The Effect of Rhizophora apiculata Bark Ethanol Extract on Burns

Healing of Rattus norvegicus Sprague Dawley Strain

Syazili Mustofa

, Delisa Mutiara Nabila

and Evi Kurniawaty

Department of Biochemistry and Molecular Biology, Medical Faculty, Universitas Lampung,

Prof. Soemantri Brojonegoro Street, Bandar Lampung, Indonesia

Medical Faculty, Universitas Lampung, Bandar Lampung, Indonesia

Keywords: Burn Wound, Rhizophora apiculata Bark Extract, Wound Healing.

Abstract: This study aimed to evaluate the ability of an ethanolic extract of Rhizophora apiculata bark to treat burn

wounds in rats. Second-degree burn wounds were induced in five groups of six rats each, and topical treatment

was done daily for 26 days. Group KN and K+ received aqua bidestilata and bioplacenton® (containing

placenta extract 10% and neomycin sulfate 5%) as a control and reference standard, and group P1, P2 and P3

were given ethanolic Rhizophora apiculata bark extract of 20%, 30% and 40% respectively. The observing

burn wounds for ± 26 days were enrolled for evaluating the effects on the burn healing phase, burn area

reduction, and burn healing time. The area of burn wounds in the groups of rats given the extract began to

shrink more quickly on the 15th day of observation. The One-way ANOVA test revealed p=0.001, and the

LSD Post Hoc test revealed p < 0.05, indicating a significant difference in the area of burns on day 15 for all

treatment groups. The healed wounds in extracts-treated rats were faster and had better wound healing time.

Wound contractions were relatively better in extract groups. In conclusion, the Rhizhopra apiculata bark

ethanolic extract positively affected wound burn healing activity.

1 INTRODUCTION

Burns ranks fourth among the world's most common

injury categories. The World Health Organization

(WHO) estimates that 180,000 people die as a result

of burn injuries out of an estimated 11 million people

who suffer from burn wounds annually. Burns are

complex wounds with a high mortality rate that are

challenging to heal, regardless of the underlying

cause (Opriessnig, 2023).

Burn injuries are generally categorized as a type of

wound where the source is one of several factors,

including heat, cold, electricity, chemicals, friction,

or radiation. Conversely, wound healing is a

complicated process, and knowledge of its biological

trend and the variations in how various wounds heal

can often lower the danger and significantly lessen

the likelihood of further harm to the injured tissue and

other organs. Burn wound healing is a dynamic

process involving various interactions of cytokines

and extracellular matrix. This healing process is

https://orcid.org/0000-0002-7646-0869

divided into three phases: inflammation,

proliferation, and tissue remodelling. Wound healing

consists of wound healing and contracture, where

regeneration of the epithelial layer occurs and the

wound shrinks (Żwierełło, 2023). Healing burn

wounds is still a challenge faced by the modern

medical era. Only a few drugs can speed up wound

healing. There is still a need to find new drugs to

speed up the healing of burn wounds. Medicines

derived from medicinal plants have great potential to

be developed as alternative treatments (Huang, 2022).

Rhizophora apiculata, commonly known as

Bakau Minyak in Indonesia, is naturally distributed in

Southeast Asia and India. The plant contains saponin,

tannins, flavonoids and terpenoids (Bulan, 2022).

Traditionally, Rhizophora apiculata is employed by

Indonesians as a medicinal plant for gastrointestinal

and skin diseases. Our earlier research revealed that

this plant might have antioxidant (Mustofa, 2023) and

anti-inflammatory properties (Mustofa, 2019). For

these reasons, we were interested in researching the

effect of Rhizophora apiculata on healing burns.

Mustofa, S., Nabila, D. M. and Kurniawaty, E.

The Effect of Rhizophora apiculata Bark Ethanol Extract on Burns Healing of Rattus norvegicus Sprague Dawley Strain.

DOI: 10.5220/0013668300003873

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Medical Science and Health (ICOMESH 2023), pages 223-227

ISBN: 978-989-758-740-5

223

2 MATERIALS AND METHODS

2.1 Plant Material

The bark was obtained from the Forest Management

Unit KPH Gunung Balak, East Lampung, Indonesia,

in June 2022. The plant was authenticated by the

Department of Biology, Faculty of Mathematics and

Natural Sciences, Universitas Lampung.

2.2 Preparation of Extracts

We have carried out the process of making ethanol

extract in two steps, namely maceration and

evaporation. Maceration is the process of soaking

simplicia using a solvent for several days at room

temperature and protection from light. After air

drying, the bark was ground into a fine powder with

a blender. We weighed 100 grams of Rhizophora

apiculata stem bark powder and put it in a 2-litre

Erlenmeyer glass. Next, soaking was done with 2000

ml of 95% ethanol solvent and stirred for

approximately 30 minutes until the powder was

dissolved entirely. Finally, this solution was left to

settle for 18 hours. After this process, the second

stage is carried out, namely evaporation. This process

uses a rotatory evaporator and water bath

(temperature 90˚C). The ethanol solution and active

substance in the flask are allowed to separate until the

ethanol flow stops dripping into the collection flask

(Mustofa, 2020).

2.3 Animals Model

For this experiment, male rats weighing 200–250 g

were employed. They were housed in a central animal

house with a temperature of 23 ± 1o C and a 12-hour

light/dark cycle. They were fed adequately and

appropriately and had unlimited access to water

(Kurniawaty, 2022). Before beginning this

experiment, the medical faculty of Universitas

Lampung approved the ethical clearance for the

research by number

4479/UN26.18/PP.05.02.00/2022.

2.4 Burn Induced

Thirty rats underwent a back shave, 70% ethanol

disinfection, and xylazine and ketamine injections to

induce anaesthesia. Afterwards, an iron plate with a

20 mm diameter that had been heated over an

electrical heater for 10 seconds was used to create a

circular burn wound (3.14 cm2) on their dorsal

portions. After that, distilled water was applied to the

wound for a minute.

2.5 Experiment Protocol

Burn induced Rats were split into five groups (n =6):

group KN got aqua bidestilata as control, group K+

received reference standard treatment

(Bioplacenton®), groups P1, P2, and P3 received

20%, 30%, and 40% (w/w) of the extract,

respectively. Rats' dorsal back wounds were treated

topically with aqua bidestilata, Bioplacenton®, and

the extract every day at 24-hour intervals. The day of

burn generation was zero, and a 26-day treatment

protocol was initiated 24 hours after the burns were

created. Wound care was given once a day, and the

burn wound was cleaned first with 0.9% NaCl, then

dried and covered using sterile gauze.

Figure 1: Induced wound burns on experiment Rats.

2.6 Macroscopic Wound Analysis

Burn wounds are treated until they heal, characterized

by scarring or scabbing and tightening and closing the

wound. Wound healing was assessed by observing for

26 consecutive days using 0.01 mm scale callipers

(see Figure 1). The area of a burn wound was

calculated by measuring the average wound diameter

from four positions and then putting it into the ¼.π.d²

formula for circle size.

Analytical statistics Mean ± S.D. was used to

express all values. After one-way ANOVA data

analysis, a post hoc test was performed. At p<0.05,

the results were deemed to be statistically different.

3 RESULTS

We have conducted a qualitative test of the extract in

the Medical Biochemistry laboratory of the Faculty of

Medicine, Universitas Lampung. This extract

ICOMESH 2023 - INTERNATIONAL CONFERENCE ON MEDICAL SCIENCE AND HEALTH

224

contains various active substances, as shown in Table

Table 1: Qualitative phytochemical screening of ethanolic

extracts of Rhizophora apiculata bark

Qualitative active

compound

Result

Saponin Positive

Steroid Ne

ative

Ter

enoids Positive

Tannins Positive

Alkaloids Negative

Flavonoid Positive

The size of burn wounds on day 4 in all groups of rats

was 3.14 cm2, still the same as the initial day of

treatment. There was no extensive reduction, and

inflammation in the form of redness was obtained.

The area of the burn wound on the 10th day appeared

to be reduced by 15-20% of the original size.

However, the mean area of burn wounds for each

group of rats was not significantly different. On day

15, treatment groups P2 and P3 had quite different

macroscopic healing compared to the control group

(p-value = 0.001). It shows that administering

Rhizophora apiculata extract has a good effect on

wound healing (see Table 2).

Table 2. Results of measuring the average area of burn

wounds per day.

Experiment

day

Average burn area (cm

)

KN K+ P1 P2 P3

3.14 3.14 3.14 3.14 3.14

2.7 2.8 2.9 2.7 2.4

1.4 1.3 1.4 0.8* 0.7*

0.17 0.12 0.64 0* 0*

(* significantly difference compared to control group)

Figure 2: the average area of burns on each day of the

experiment.

The reduction in the area of burn wounds occurred

more quickly in the group of mice given the extract.

Burns were no longer visible on day 18 in group P2

and day 19 in group P3. Meanwhile, burns were no

longer visible on day 21 in the control group (see

Figure 2).

Figure 3: The averages complete burn wound healing time.

The results of macroscopic observations found that

the groups given the extract, namely P2 and P3,

experienced faster wound healing, namely on day 19

and day 20. Meanwhile, the control group only

experienced the healing of burn wounds on days 23

and 24 (see Figure 3).

4 DISCUSSIONS

The complicated process of burn healing causes

dermal and epidermal tissue degradation. It triggers a

few physiological reactions at the injury site, such as

an immediate inflammatory response followed by a

protracted, intense tissue creation phase.

Granulocytes or Polymorphonuclear leukocytes

leucocytes (PMNs) mediate this acute inflammation.

In this stage, the inflammatory site produces free

radicals that have the potential to harm tissues and

hinder the healing process of wounds (Żwierełło,

2023).

Mast cells and macrophages are immune cells

involved in the inflammatory phase. Numerous

growth factors and cytokines are secreted by these

two cells. Nerve Growth Factor (NGF) is a significant

cytokine that contributes to inflammatory responses.

Next, during the proliferation phase, NGF causes

endothelial cells to express Vascular Endothelial

Growth Factor (VEGF) and Fibroblast Growth Factor

(FGF), increasing keratinocyte proliferation and

angiogenesis (El Baassiri, 2023).

Therefore, tissue development, re-epithelization,

and epidermis differentiation can be hastened using

an anti-oxidant to minimize inflammation. Preventing

some of the frequently fatal infections in the wound

area is another critical step in healing. The

antibacterial action can promote wound healing and

The Effect of Rhizophora apiculata Bark Ethanol Extract on Burns Healing of Rattus norvegicus Sprague Dawley Strain

225

partially inhibit the growth of pathogenic microbes on

the skin. Thus, natural ingredients of antibacterial,

anti-inflammatory, and antioxidant properties can be

fundamental in the healing process of burns (Huang,

2023). The extract of Rhizophora apiculata prevented

rat hepatocyte and pancreatic cell necrosis caused by

cigarette smoke in an animal model assessment of its

anti-inflammatory action in rats (Mustofa, 2018).

Moreover, Rhizophora apiculata possesses a broad

antibacterial activity (Acharya, 2023). Thus, the

antibacterial, anti-inflammatory, and anti-

inflammatory properties of Rhizophora apiculata

may partially explain its favourable benefits in burn

healing acceleration and inflammation reduction

(Nisar, 2019). These effects could be attributed to

phytochemical components. Previous research and

our phytochemical analysis indicate that terpenoids,

flavonoids, tannins, and saponins are some of the

significant constituents of Rhizophora apiculata

(Chan, 2022).

One way to think of phenolic phytochemicals as

the main classes of secondary metabolites with

antimicrobial properties is flavonoids and tannins.

Through various mechanisms, including their

astringent, antibacterial, antioxidant, and angiogenic

effects, tannins can hasten the healing of wounds

(Ramya 2023). Terpenoids promote the breakdown of

stored extracellular proteins and prevent

prostaglandin formation, which can reduce tissue

oedema and inflammation. Because of their

antioxidant potential, they can avoid damage caused

by free radicals, just as flavonoids do through other

processes such as direct radical scavenging (Chan,

2022).

Because they regulate inflammation-related cells

and have an antioxidative action, flavonoids have

anti-inflammatory properties in vivo. Additionally,

reducing lipid peroxidation can improve collagen

strength and viability, boost vascularity and

circulation, and stop cell damage and necrosis. These

arguments suggest that aqueous extract's superior

antioxidant and anti-inflammatory properties may

contribute to its comparatively more significant effect

on burn healing, but they are not the only ones. The

results of the qualitative phytochemical screening

indicated that the ethanolic extract contained more

flavonoids. Thus, the higher flavonoid content of the

ethanolic section explains its more decisive

antioxidant action (Nisar, 2019). However, further

quantitative experiments are needed for a more

accurate assessment.

5 CONCLUSIONS

Rhizophora apiculata extracts significantly

accelerated the healing of burn wounds in rats. This

effect may result from several mechanisms, including

a faster rate of vascularization and re-

epithelialization, the inhibition of harmful free

radicals, a decrease in oedema and inflammation, and

the ability of this plant's antioxidant, anti-

inflammatory, and antimicrobial components to

control infection. More research utilizing purified

components is necessary to fully comprehend the

mechanism underlying the burn-healing ability of

Rhizophora apiculata.

ACKNOWLEDGEMENTS

This research was financially supported by HETI

Project Universitas Lampung.

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