Althea Medical Journal. 2015;2(2)

199

Effect of Neem Leaves Extract (Azadirachta Indica) on Wound Healing

Naveen Kumar Chundran,1 Ike Rostikawati Husen,2 IrraRubianti3
1Faculty of Medicine Universitas Padjadjaran, 2Department of Pharmacology and Therapy 

Faculty of Medicine Universitas Padjadjaran, 3Department of Plastic Surgery Faculty of Medicine 
Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital Bandung

Abstract

Background: Neem leaves (Azadirachta Indica) have active ingredients such as nimbidin and sodium 
nimbidate which possess/possesinganti-inflammatory, antibacterial, antifungal and antiviral properties 
that help in healing process and also contains an excellent nutrition which plays/playing a vital role in 
formation of collagen and formation of new capillaries. The aim of this experimental study is to evaluate 
healing activity of neem leaves.
Methods: This experiment was conducted in Pharmacology Lab of Universitas Padjadjaran on October 2012. 
Twenty seven rats were grouped randomly into 3 groups and 1.5cm of excision wound was created. Negative 
control group was treated with a topical application of saline solution (sodium chloride0.9%), treatment 
group with a topical application of neem leaves extract and positive control group had been treated with a 
topical application of povidone-iodine for 15 days. Healing was assessed by the longest diameter of the raw 
surface of wound on days 0, 5, 10 and 15. The data were then analyzed using Mann-Whitney U test.
Results: There was a significant reduction in the longest diameter of wound in group of neem leaves extract, 
compared with group sodium chloride 0.9%,.and there was no significant difference in the longest diameter 
of wound between neem leaves, extract and povidone iodine. 
Conclusions: Neem leaves extract has the same wound healing rate compared to povidone iodine. A further 
study in human should be conducted in the future. [AMJ.2015;2(1):199–203]

Keywords: Neem, povidone iodine, wound healing

Correspondence: Naveen Kumar Chundran, Faculty of Medicine, Universitas Padjadjaran, Jalan Raya Bandung-
Sumedang Km.21, Jatinangor, Sumedang, Indonesia, Phone: +6287827969020 Email: naveen_160888@yahoo.com

Introduction

The skin, as the body’s largest organ consists 
of two layers, epidermis and dermis.1  Human 
skin provides protection for the body from 
external environment, preventing dehydration, 
regulates body temperature, detects cutaneous 
sensation, and synthesizes vitamin D.2 The skin 
barrier can be broken, such as from a wound.3 
There are many types of wound which have 
been classified into two major groups,  open 
and closed wound.4

Healing is a process to replace damaged 
tissue, organs, and biological system to normal 
function. Injury will initiate a series of event to 
repair the damaged skin and reform its normal 
structure. There are four phases in wound 
healing process, haemostasis, inflammation, 
proliferation, and remodeling.5-8 

Neem (Azadirachta Indica) is a well known 

medicinal plants, having a wide spectrum of 
biological activity as a traditional medicine 
for household remedy against various human 
ailments in ayurveda and homoeopathic 
medicine. Recent studies and research show 
that neem contains many therapeutic effects 
such as anti-inflammatory, anti-diabetic, 
antifungal, antiviral, antibacterial, and 
anti-malarial. Neem contains many active 
ingredients such as nimbidin, nimbin, and 
nimbidol with anti-inflammatory, antibacterial, 
antifungal and antiviral properties that may 
help it accelerating the wound healing process. 
In addition, neem contains an excellent amount 
of amino acid, vitamin and mineral that is 
very important in wound healing processes 
in proliferation phase. 9 So, there is a possible 
effect that neem can accelerate the  process. 
The aim of this study is to find out whether 
neem leaf extract can accelerate the duration 
of wound healing.



Althea Medical Journal. 2015;2(2)

200     AMJ June, 2015

Methods

An experimental study was conducted using 
27 rats (Rattus norvegicus) strain wistar. 
These rats were all male, aged between 2-3 
months and weighing about 200-250 grams. 
The rats were divided randomly into 3 groups. 
Each group contained 9 rats and was given 
intervention as followed: group 1 acted as 
the negative control group with a topical 
application of saline solution (sodium chloride 
0.9%), group 2 was the treatment group with 
a topical application of neem leaves extract, 
group 3 acted as the positive control group 
with a topical application of povidone-iodine 
solution 

During the experiment period, rats were 
given foods and water ad libitum (free access 
to foods or water thereby allowing animals to 
self-regulate according to its biological needs). 
The neem leaves extract was made by using 
1kg of neem leaves (AzadirachtaIndica). The 
leaves were washed with water and air-dried 
at room temperature. The leaves then were 
powdered by mixer, macerated in 10 liter 
of ethanol 96% and then kept for 72 hours 
away from lights and high temperature. It was 
filtered, evaporated, and dried with Rotary 
evaporator.

Rats were anesthetized before and during 
the creation of the wounds, with 1mL of 

intravenous ketamine. The hairs around the 
area of dorsal midline region were shaved 
clean using a shaver. Then, 1.5cm diameter 
was measured and marks were made on the 
rat. Using a permanent marker, the marks of 
the diameter were all joined to make a circle. 
Using a scalpel, excision was made all around 
the marked circle. Then, using an anatomical 
pin set and a curved hemostat scissors, a full 
thickness of excision wound for 2 mm depth 
was created following the marked circle. 
Wound was left uncovered throughout the 
experiment. Then, apply either saline solution, 
neem leaves extract or the povidone-iodine 
solution topically according to the group the 
rats were placed. The intervention applied 
sufficiently until it covered all wound area. 
The wound was left uncovered and cleaned 
using water with gauze and applied with daily 
intervention.10

The longest diameter of the raw surface 
wound was calculated on days 0, 5, 10, 15 
and the mean diameter of the raw surface of 
wound (red colour area) was compared to 
a control group at each time intervals. Raw 
surface of the wound can be differentiated 
from epithelisation surface by the colour, wet 
look and shininess.

Epithelisation surface would be pink in 
colour with the formation of a new epidermis 
while raw surface would be red in colour. Other 
than that, raw surface would be wet and shine 

Table 1 Longest Diameter of the Raw Surface of Wound after Topical Administration of 
                Sodium Chloride 0.9%

No
Longest Diameter Group 1 (Sodium Chloride) (cm)

Day 0 Day 5 Day 10 Day 15
1 1.4 1.1 1.5 1.2
2 1.5 1.4 1.3 1.0
3 1.6 1.8 1.9 1.6
4 1.7 1.4 1.0 1.7
5 1.5 1.7 1.5 1.2
6 1.5 1.2 1.4 0.9
7 1.5 1.3 2.0 1.8
8 1.5 1.7 2.0 1.6
9 1.5 1.1 1.2 1.2
Median 1.5 1.4 1.5 1.2
Mean 1.52 1.41 1.53 1.36
Minimum 1.4 1.1 1.0 0.9
Maximum 1.7 1.8 2.0 1.8



Althea Medical Journal. 2015;2(2)

201Naveen Kumar Chundran, Ike Rostikawati Husen, Irra Rubianti: Effect of Neem Leaves Extract (Azadirachta 
Indica) on Wound Healing

if it is exposed to lights or the sun meanwhile 
epithelisation surface would be dry and had a 
pale appearance.

The data were then analyzed between 
the groups using Mann-Whitney U test to 
compared the efficacy and potential of each 
treatment group. This statistic test used a 
significance level (α) = 0.05, two-tailed test, Z 
table = 1.65 with 95% confidence level.

This experiment was conducted in 
Pharmacology Lab of Universitas Padjadjaran 
on October 2012. The study was carried out 
with prior approval of the animal Ethical 
Committee, Faculty of Medicine,Universitas 
Padjadjaran.

Results

On day 0 the longest diameter on wound in 
group 1 was 1.52 cm, whereas in group 2 Was 
1.62 cm and in group 3 was 1.57 cm. On day 5, 
the longest diameter on wound in group 1 was 
1.41 cm, whereas in group 2 was 1.3 cm and 
in group 3 was 1.34 cm (Table 1, 2, 3). On day 
0 and day 5 there was no significant change in 
the longest diameter of wound between group 
sodium chloride 0.9% and group neem leaves 
extract (p> 0.05), where it can be seen from 
the p-value being 0.161 and 0.489.

However, on days 10 and 15 there was a 
significant change in the longest diameter of 

wound from group sodium chloride 0.9% (1.53 
and 1.36 cm) and group neem  leaves extract 
(1.11 and 0.31 cm), where it can be seen from 
the p-value being 0.008 and 0.000 (p < 0.05).

On day 0 and day 5 there was no significant 
change in the longest diameter of wound 
between groups sodium chloride 0.9% (1.53 
and 1.41 cm) and group povidone iodine 
(1.57 and 1.34 cm), where it can be seen 
from the p-value being 0.546 and 0.730 (p > 
0.05). However, on day 10 and 15 there was 
a significant change in the longest diameter 
of wound from group sodium chloride 0.9% 
(1.53 and 1.36 cm) and group povidone iodine 
(1.31 and 0.37 cm), where it can be seen from 
the p-value being 0.003 and 0.000 (p < 0.05).

On days 0, 5, 10 and 15 there was no 
significant change in the longest diameter of 
wound between group 2 and group 3, where 
it can be seen from the p-value being 0.387, 
0.666, 0.489 and 0.546 (p > 0.05).

Discussions

Wound healing is a process that involves several 
phases. The initial vascular response involves 
a period of vasoconstriction and haemostasis. 
The first phase is inflammation. Inflammation 
will cause an increase in vascular permeability 
and resulting in migration of neutrophils 
and monocytes into the surrounding tissue. 

Table 2 Longest Diameter of the Raw Surface of Wound After Topical Administration of  
                Neem Leaf Extract

No
Longest Diameter Group 2 (Neem Leaf Extract) (cm)

Day 0 Day 5 Day 10 Day 15
1 1.7 1.4 1.0 0.2
2 1.5 1.5 1.3 0.1
3 1.5 1.2 1.1 0.4
4 1.5 1.2 1.1 0.3
5 1.5 1.1 1.3 0.3
6 1.6 1.3 1.2 0.4
7 1.6 1.2 1.1 0.6
8 1.7 1.3 0.8 0.2
9 2.0 1.5 1.1 0.3
Median 1.6 1.3 1.1 0.3
Mean 1.62 1.3 1.11 0.31
Minimum 1.5 1.1 0.8 0.1
Maximum 2.0 1.5 1.3 0.6



Althea Medical Journal. 2015;2(2)

202     AMJ June, 2015

Infiltrating neutrophils cleanse the wounded 
area of foreign particles and bacteria. Second 
phase is the proliferative phase which occurs 
from 2 days to 3 weeks’ time. In this phase 
formation of granulation tissue, contraction 
and epithelialization will take place. The 
final phase of wound healing is wound 
remodelling, including a reorganization of 
new collagen fibers, forming a more organized 
lattice structure that progressively continues 
to increase wound tensile strength. The 
remodelling process continues up to two 
years.5-8

In this study the wound contraction process 
did not begin before day 10 in all groups. On 
days 10 and 15, there was a significant change 
in wound healing between group 1 and 2, it 
was proved by the reduction of diameter of 
the wound in group 2.  This shows that neem 
leaves extract has an active compound that has 
effects as anti-microbial, antioxidant and anti-
inflammatory.  

This proves that neem accelerates the 
healing process by prevent the infection which 
can prolong the healing duration. The anti-
inflammatory effect of neem can be seen more 
accurately if the histological examination had 
been performed.

Neem is also an excellent wound healer 
that helps the body to rapidly create collagen 
fibers to close the wound. On  day 10 and 
15, neem showed significant changes in the 
healing process by enhancing the proliferation 

phase. Neem contains an excellent amount 
of amino acid, vitamin and mineral that is 
very important in wound healing process in 
proliferation phase. Thus, neem accelerates 
the healing process by speeding up the 
proliferation phase. The result proves that 
neem leaves extract speeds up the proliferation 
phase in the entire wound healing process.

As a positive control, povidone iodine is an 
uniquely effective antiseptic and used widely 
for the prevention and treatment of infection. 
The prolonged non selective antimicrobial 
action of povidone iodine is unparalleled for the 
surface microbiocidal activity and particularly 
effective in treating mixed infection. Povidone 
iodine is suitable for hand disinfection, surgical 
hand disinfection, as well as preoperative 
and pre-puncture skin disinfection.11,12 The 
result proves that povidone iodine indeed 
increases the rate of wound healing by being a 
disinfectant to the wound.

Neem leaves extract and povidone iodine, 
both accelerate the rate of wound healing. The 
results shows that neem leaves extracts have 
the same wound healing rate compared to 
povidone iodine. Thus, neem leaves extract can 
be considered to be made an alternative choice 
to povidone iodine because both groups give 
the same outcome. 

This study has limitation, histological 
examination of the wound was not examinated 
to see the inflamantory effect of neem.

Table 3 Longest Diameter of the Raw Surface of Wound after Topical Administration of 
                Povidone-Iodine (Betadine) Solution

No
Longest Diameter Group 3 (Povidone Iodine) (cm)

Day 0 Day 5 Day 10 Day 15
1 1.6 1.2 0.6 0.3
2 1.6 1.4 1.3 0.2
3 1.5 1.3 1.1 0.1
4 1.5 1.2 0.9 0.4
5 1.5 1.3 1.3 0.7
6 1.5 1.4 0.8 0.6
7 1.9 1.7 1.2 0.3
8 1.5 1.4 0.8 0.3
9 1.5 1.2 1.1 0.4
Median 1.5 1.3 1.1 0.3
Mean 1.57 1.34 1.01 0.37
Minimum 1.5 1.2 0.6 0.1
Maximum 1.9 1.7 1.3 0.7



Althea Medical Journal. 2015;2(2)

203Naveen Kumar Chundran, Ike Rostikawati Husen, Irra Rubianti: Effect of Neem Leaves Extract (Azadirachta 
Indica) on Wound Healing

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Figure 1 Mean of the Longest Diameter of The Wound in Three Groups on Day 0, 5, 10, and 
    day 15