Effectivity Test of Loquat (Eriobotrya japonica (Thunb.) Lindl.)

Leaves Extract on the Incision Wound Healing in Mice

(Mus musculus L.)

Emita Sabri and Raysa Zahra

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Eriobotrya japonica (Thunb.) Lindl., Incision Wound, Mice, Wound Healing.

Abstract: The wound healing process is divided into four phases; 1) coagulation 2) inflammatory 3) proliferation 4)

remodelling. Loquat (Eriobotrya japonica (Thunb.) Lindl.) leaf contains triterpenoid, flavonoid, alkaloid,

and tannin as anti-inflammation, antibacterial, anti-allergic that can accelerate the wound healing process.

The aims of this research were to find out the effectiveness of ethanol extract of loquat leaves and to

examine the histological appearance of wound healing process on mice’s skin in day 14

. This research

used 25 male mice (Mus musculus L.) that were divided into five different treatment groups. The treatment

groups were treated with two control groups; povidone-iodine (K+) and without treatment (K-) and loquat

leaves ethanol extract with three concentration of 15% (PI), 30% (PII), and 45% (PIII). Incision wound was

made into 1 cm length, the ointments were applied onto the wound and observed for twice a day in 14 days.

Histological preparation was made to calculate epithelial thickness, fibroblasts, and lymphocytes. The data

were analyzed statistically using SPSS. The result of this research showed significant difference of average

time span of wound healing of each group's, K-, K+, PI, PII, and PIII was subsequently 8 days, 9.2 days, 7.6

days, 8.4 days, and 9.4 days, with p=0.048 (p<0.05). Histological observations showed the average of

epithelial thickness, fibroblasts, and lymphocytes in the treatment group PI is higher than the other treatment

groups. In conclusion, loquat leaves ethanol extract with concentration 15% (PI) was the most effective to

accelerate the wound healing process.

1 INTRODUCTION

A wound is defined as damage or disturbance that

occurs in the structure and function of normal

anatomy tissue caused by a defense, chemical or

trauma. Incision wound is a wound that can be

healed properly if complications do not occur. The

criteria for an incision wound are new, sudden and

fast to healed (Perdanakusuma, 2007). Wound

healing is a natural process that occurs after an

injury, predictable progression of steps used by the

body to resolve impaired tissue integrity (Szycher

and Lee, 1992).

Wound healing is a complex process that

requires a series of steps, some of the steps are

granulation, collagen maturation, and scar

formation. (Zheng and Qin, 2007) which divided

into four phases: 1) coagulation and hemostatis; 2)

inflammation; 3) proliferation; 4) remodeling of a

wound (Velnar et al., 2009).

The hemostatic phase will begin the healing

process where the skin slashed then blood clots

containing fibrin and blood cells fill a narrow space

at the edge of the incision. Followed by an

inflammatory process, which starts 24 hours after

the incision occurs (Ross and Pawlina, 2011). The

inflammatory phase is the phase of the formation of

immunity such as leukocytes which plays an

important role for the initial inflammatory response

after the occurrence of wounds that can prevent the

entry of microorganisms to avoid acute injuries

(Robson et al., 2001). Proliferative phase is

characterized by the formation of granulation tissue

within the wound bed, composed of new capillary

network, fibroblast, and macrophages in a loose

arrangement of supporting structure (Prasettyono,

2009).

Indonesia has many plants that have various

benefits, one of them as medicine. The use of plants

as traditional medicine is widely used as an

162

Sabri, E. and Zahra, R.

Effectivity Test of Loquat (Eriobotrya japonica (Thunb.) Lindl.) Leaves Extract on the Incision Wound Healing in Mice (Mus musculus L.).

DOI: 10.5220/0010138100002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 162-170

ISBN: 978-989-758-556-2

alternative for healing because it is easy to obtain,

use and safer in terms of side effects.

The loquat (Eriobotrya japonica (Thunb.) Lindl.)

leaves have been used as a component in Chinese

medicine. Loquat is grown well in Kabanjahe,

Kabupaten Karo, North Sumatera, Indonesia, has

potential genetic compares with another loquat. The

fruit is large, has a very sweet taste, and becomes

multifunction plants. The leaves and the seeds are

containing bioactive compounds that are commonly

used for Karo traditional medicine to cure diabetic

disease and for expectorant to relieve cough

(Nurwahyuni et al., 2017).

Loquat leaves contain various triterpenes,

sesquiterpenes, flavonoids, tannins, and

megastigmane glycosides and these compounds have

been reported as anti-inflammation, anti-viral,

antioxidant, anti-mutagenic, and anti-tumor (Banno

et al., 2005), anti-bacteria and anti-allergic (Tan et

al., 2017). In a study conducted by Zhang et al.

(2015) founded 19 secondary metabolite contents in

loquat leaves for various bioactivities, such as anti-

inflammatory, antioxidant, and anti-cancer

properties. Mong these 19 ingredients, there are 15

components, most of which are classified as

triterpenoids and flavonoids that show

antiinflammatory activity.

Various pharmacological and biology reports

showed that the medicinal value of plants lies in

bioactive phytochemical constituents that produce

definite physiological action on the human body.

The herbal extracts promote fast wound healing than

control and non medicated group in different in vivo

studies, the process is promoted by several herbal

extracts, which are composed of active agents like

triterpenes, alkaloids, flavonoids, tannins, saponins,

anthraquinones, and other biomolecules (Thakur et

al., 2011). According to the research of Kimura et al.

(2008), Lee et al. (2006) and Liu et al. (2008) that

asiaticoside, a triterpenoid component can accelerate

the wound healing process in mice by increasing

antioxidant activity, collagen synthesis, and

angiogenesis. Two triterpenes compounds,

asiaticosides and madessicosides, showed better

result wound healing pattern in a view of

histological examination (Wu et al., 2012).

From the various research data above, it shows

that the loquat leaves have the potential to heal the

wounds due to the content of secondary metabolites

which have anti-inflammatory, antibacterial,

antiallergic and antioxidant effects that an accelerate

wound healing. In this case, a study was conducted

to test whether ethanol extracts of loquat leaves can

accelerate wound healing in mice (Mus musculus).

2 MATERIALS AND METHODS

2.1 Grouping and Dosing of Animals

This study was taken in Laboratory Animal

Structure and Physiology, Faculty of Mathematics

and Natural Science, Sumatera Utara University.

Twenty-five male mice (Mus musculus L.) were

used for the study. Mice weighed 25-30 grams and

aged 8-10 weeks. Mice were housed in cages and

provided with adlibitum pellet and water. The study

protocol was approved by the Ethics committee of

the Faculty of Mathematics and Natural Science,

Sumatera Utara University, AREC. Mice were

divided into five groups. The treatment groups were

treated with two control groups; povidone-iodine

(K+) and without treatment (K-) and loquat leaves

ethanol extract with three concentration of 15% (PI),

30% (PII), and 45% (PIII).

2.2 Plant Extraction

Loquat leaves were dried for 3 three weeks under the

shade. The dried leaves were grinded to coarse

powder. The powder was macerated with 70%

ethanol as a solvent in 6 hours with occasional

shaking and stirring, then soaked for 18 hours. The

extract was then filtered. The maceration process is

repeated once again. The filtrate was then combined

and evaporated in a rotary evaporator at 40°C and

concentrated in water bath until a thick extract is

obtained (Kemenkes RI, 2013).

2.3 Ointment Formulation

Simple ointment of the loquat leaves ethanol extract

with vaseline was prepared following the formula

(Table 1) based on Kusumawardhani et al. (2015):

L





𝑥 100 % (1)

L : Ointment concentration (%)

a : Loquat leaves ethanol extract (gram)

b : Ointment (50 gram)

Table 1: Ointment formulation.

Ingredients

Ointment (gram)

15% 30% 45%

Vaseline 42,5 35 27,5

Ethanol extract

of loquat leaves

7,5 15 22,5

Effectivity Test of Loquat (Eriobotrya japonica (Thunb.) Lindl.) Leaves Extract on the Incision Wound Healing in Mice (Mus musculus L.)

163



2.4 Incision Wound Model

After acclimatization in a week, mice were

anesthetized in the same manner to reducing the

pain. The dorsal of each mouse was then shaved and

a 1 cm long incision was made through the skin. The

scalpel was marked on the tip for the depth of the

wound. Mice were treated under grouping dosing

section and the ointment formulation as described.

Treatments were started from day 1

until day 14

Observed in twice a day, at 08.00 AM and 05.00

PM.

2.5 Histological Assessment

The skin samples were taken in day 14th.

Histological assessment using paraffin methods and

stained by Hematoxylin-Eosin stain (Suntoro, 1983).

2.6 Data Parameters

Data parameters were divided into macroscopically

and microscopically. Macroscopical data was the

average time span of wound healing in each group.

Microscopical data was average of epithelial

thickness, fibroblasts, and lymphocytes on day 14

in skin mice. Microscopical examination used

OptiLab Microscope Camera with magnification

100x for epithelial thickness and 400x for

calculating fibroblast and lymphocyte cells.

The data

were analyzed using SPSS version 22.0.

3 RESULTS

3.1 The Average Time Span of Wound

Healing

The effect of ethanol extract of loquat leaves on

wound healing day was marked by the wound

having healed. Data on the average time span of

wound healing day of mice can be seen in Table 2.

Table 2: Average time span of wound healing.

Groups Average time span ± SD

- 8,0

± 0,70

K+ 9,2

± 0,83

PI 7,4

± 1,14

PII 8,4

± 1,14

PIII 9,4

± 1,14

Data were tested with One Way ANOVA and

obtained significant results of 0.028 (p <0.05),

followed by Duncan test and the results showed that

the treatment group of ethanol extract of leaves of

15% concentration (PI) was the most potent

treatment on wound healing seen from average time

span of wound healing in mice.

Wounds treated with a concentration of 15% (PI)

provide a faster healing effect when compared to

other treatments and treatment with a concentration

of 45% (PIII) provides the longest healing effect of

all treatments. Normal wound healing can take place

naturally without assistance such as healing that

occurs in negative control treatments (K-), but herb

extracts such as at a concentration of 15% (PI)

loquat leaves will help in accelerating wound

healing because it contains secondary metabolites

that have an effect anti-inflammatory and

antibacterial properties such as terpenoids and

alkaloids.

Accordance to Rahman et al. (2013) said that the

speed of the wound healing process can be

influenced by the compound of secondary

metabolites such as terpenoid, alkaloid, and tannin

which have the function to improve the repair and

strengthen the skin cells and stimulate the growth of

connective tissue. Krishnaiah et al. (2009) said that

triterpenoids are components that have an active role

in wound healing. Triterpenoid help strengthens the

skin structure, increase the concentration of

antioxidants and restore inflammatory or inflamed

tissue by increasing blood supply to the wound area

and accelerate the process of wound healing.

Loquat leaves have secondary metabolite

compounds such as triterpenes, flavonoids, tannins,

which have an affect as an antiinflammatory,

antiviral, antioxidant (Banno et al., 2005),

antibacteria, antiallergic (Tan et al., 2017).

Triterpene compounds in Centella asiatica, mainly

including two glycosides are considered to facilitate

burn wound healing via activating growth factors

such as TGF-β that favors fibroblast proliferation

and could elevate collagen synthesis. The

compounds can accelerate the time span of the

wound healing process (Wu et al., 2012).

The hemostasis/inflammation phase of acute

wound healing reflects the time recruit the many

celullar elements in several days to weeks that are

activated during early repair and the absence of

mechanical strength (Robson et al. 2001). So that, PI

has a potential effect on wound healing to the

average time span of wound healing.

Macroscopical examination of wound area of

each group (Mus musculus L.) in the day 1

, day 7

day 14

can be seen in Figure 1.

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164

Groups

Day 1

Day 7

Day 14

K(-)

(Negative

control)

K+

(Positive

control)

PI (loquat leaves

extract with

concentration

15%

PII (loquat

leaves extract

with

concentration

30%)

PIII (loquat

leaves extract

with

concentration

45%)

Figure 1 : macroscopical examination of wound area of each group of treatment.( ) day 1

, wounds red and swollen,

( ) day 7

, wounds shrink and dry, ( ) day 14

, wounds healed.

Effectivity Test of Loquat (Eriobotrya japonica (Thunb.) Lindl.) Leaves Extract on the Incision Wound Healing in Mice (Mus musculus L.)

165

3.2 The Average Number of Fibroblast

Cells

Histological observations of the fibroblast cells on

the histological preparations of the mice skin with a

magnification of 400 x. Data on the average number

of fibroblast cells in each group on the day 14

can

be seen in Table 3.

Table 3: The average number of fibroblast cells.

Grou

s Fibroblasts ± SD

(

)

6,42

± 4,36

K(+) 12,26

± 2,93

PI 19,16

± 5,83

PII 15,42

± 3,85

PIII 14,74

± 3,55

Based on Table 3, it can be seen that the highest

average number of fibroblasts on the day 14

was in

the treatment group, a concentration of 15% (PI)

with 19.16, while the lowest was in the negative

control treatment group (K-) with 6.42.

Based on the statistical test, data was obtained

significant results 0.002 (p <0.05). Followed by the

Duncan test and the result is that the treatment group

concentration 15% (PI) is the group that the most

potential on wound healing seen from the number of

fibroblasts.

The effect of ethanol extract of loquat leaves on

the number of fibroblasts is shown from the lower

extract concentration, the higher the number of

fibroblasts. The use of ethanol extracts of loquat

leaves might effect the addition of nutrients derived

from loquat leaves in the wound area which can

optimize wound healing by increasing the number of

fibroblasts.

Alkaloid compound can accelerate soft tissue

repair. Reyes et al., (1993) presented evidence that

alkaloid compound, taspine, extracted from Croton

lechleri by its chemotactic process toward

fibroblasts that migrate into the wound from local

tissues and increasing extracellular matrix synthesis

due to their increased number.

Two glycoside triterpene compounds were able

to enhance collagen type I and type III synthesis

mainly through activating skin fibroblasts (Wu et al.,

2012). Collagen fibers type III is synthesized first in

the proliferation phase by fibroblasts that are

stimulated by growth factors TGF-β from fibroblast

cells and macrophages itself (Sabirin et al., 2013).

The large amount of connective tissue in the

wound area could help accelerate the wound

contraction. So, the wound side will be contracted

and caused the wound area to be smaller (Prasetyo et

al., 2010).

Fibroblasts synthesize and release

glycosaminoglycans and proteoglycans, which are

also important components of the extracelullar

matrix of granulation tissue. Simultaneously,

vascular generation (angiogenesis) occurs with the

use of the maturing matrix. The acute wound

fibroblast density reaches a maximum between 7 and

14 days after injury (Robson et al., 2001).

Fibroblast is one indicator that healing process

occurred faster. Increasing of fibroblast as a result of

lymphokines induced which secreted by CD4

lymphocytes, which has a role as healing promotor

to cellular immune response. Depletion of CD4

lymphocytes can decrease skin tension, angiogenesis

and extracellular matrix component (Prasettyono,

2009).

3.3 The Average Number of

Lymphocyte Cells

Histological observations of the lymphocyte cells on

the histological preparations of the mice skin with a

magnification of 400 x. Data on the average number

of lymphocyte cells in each group on the day 14

can be seen in Table 4.

Table 4: The average number of lymphocyte cells.

Grou

hoc

te ± SD

(

)

5,10

± 2,96

K(+) 4,70

± 2,04

PI 18,08

± 3,97

PII 11,22

± 3,63

PIII 13,38

± 4,98

Based on Table 4, it can be seen that the highest

average number of lymphocytes on the day 14

was

in the treatment group, a concentration of 15% (PI)

with 18.08, while the lowest was in the positive

control treatment group (K+) with 4.70.

Based on the statistical test, data was obtained

significant results 0.000 (p <0.05). Followed by the

Duncan test and the result is that the treatment group

concentration 15% (PI) is the group that the most

potential on wound healing seen from the number of

lymphocytes.

The effect of ethanol extract of loquat leaves on

the number of lymphocytes is shown from the lower

extract concentration, the higher the number of

lymphocytes. Secondary metabolite compounds in

loquat leaves might able to accelerate the

inflammatory phase as seen from the high number of

lymphocytes in the treatment group of loquat leaves

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166

extract which can inhibit the growth and kill

microorganisms in the wound area.

Loquat fruit and leaves have high concentration

of Vitamin A. Vitamin A can play a role in

accelerating the inflammatory phase to the

proliferation phase by increasing monocytes,

lymphocytes and macrophages to the wound area

which will eliminate bacteria from the wound area

and produce growth factors needed for proliferation

of fibroblast cells and angiogenesis (Kumar et al.,

2014; Negara et al., 2014).

Lymphocytes were present within the wound at

one day, increased to peak numbers between days 8

and 14 post-wounding and remained present.

Lymphocytes are an important regulator of

fibroblast activity both directly and indirectly

through the macrophages during wound healing.

Martin and Muir, 1990).

On the day 7th, inflammatory cells and

macrophages begin to migrate together with

fibroblast cells into the wound tissue. Fibroblasts

will proliferate with the help of growth factors,

especially transforming growth factor -β (TGF-β)

and basic fibroblast growth factor (bFGF) which are

secreted by platelets and macrophages. Macrophages

will experience a reduction in the number as a result

of tissue repair which process will be followed by

fibroblast, endothelial cells and (Sabirin et al.,

2013

On proliferation phase, CD4

lymphocytes

induce keratinocytes to release IL-1 in the wound

area. Keratinocytes have a potential role on

epithelization, proliferation, and maturation of

epidermis. IL-1 that has been released by

keratinocytes induces endothelial cells to form

angiogenesis and fibroblast to form extracellular

matrix (Prakoso and Kurniasih, 2018).

The histological figure of fibroblast and

lymphocytes of mice skin on day 14

with 400x

magnification can be seen in Figure 2 below:

Figure 2: Microscopical examination of fibroblasts and lymphocytes.

(a) negative control (K-); (b) positive control (K+); (c) loquat leaves extract with concentration 15% (PI);

(d) loquat leaves extract with concentration 30% (PII); (e) loquat leaves extract with concentration 45% (PIII), ( )

fibroblas ( ) limfosit.

According to Balqis et al., (2014) that on the 14

day of the histological slide, infiltration of

inflammation cells was still visible and collagen

fibers have spread. In normal tissue, fibroblast cells

are rarely found. After the injury, fibroblast will

actively migrate to the wound area, will proliferate

collagen which plays a role in new tissue formation

until the skin returns to normal.

Effectivity Test of Loquat (Eriobotrya japonica (Thunb.) Lindl.) Leaves Extract on the Incision Wound Healing in Mice (Mus musculus L.)

167



3.4 The Average Number of Epithelial

Thickness

Histological observations of the epithelial thickness

on the histological preparations of the mice skin

with a magnification of 100x. Observation of

epithelial thickness is measured from the stratum

corneum layer to the stratum basale. Data on the

average epithelial thickness in each group on the

day 14

can be seen in Table 5.

Table 5: The average number of epithelial thickness.

Groups Ephitelial thickness (µm) ± SD

- 88,18

± 27,71

K+ 110,60

± 41,99

PI 171,61

± 35,62

PII 166,12

± 40,85

PIII 156,88

± 48,59

Based on Table 5, it can be seen that the highest

average number of epithelial thickness on the day

was in the treatment group, a concentration of

15% (PI) with 171,61 µm, while the lowest was in

the positive control treatment group (K+) with 88,18

µm.

Based on the statistical test, data was obtained

significant result 0.010 (p <0.05). Followed by the

Duncan test and the result is that the treatment group

concentration 15% (PI) is the group that the most

potential on wound healing seen from the number of

epithelial thickness.

Group PI, PII and PIII has shown the highest

number. The epithelial thickness can indicate the

faster process of reepithelization, so that can

accelerate the wound healing. The faster process in

the treatment group might be affected by loquat

leaves extract. Loquat leaves contain tannin, vitamin

A, Pratiwi et al. (2015) suggested that vitamin A, C,

E, tannins. and saponins in clove flower bud extract

can help the process of reepithelization by increasing

the differentiation of epithelial cells.

Loquat contain secondary metabolites such as

alkaloids which have antibacterial and antioxidant

effects that are high enough to maintain skin

integrity (Kumar et al., 2014), and enough to play a

role in the wound healing process by increasing

collagen formation, differentiation of epithelial cells

and increasing immunity (Negara et al., 2014). The

antioxidant effect can thicken the epithelial layer.

Substitution epithelial tissue occurs on the surface of

epithelial cells that continue to experience cell death

(Yohana, 2015).

Microscopical examination of epithelial

thickness with 100x magnification can be seen in

Figure 3.

Figure 3: Microscopical examination of ephitelial thickness.

(a) negative control (K-); (b) positive control (K+); (c) loquat leaves extract with concentration 15% (PI); (d) loquat leaves

extract with concentration 30% (PII); (e) loquat leaves extract with concentration 45% (PIII), ( )fibroblas ( ) limfosit.

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168

4 CONCLUSIONS

The average time span of wound healing for each

group K-, K +, PI, PII, and PIII were subsequently 8

days, 9.2 days, 7.6 days, 8.4 days, and 9.4 days.

Wounds treated with 15% concentration of loquat

leaf ethanol extract (PI) showed the fastest healing

effect while the 45% concentration of loquat leaf

ethanol extract (PIII) showed the longest healing

effect. The average number of epithelial thickness,

fibroblasts, and lymphocytes in the treatment group

of loquat leaf ethanol extract with a concentration

15% (PI) is higher than the other treatment groups

with the significant results of statistical analysis.

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