ointment
The mice were anesthetized with inhaled ether, and then the hair on the right
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side of the cutting area was shaved before
the creation of the wound. Incision wounds
were created on all mice by cutting the dorsolateral surface of skin ± 1.5 cm from the
shaven area using sterile scissors (full-thickness type extending up to the adipose tissue).
The ointment treatment was smeared twice
a day in the morning and afternoon for
14 days consecutively. The animals were
allowed to consume food and water ad libitum
(Chen et al., 2005; Winarsih et al., 2012).
Histological Preparation and Examination
On the 15th days, after an overnight fast,
the animals were weighed and sacrificed by
cervical dislocation, wounds were harvested
and fixed in 10% of neutral buffered formalin.
The tissue was dried using a serial of N-butanol-xylene solution, inserted in paraffin, sectioned at 5-7 μm thickness, mounted on glass
slides, stained with hematoxylin and eosin as
well as Trchrome Heidenhains Azan. The histological examination included the percentage
of reepithelization, formation of the granulation tissue, number of blood capillaries, and
density of collagen in five areas per slide.
Data Analysis
The results were expressed as mean
values ± standard deviation (S.D). Statistical significance for the granulation tissue
area, and the number of blood capillaries
was analyzed using one-way ANOVA followed by Duncan’s multiple range test.
Whereas the density of collagen was analyzed by the Kruskal-Wallis test. P values
less than 0.05 were considered significant.
RESULTS AND DISCUSSION
The Effect of PLGA Nanoparticle Ointment
-EEJFP on Granulation Tissue
Histological observations on granulaMalini et al.
tion tissue showed that negative control treatments had the narrowest granulation tissue,
whereas positive control treatments had the
most extensive granulation tissue, compared
to other treatments (Table 1). It happened
because the vaseline does not contain active
ingredients that can accelerate the wound
healing process, causing the granulation tissue
constriction to occur longer (Yanhendri &
Yenny, 2012). The treatment of Ethanol extract of Jengkol Fruit Peel (EEJFP) ointment
and PLGA nanoparticle ointment-EEJFP has
granulation tissue that was narrower than the
comparative treatment using betadine and
positive control treatment (Figure 1). This
is due to the fruit peel of jengkol containing
phytochemical compounds such as saponins
and tannins.
Tannins contained in jengkol fruit
peel are thought to act as an antibacterial by
denaturing cell proteins. The hydrogen bonds
formed between tannins and bacterial proteins
cause the structure of the protein to become
damaged. The hydrogen bond affects the permeability of the cell wall and cytoplasmic
membrane, both of which are composed of
proteins. The permeability of the disrupted
cell wall and cell membrane can cause an imbalance of macromolecules and ions in the
cell resulting in cell microbial lysis (Sari &
Sari, 2011).
Flavonoids are powerful antioxidants
that can control free radicals, protect the body
against reactive oxygen species (ROS), enhance endogenous antioxidant functions, and
increase antioxidant enzymes in granulation
tissue (Keller et al., 2006). Flavonoids play
a role in inhibiting inflammatory mediators
such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) produced by macrophages
and cytokine receptors that are generally seen
in pain suppression and tissue damage (Saroja
et al., 2012).
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Table 1. The Average Area of Granulation Tissue, The Number of Blood Capillaries, and Collagen Fiber Density
Scores in Diabetic Mice Skin Lesions
Average Area of
Average The
Average Collagen
Treatment
Granulation Tissues
Number of Blood
Density
Score ± SD
± SD (μm2)
Capillaries ± SD
Negative
control
Without STZ + Vaseline
Induction
18.50 ± 1.29a
4.00 ± 0.00a
2054.83 ± 164.31a
Positive
control
Induced STZ + Vaseline
4.50 ± 1.29d
1.62 ± 0.47c
5123.61 ± 242.47e
Induced STZ + Beta11.50 ± 1.29c
2.00 ± 0.40c
3679.48 ± 58.62d
dine Ointment
Induced STZ + 10 % of
Treatment 1
14.25 ± 0.95b
3.50 ± 0.40b
2885.91 ± 95.30c
EEKBJ Ointment
Induced STZ + 5 % of
Treatment 2 PLGA-EEKBJ Nano17.25 ± 2.06a
4.00 ± 0.00a
2058.84 ± 50.30a
particles Ointment
Induced STZ + 2,5% of
Treatment 3 PLGA-EEKBJ Nano14.50 ± 1.29b
3.68 ± 0.23b
2655.29 ± 64.09b
particles Ointment
Note: The numbers followed by the same letters show no significant difference based on Mann Whitney U test for
granulation tissue test at the 95% significance leveland the number of blood capillaries. Whereas the density of collagen was analyzed by the Kruskal-Wallis test. P values less than 0.05 were considered significant.
Comparative
Figure 1. Photomicrograph of the cross section of mice skin from each treatment groups after treatments for 14
days. Hematoxylin Eosin stain. M.100×. (A). Negative control ( non-diabetic mice +vaseline); (B) Positive control (diabetic mice +vaseline); (C) Comparative (diabetic mice +Betadine ointment); (D) Treatment 1 (diabetic mice +10 % EEJFP ointment); (E) Treatment 2 (diabetic mice + 5 % PLGA nanoparticles ointment- EEJFP); (F) Treatment 3 (diabetic mice + 2,5% PLGA nanoparticles ointment- EEJFP).
Jurnal Biodjati 5(2):214-222, November 2020
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The Effect of PLGA Nanoparticle Ointment-EEJFP on The Number of Blood
Capillaries
The results of Duncan's multiple range
test analysis (α < 0.05) showed that the positive control treatment was significantly different from the negative control. The positive control treatment had the lowest number
of blood capillaries, whereas the negative
control treatment had the highest number of
blood capillaries compared to other treatments
(Table 1). In the positive control, mice experienced hyperglycemia which caused a reduced
supply of nutrients and oxygen in the injured
area for the formation of new blood vessels
(neovascularization).
The treatment of PLGA nanoparticle
with a concentration of 5% had the narrowest
granulation tissue area as well as negative
control treatments that were not induced by
diabetes (Figure 1). This is caused by phytochemical compounds found in the jengkol
fruit peel which play a role in the formation of
new blood vessels. As one of the compounds,
flavonoids can help to repair damaged blood
vessels by inhibiting the action of ROS, where
one free electron contained in the flavonoid
binds to one free electron contained in the
ROS. Therefore there is no tissue damage
caused by ROS and the neovascularization
process takes place quickly (Keller et al.,
2006).
The Effect of PLGA Nanoparticle Ointment
-EEJFP on Collagen Density
The results of the Mann Whitney U test
analysis (α <0.05) showed that the positive
control treatment was significantly different
from the negative control (Table 1). This is due
to the condition of high glucose levels in the
blood that can inhibit the wound healing process so that the process of formation of collagen
fibers takes place slowly. According to Robert
Malini et al.
et al. (2006), the low collagen density in diabetic wounds is due to an abnormal process in
the wound healing process, namely the poor
circulation of nutrients, oxygen, hormones,
and growth factors due to the presence of hyperglycemia.
The treatments of jengkol fruit peel
ointment both in extract form and in the
form of nanoformulation were significantly
different from the comparison treatments. This
shows that herbal medicines derived from nature were more effective than antiseptic (povidone-iodine). The treatment of jengkol fruit
peel in the form of extracts and nanoformulations had a high value of collagen fiber density compared to the comparative treatments
(Figure 2), this is because jengkol fruit peel
contains several phytochemical compounds
that play a role in the process of formation of
collagen fibers.
One of the antioxidants found in the
body is ascorbic acid, which is a material
needed in collagen synthesis (Hartanto,
2013). One of the phytochemicals contained
in jengkol fruit peel is flavonoids. It contains
antiscorbutic which can protect ascorbic acid
from ROS (Nisa et al., 2013). Flavonoids
protect ascorbic acid from ROS by inhibiting
cyclooxygenase and lipoxygenase, so there is
a limitation on the number of inflammatory
cells that migrate to wound tissue (Napangala
et al., 2012). This will cause an inflammatory
reaction and the time of exposure to wound
tissue to ROS to be shorter so that the levels
of ascorbic acid in the body can be maintained
and the process of collagen synthesis can take
place quickly.
Saponins contained in jengkol fruit peel
play a role in stimulating the synthesis of fibronectin by fibroblasts (Gurtner, 2007), by
increasing the ability of TGF-β receptors contained in fibroblasts to bind to TGF-β which is
a growth factor needed by fibroblasts in citing
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collagen (Agarwal et al., 2009). This causes
the migration of fibroblasts by fibronectin will
be faster so that there will mire collagen synthesized by fibroblasts.
The collagen density of the treatment
of PLGA Nanoparticle Ointment-EEJFP in
a concentration of 5% was significantly different from the treatment of EEJFP a concentration of 10% but was not significantly
different from the negative control treatment
(Table 1). This shows that nanoparticle carriers can reduce the use of ointment concentrations (1/2 of the usual concentration). It is
supported by Oktaviana et al. (2016), which
stated that the use of PLGA nanoparticles can
increase the bioavailability profile of herbal
medicines and encapsulate extracts to minimize the degradation of extracts and easily
absorb them more quickly to increase the effectiveness of diabetes wound therapy. Nano-
technology provides a safe drug delivery system because all concentrations of therapeutic
agents are delivered directly into cells, tissues, and organs in certain periods (Sahoo
& Labhasetwar, 2003). In general, nanoparticle drugs have several advantages, including
increasing solubility and bioavailability, pharmacological activity, and distribution of macrophages in tissues, preventing physical and
chemical degradation (Raffa et al., 2010).
ACKNOWLEDGEMENTS
The research was supported by the
Directorate of Research, Technology and
Higher Education of the Republic of Indonesia. In this opportunity, we gratefully
acknowledge the financial support of the
PUPT with contract number No. 3916/UN6.
RKT/KP/2015 budget year 2018.
Figure 2. Photomicrograph of the cross-section of mice skin from each treatment group after treatments for 14 days.
Heidenhain’s Trichrome Azan stain. M.100×. Note Blood capillary (arrow); collagen in blue color. (A).
Negative control (non-diabetic mice +vaseline); (B) Positive control (diabetic mice +vaseline); (C) Comparative (diabetic mice +Betadine ointment); (D) Treatment 1 (diabetic mice +10 % EEJFP ointment); (E)
Treatment 2 (diabetic mice + 5 % PLGA nanoparticles ointment- EEJFP); (F) Treatment 3 (diabetic mice +
2,5% PLGA nanoparticles ointment- EEJFP)..
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