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Influence of Aloe Vera Gel on Dermal Wound Healing Process in Rat

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Influence of Aloe Vera Gel on Dermal Wound Healing Process in Rat

Nasrin Takzare a; Mir-jamal Hosseini b; Gholamreza Hasanzadeh a; Hamideh Mortazavi c; Alireza Takzare a;
Parisa Habibi d
a
Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran b
Departments of Toxicology and Pharmacology, School of Pharmacy, Zanjan University of Medical Sciences,
Iran c Department of Midwifery, School of Nursing and Midwifery, Tehran University of Medical Sciences,
Tehran, Iran d Department of Animal Biology, School of Biology, College of Science, University of Tehran, Iran

First Published:January2009

To cite this Article Takzare, Nasrin, Hosseini, Mir-jamal, Hasanzadeh, Gholamreza, Mortazavi, Hamideh, Takzare, Alireza and Habibi,
Parisa(2009)'Influence of Aloe Vera Gel on Dermal Wound Healing Process in Rat',Toxicology Mechanisms and Methods,19:1,73 —
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 Toxicology Mechanisms and Methods, 19:73–77, 2009
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ISSN: 1537-6516 print; 1537-6524 online
DOI: 10.1080/15376510802442444
Influence of Aloe Vera Gel on Dermal Wound Healing
Nasrin Takzare
Department of Anatomy,
School of Medicine, Tehran
University of Medical Sciences,
Tehran, Iran
Mir-jamal Hosseini
Departments of Toxicology and
Pharmacology, School of
Downloaded By: [Karolinska Institute] At: 11:47 26 December 2008
Pharmacy, Zanjan University of
Medical Sciences, Iran
Gholamreza Hasanzadeh
Department of Anatomy,
School of Medicine, Tehran
University of Medical Sciences,
Tehran, Iran
Hamideh Mortazavi
Department of Midwifery,
School of Nursing and
Midwifery, Tehran University of
Medical Sciences, Tehran, Iran
Alireza Takzare
Department of Anatomy,
School of Medicine, Tehran
University of Medical Sciences,
Tehran, Iran
Parisa Habibi
Department of Animal Biology,
School of Biology, College of
Science, University of Tehran,
Iran
Received 14 July 2007;
accepted 22 November 2007.
Address correspondence to Nasrin
Takzare, Department of Anatomy,
School of Medicine, Tehran University
of Medical Sciences, PO Box:
13145–784, Tehran, Iran. E-mail:
takzare@yahoo.com;
m hosseini@razi.tums.ac.ir
Process in Rat
ABSTRACT In this topical study the influence of Aloe Vera, on the wound
healing process was investigated in 63 male rats with microscopic and cell
count methods. On the day of surgery a round wound, of diameter 20 mm, was
created on the back of rats necks under sterile conditions. The surgery day was
determined as day zero (0). Then the rats were divided randomly into control
and experimental groups 1 and 2. Animals in each group were sub-divided to
three smaller groups, investigated every 4, 7, and 14 days. From day 0, wound
surfaces were covered with gel once daily in experimental group 1 and twice
daily, for 12 h interval, in experimental group 2. Each rat received 30 g of the
gel. The wound surface and healing were assessed on days 4, 7, and 14, and
then a sample from the wound was prepared and investigated microscopically.
The results show that the number of neutrophil, macrophage, and fibroblast
cells and the wound thickness in the control group were statistically different
from the experimental groups. It was found that the wound diameter thickness
in the experimental group was greatly lower due to twice administration of gel
and the power of wound healing was more than other groups.
KEYWORDS Aloe Vera; Gel; Wound Healing; Natural Medicines; Rats; Microscopic
INTRODUCTION
The aim of wound healing, in medical science is a short time for repair and with few side
effects. Acceleration in wound healing with an extra stimulation of a factor can decrease the
time of patient motionlessness and therapeutic costs. It causes rapid return of the wounded
to their daily activities. Natural medicines, especially pharmaceutical herbs, have been
considered the principal cure for several years. Today, further tendency to pharmaceutical
herbs usage is made. These have been used in the treatment of a variety of disorders
including wound and burns since ancient times.
Wound healing, a highly complex process, and in short time with less side effects,
is one of the objectives of medical science (Chithra et al. 1998). Since the ancient
times, Egyptian, Greek, Indian, and European physicians had been attempting to treat
wounds in a shorter time with less side effects (Goutoid et al. 1996), concerning the
importance of wound healing and because infection and cancer occur in cases of not
healing the wound (Adzick 1996). Various researches have been done on wound healing,
consequently different substances have been made as wound healing and introduced
that often are herbal compounds and sometimes are chemical ones (Somboonwong et
al. 2000; Adzick 1996). But none of them can be offered as an effective medicine till
now. The effects of Ca, Cu, Zn ions, ultra sound waves, and the effects of chemical
substances such as hydrocortisone, Vit A, Ascorbic Acid, phenition, salt serum, and growth
factors can be mentioned as some examples (Stanford and Rappole 1969; Barnett and
Varly 1987; Brown and Mcdonnell 1988; Mantle et al. 2001; Kumar and Jagetiz 2005).
73
 Natural drugs, especially pharmaceutical herbs, were considered
the principal treatment and even the only healing method
in some cases for several years and meanwhile the primary
substance of them were used in the pharmacy industry (Mantle
et al. 2001). Today further tendency to pharmaceutical herbs
usage is made due to less side effects, effective compounds,
and expansion-dependent industries to pharmaceutical herbs
cultivation, prevention from foreign exchange exit, job creation,
and especially WHO recommendation for using pharmaceutical
herbs. Injured tissue healing is of highly complex homeostatic
body activities including a series of complicated and regular
processes. Rate of wound healing is affected by various factors
such as nutrition, vitamins, steroidal hormones, oxygen, and
environmental factors (Brown and Mcdonnell 1988; Linch
et al. 1989; Kagan and Warden 1994; Atiyeh Bishara et al. 2003).
Aloe Vera is one of the pharmaceutical herbs belonging to the
liliaceae family. It has been used in the treatment of a variety
of disorders including infections dermatologic conditions and
used as a laxative since ancient times. This plant has long meaty
thick leaves with twisted sides which end in thorns (Grindlay and
Reynold 1980; Heggars et al. 1993). The substance inside the leaf
called gel consists of 99% water with long chain polysaccharide,
of Acetylated glucoannan kind, and other carbohydrates. It also
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contains the complex of Amino Acids, salisilic Acid, Ascorbic
Acid, Vit A, and Vit E with anti-oxidant properties (Grindlay
and Reynold 1980; Davis et al. 1988; Heggars et al. 1993;
Chithra et al. 1998). This gel prevents skin dryness due to
the high volume of water. The high percentage of glucose
present in gel prevents bacterial growth due to the high osmotic
virtue (Adzick 1996). Prostaglandin and bradykinin hydrolyzing
enzymes in Aloe Vera reduce pain and inflammation. The
existence of amilaze enzyme in Aloe annihilated the necrosal
tissue, aloctin-A, which has a cell division and mitosis effect and
causes the acceleration in healing and stimulating macrophage
to excrete the dead tissue. Amino Acids present in plant gel
are used to produce protein, causing tissue growth and healing.
Vitamins including β-carotene, Vit E, Vit C, and B complex,
used in cell reaction, are consumed as antioxidants in strength-
ening the body immune system (Vazquez et al. 1996). Plant
juice, antrakinons with anti-microbial, antiviral, antifungal, and
anti-inflammatory properties and the saponins with antiseptic
properties are effective in preventing infections (Brown and
Mcdonnell 1988; Vazquez et al. 1996; Kumar and Jagetiz 2005).
Concerning the above information, complementary research
to study the influence of different doses of Aloe Vera gel on the
dermal wound healing process was performed.
MATERIALS AND METHODS
Experimental Animals
In this experimental study, 63 male wistar rats, weighing
200–250 g, 10–12 weeks old, were used. Rats were housed
on a 12-h light and 12-h dark cycle with unrestricted access
to food and water. They were kept in individual cages in a
controlled room (temperature 20–25◦ C; humidity 70–80%).
The animal room was under standard conditions. All exper-
iments were conducted in the Tehran University according
to the recommendations of the ethics committee on animal’s
experimentation of medical school.
Nasrin Takzare et al.
Wound Creation
On the surgery day, rats were anaesthetized. Ketamin
hydrochloride 5 mg/100 g body weight (Gedeonrichter Factory,
German) and diazepam 0.45 mg/100 g body weight (Kimidaroo
Company, Iran) and pentazocin 0.04 mg/100 g body weight
(Toliddaroo Company, Iran) were used as anesthetic drug. All
the anesthetic drugs were injected intra muscularly. Under the
general anesthesia, the back of the rat’s necks were shaved
and antisepticized with povidin–Iodine (Toliddaroo Company,
Iran) under sterile conditions. From day 0, wound surfaces were
covered with gel once daily in experimental group 1 and twice
daily, for 12 h intervals, in experimental group 2. Each rat
received 30 g of the gel.
The surgery day was determined as day zero (0) and the
following days were 1, 7, and 14. All wounds were of full-
thickness type extending up to the adipose tissue. Excision
wounds of size 4 cm2 were made by cutting out 2 cm ∗ 2 coppice
of skin from the shaven area (a round full-thickness wound, of
diameter 20 mm was made). The randomized method was used
to divide the rats in the control group and experimental groups
1, 2. Animals in each group were sub-divided into three smaller
groups and were evaluated every 4, 7, and 14 days. From day
zero, wound surfaces were covered with Aloe Vera gel once daily
in experimental group 1 and twice daily in experimental group
2. Nothing was used on wounds in the control group.
On days 4, 7, and 14 after the treatments of rats in
experimental groups, animals were sacrificed by ether inhala-
tion, then a sample from the wound matrix and healthy
skin beside that was made, Then, after tissue processing and
fixation (Formaldehyde 4%) and staining they were observed
microscopically for histological changes.
Sacrificed animals on the 1st, 7th, and 14th days after wound
creation, and the entire wound on each animal was cut out
and stored at −70◦ C until analysis. All of the samples were
colored with the Hematoxylin-Eosin method. Ten parts of the
wound were assessed by using the optic part of a photomicro-
scope. In this evaluation, fibroblasts, macrophage, neutrophil,
and vessels endothelium were counted.
Wound Healing Assessment Method
Wound healing was assessed by various parameters such
as evaluation of wound surface, percentage of wound healing,
duration of healing, and complete healing. Wound surface on
days 4, 7, and 14 after creation was evaluated, of cm2 unit,
and the percentage of healing was normalized by the following
formula:
Percentage healing
Wound surface on day 1 − wound surface on day x × 100
=
Wound surface on day 1
where x is the day when wound surface is evaluated.
Preparation of Aloe Vera Gel
The important matter in this study was preparation of the
pure and effective substance of Aloe Vera gel. According to
recent studies pharmaceutical usage of Aloe Vera plant are
divided into two parts: Aloe Resins or Aloe Antrakinons, which
74
 is yellow and bitter and is placed on the outer surface of
Aloe skin and consist of Aloin, Isobarbaloin, and chrysaphanic
Barbaloine. These compounds are semi-toxic. The second part
is Aloe gel, which is a substance in the inner part of the plant
leaf and consists of water with a polysaccharide, of acetylated
glucomannan kind, and other carbohydrates. Also gel contains
the complex of amino acid, salicylic acid, Ascorbic Acid, Vit A,
and Vit E with Antioxidant properties. Aloe leaves can activate
the macrophage and control cell immune system, fibroblasts,
and granulocytes. In our study, leaves were just used so that the
pure gel preparation needed specialty and direct supervision.
The gel preparation process was performed in some phases.
At first, the healthy plant leaves of Aloe Vera (3 years) were
collected from Karaj farmland (West of Tehran, Iran), then
were cleaned and antisepticized. In the next phase, full size
mature leaves were cut from the plant and the rind removed.
But, due to the sensitivity of the investigation, the effective
factors in wound healing abandoning the toxic compounds
of the plant gel extraction was prepared as incision (slice). FIGURE 1 The number of macrophage of aloe vera treated and
untreated wound tissue in rats. Data are given as mean ± SD for
Longitudinal incision was made on the plant leaf and the
seven animals in each group.
gel present in the leaf was extracted for gel preparation. The
extracted gel was heat at a temperature 50◦ C and filtered to be
used in formulation. To make the gel constant it was mixed with
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some glycerin and triethanolamin and heated at a temperature

of 75◦ C. After completing the mentioned phases, a gel of 95%
purity was obtained.

Grouping of Animals
After wound creation, experimental animals were divided
into three groups of 21 rats. Then each group was sub-divided
to three smaller groups, investigated every 4, 7, and 14 days
as follows: Control groups—wounds untreated, Experimental
group1—wound treated once daily, and Experimental group
2—wound treated twice daily in 12 h interval.

Statistical Analysis
FIGURE 2 The number of neutrophil of aloe vera treated and
Experimental groups 1 and 2 were compared with the untreated wound tissue in rats. Data are given as mean ± SD for
control group. Student’s t-test used to identify differences seven animals in each group.
between groups, and data were considered significant at
p < 0.05, p < 0.01 and p < 0.001. To count the wound cells, a
photomicroscope (×40) was used.

RESULTS
The results obtained from the study are shown in Fig. 1–5.
The results show that number of neutrophil, macrophage,
fibroblast cells, and the wound thickness in the experimental
group had statistically significant differences. On the 4th
day of the experiment, number of fibroblast, macrophage
and neutrophil cells, and the vessel endothelium were sig-
nificantly different between experimental and control groups
(p < 0.0001).
The results on the 7th day demonstrated that number of
fibroblast, neutrophil and vessels endothelium, and wound
thickness in the experimental and control groups were signifi-
cantly different (p < 0.001).
The results on the 14th day showed that number of FIGURE 3 The number of fibroblast of aloe vera treated and
fibroblast was significantly different between control group untreated wound tissue in rats. Data are given as mean ± SD for
and experimental group 1 (p < 0.001). However, there was seven animals in each group.

75 Influence of Aloe Vera Gel on Wound Healing


FIGURE 4 The number of macrophage of aloe vera treated and
untreated wound tissue in rats. Data are given as mean ± SD for
seven animals in each group.
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FIGURE 5 The thickness of wound in aloe vera treated and
untreated wound tissue in rats. Data are given as mean ± SD for
seven animals in each group.
no significant difference in the number of macrophage, Neu-
trophil, and vessels endothelium between the control group and
experimental group 1.
No significant difference was found in wound thickness
between the control group and experimental group 1 on the
14th day, but there was a significant difference between control
group and experimental group 2 (p < 0. 05). The mentioned
difference was found between experimental groups 1 and 2
(p < 0.05).
DISCUSSION
Aloe Vera, of the pharmaceutical herbs, has been used in the
treatment of many disorders including infections, dermatologic
condition, and used as a laxative since ancient times. In
the present study, the influence of Aloe Vera gel on dermal
wound healing is evaluated. The findings of the present study
confirm the significant acceleration of dermal wound healing
in rats following administration of gel twice daily. The result
of this study is similar to previous studies and shows that the
Nasrin Takzare et al.
application of gel accelerates the process of tissue bud creation
and wound healing.
Another study mentioned that wound healing is different
when Aloe Vera gel is administered, but the concentration of
consumed Aloe Vera gel was not clear and it was mentioned
that various concentrations of gel can be different in wound
healing acceleration (Visuthikosol and Chowchuen 1995)
In the present study, the number of fibroblasts in both
experimental groups is more than in the control group. This
increase is due to the significant acceleration in the healing
process in experimental groups and the proliferation phase of
the healing process has begun earlier during that the same day,
and, in the experimental groups, the number of macrophages is
more than in the control group. Also the number of Neutrophils
in experimental group 2 is more than in the control and
experimental group 1. Wound diameter in experimental group
2 was lower than the other two groups and this is due to
the increase in number of fibroblasts in experimental group
2 compared to the control group. The role of these cells in
extra cellular substance synthesis can be justified and the twice
application of gel during the day was more effective. In the
same researches (Atiyeh Bishara et al. 2003; Dill and Lacopino
2004), in order to know the reason and effective mechanism of
Aloe Vera gel, rate of wound healing is mentioned to be due
to the increase in collagen synthesis by fibroblasts. Aloe Vera
gel contains various compounds such as polysaccharides and
special Amino Acids possessing power of stimulation (Crindly
and Reynolds 1986; Chithra et al. 1998).
After administration of gel on the wound, the number of
fibroblasts increased compared to the control group and this
increase caused the creation of tissue bud with excess rate.
Also the rate of inflammation on wound reduced (macrophage
& Neutrophil increase) compounds present in gel cause the
rapid fibroblast transformation. Further administration of gel
experimental seems to increase DNA and tissue power and
stimulates the formation of new vessels and epithelization.
Application of gel causes the increase in collagen synthesis and
thus the healing is performed quicker (Crindly and Reynolds
1986; Brown and Mcdonnell 1988; Chithra et al. 1998;
Takamiya et al. 2003). On the 7th day, which is considered as the
proliferation phase of the healing process, increase in number
of fibroblasts in experimental group 1 is decelerated, but the
number of fibroblasts in experimental group 2 increases. These
results show the capacity which potentially exists in the wound
healing process and twice application of the gel activates this
reaction more. The wound diameter on the 7th day is lower in
experimental group 2 and the wound heals better. The structure
renewal of the wound healing process occurs after the significant
acceleration in proliferation phase.
On the 14th day, this is considered as structure renewal of
the wound healing process, in the experimental groups, excess in
the number of fibroblasts as compared with the control group
and even as compared with day 7, mentions the continuous
proliferation phase of healing process till day 14. Significant
and simultaneous increase in fibroblasts and decrease in the
wound thickness of the experimental groups shows the effect of
gel on increasing the proliferation and fibroblast metabolism.
Decrease in number of macrophage in experimental group 2
was in agreement with the condition of this day, because of the
application of gel, the extra gel particles present in the wound
matrix, the number of neutrophils in experimental groups,
especially experimental group 2, increases and the number of
76
 vessels and endothelium in experimental group 2 decreased.
Wound diameter in experimental group 2 decreased greatly
because of twice administration of gel and the power of wound
healing was more than the other groups. Administration of the
Aloe Vera gel twice daily on dermal wound in wistar rat causes
significant and better results compared to administration once
daily.
Declaration of interest: The authors report no conflicts of
interest. The authors alone are responsible for the content and
writing of the paper.
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Influence of Aloe Vera Gel on Wound Healing