Many of the spices and herbs used today were known to the people
of the ancient cultures throughout the world and they were valued for
their preservative and medicinal powers as well as their flavor and odor
qualities (Zaika, 1988). Garlic is one of the edible plants, which has
generated a lot of interest throughout human history as a medicinal panacea.
Garlic (Allium sativum Linn.) a member of the Liliaceae family
is used a public spice extensively in many parts of the world (Baghalian
et al., 2005). Many species of genus Allium have been used for
thousands years as vegetables, spices and as medicinal plant for the treatment
of various diseases (Baruchin et al., 2001; Haciseferogullari et
al., 2005; Sharma and Prasad, 2001).
Avecina, the famous ancient Persian physician, had suggested the usage
of garlic extract in the curing of numerous infection diseases (Hosseini,
The various powerful flavors of these plants and their possible medical
applications have attracted the attention of chemists and plant physiologists
(Akgul, 1993; Graham and Graham, 1987). The most of its therapeutic effects
are attributed to specific oil and water-soluble organosulfur compounds
which are responsible for the typical odor and flavor of garlic (Sivam,
Garlic has an unusually high concentration of sulfur-containing compounds
(1-3%) and its therapeutic properties are largely due to one particular
class of sulfur-containing compounds, the thiosulfinates (Koch and Lawson,
1996; Lawson, 1996).
The thiosulfinate structure appears to be essential for the bactericidal,
antifungal and antiprotozoal properties of garlic, likely reacting with
SH-containing enzymes of these pathogens (Reuter et al., 1996).
One of the important of these thiosulfinates is allicin, which name taken
from the Latin name of the garlic plant, Allium sativum (Ankri
and Mirelman, 1999). Other studies were also carried out on the antimicrobial
and antioxidant properties of garlic and its derivatives such as essential
oil and oleoresin (Akgul, 1993; Zaika, 1988).
These bioactive components have been isolated from aqueous, ethanolic
and fermented extracts of crushed garlic and have the potential to interact
with a number of cellular targets, particularly those exhibiting reactive
sulfhydryl moieties, whose functions range from control of cell cycle
to expression of crucial antioxidant and detoxification enzymes. Interactions
with these processes may underlie garlic`s putative therapeutic potential
(Cooper and Pinto, 2005).
With attention to widespread usage of garlic in curing of various illnesses
particularly infectious diseases, there is an absence of scientific investigation
of application of this medicinal plant in wound healing. The present study
is a clinical and microbiological evaluation of topical application of
garlic extract in treatment of burn wound healing.
MATERIALS AND METHODS
The garlic extracts were freshly prepared everyday. The garlic was
purchased from local vegetable market (Gilan, Iran). The garlic bulbs
were peeled and ground to form a paste in 1 g quantity. The paste was
then dissolved in 100 mL distilled water in a sterile tube. The solution
was then centrifuged at 6000 rpm for 20 min at room temperature. The pellet
was discarded and supernatant was diluted 10 times with distilled water
to get 1% of garlic extract. The concentration of garlic extract used
was based on Shukla and Taneja (Shukla and Taneja, 2002).
The extract was subjected to silica gel Column Chromatography (CC). On
elution with hexane-ethyl acetate in a gradient in order to increase polarity
(from 0 to 100% EtOAc and afterwards by washing the column with methanol)
4 fractions were obtained. These fractions were analyzed by GC-MS.
The prepared samples were irradiated with 25 KGy of gamma ray. Irradiation
process was performed in the Atomic Energy Agency of the Islamic Republic
This study was performed on ten apparently healthy adult mongrel dogs
of both sexes, 4 to 5 years old with mean weight of 20 ± 2.25 kg.
Animals were categorized randomly into control and garlic treated groups.
The dogs were housed in individual cages and had access to water and food
ad libitum. The investigators adhered to the Animal Welfare Act and the
experimental protocol was also approved by the Animal Ethics Committee
of the university. The model of the burn wound was produced according
to Hoekstra standard (Brans et al., 1994). Food was withheld for
12 h before surgery. Dogs were premedicated with acetylpromazine (Hoogsrraten,
Belgium) (0.1 mg kg-1, intravenously), anesthetized with sodium
thiopental (Biochemie GmbH, Vienna, Austria) (10 mg kg-1, 2.5%,
intravenously) and maintained with halothane (ICI Pharmaceuticals, Cheshire,
England) in oxygen in a semiclosed circle system. Lactated Ringer`s solution
(Shahid Ghazi Co., Tabriz, Iran) (10 mL kg-1 h-1,
intravenously) was administered during the surgical procedure. Dogs were
positioned in ventral recumbency and the area just behind the shoulders
was shaved backwards and was prepared for aseptic surgery.
All animal were subjected to the rectangular burn wounds (2x3 cm2)
using a hot (180°C) brass brick weighing 500 g which was pressed against
the shaved skin for 10 sec on either side of the spine. Left side defect
assigned to as experiment and the right one as control, so that each animal
served as its own control. The experiment wounds received 0.5 mL of 1%
aqueous garlic extract, in aseptic conditions, every three other days
for 21 days. But in the control group, the wounds were washed with normal
saline at same frequency and time of day. All the wounds were bandaged
with routine sterile dressing, held in place with an elastic wrap. No
antibiotic was used as a pre- or post-operative prophylaxis. To manage
the pain and discomfort, Tramadol (KRKA, d. d., Novo mesto, Slovenia)
(0.2 mg kg-1, IM) was administered every 3 h after surgery
for 24 h and continued as needed.
Clinical and microbiological [quantitative (total plate count) and qualitative
(using specialized microbial medias] examinations of the burn wounds were
carried out on 0, 7, 14 and 21 days of the experiment. The clinical evaluation
was including the general health conditions and the reaction to environment.
The process of burn wound healing was carefully assessed as well as the
granulation tissue formation and the progression of scar formation.
The wounds were photographed and all the photographs were scanned and
wound areas were measured using digital scanning software (Sigma Scans
Pro 5.0, SPSS Science, Chicago, IL). Time elapsed for wound healing was
considered in both groups. The rates of wound contraction (percent decrease
of wound area) (wound area on day 0 min wound area on day n, divided by
the wound area on day 0 expressed as a percentage) were analyzed.
The results of total bacterial count, time taken for healing and rates
of wound contraction were analyzed with a paired Student`s t-test. Differences
were considered significant if p<0.05 (SigmaStat for Windows, version
2.03, Jandel Corporation, San Rafel, CA).
Results of biochemical analysis of the garlic extract were shown
in Table 1. Also, from point of view of clinical evaluation,
healing process of burn wounds was without any major complications during
the study. Throughout the days after the experiment the animals showed
a normal reaction to the environment and displayed no signs of suffering
due to burn wounds. The rates of wound contraction (percent decrease of
wound area) in experiment and control wounds were shown in Table
The purpose of bacteriological examination was to evaluation of contamination
of wound surface before and after topical application of garlic extract.
The growth of Candida albicans, Staphylococcus aureus, Streptococcus
pyogenes and Escherichia coli strains were observed during
the microbiological examination of the skin before use of the preparations.
The total number of the strains on the skin were 3 ± 0.5x104
cm-2 in control group and 5 ± 0.4x104 cm-2
in the experiment group.
In the control group, the number of microbes existing on 1 cm2 of
the wound in the first day, were 3 ± 0.8x104 cm-2
and gradually increased during the following days to the value of 6 ± 0.6x105
cm-2 on day 7. In the 14th day the number of microbes imperceptibly
to decrease until day 21, when reached to 2 ± 0.5x105
cm-2. In this group, Staphylococcus aureus, Streptococcus
pyogenes, Escherichia coli and Candida albicans were
also isolated on 7th day of the study. On the 14th day, the growth of
Escherichia coli and Candida albicans was not observed.
On 21st day, just Staph. aureus was isolated.
||The fraction composition of the garlic extract
||Comparison of wound area (mm2) (mean ± SD) and percent
decrease in the experiment and control wounds (n = 5 dogs)
|*: Significant difference (p<0.05) Fig.
1: Results of quantitative microbiological examinations
||Results of quantitative microbiological examinations.
In the garlic treated group, the numbers of microbes were 5 ± 0.6x104
cm-2 in the first 24 h of the experiment. On the 7th day of
the experiment this count was 4 ± 0.8x105 cm-2
, however, on day 14 it decreased significantly to the value of 2 ± 0.6x105
cm-2. This value decreased significantly on the 21st day and
reached to the 4 ± 0.5x104 cm-2. In this group,
on the 7th day of the study, Streptococcus pyogenes, Staphylococcus
aureus and Escherichia coli were isolated. Also on 14th day
the growth of these microorganisms were observed, but just S. pyogenes
was isolated on 21st day of the experiment.
Results of microbiological examinations was presented in Fig.
Today usage of garlic as a natural and traditional therapy appears
to become popular. There are many reports about therapeutic application
of garlic in treatment of various diseases (Chowdhury et al., 1991;
Dietz et al., 2004; Harris et al., 2001; Shadkchan et
al., 2004; Sivam, 2001; Shukla and Taneja, 2002). But there are few
researches on the accelerating effect of garlic on wound healing (Saifzadeh
et al., 2006; Sardari et al., 2006a, b). The aim of this
investigation is clinical and microbiological evaluation of efficacy of
Iranian garlic extract on healing of burn wound.
The results of present study showed that the decrease in wound area was
significantly greater (7, 14 and 21 days) in the experiment wounds compared
to the control (p<0.05) (Table 2). These findings
demonstrated that the rates of wound healing were affected significantly
by 1% aqueous garlic extract treatment. These results are in agreement
with other researches in this field (Saifzadeh et al., 2006; Sardari
et al., 2006a, b). Although, these studies were accomplished on
incisional wounds while our study was carried on burn wounds.
Most of the therapeutic effects of garlic are known to referable to its
sulfur-containing compounds Biochemical analysis of garlic extracts in
this study were revealed that concentration of two sulfur-containing compounds
(Allicin and Methyl sulfonyl methane) which very important in healing
accelerating efficacy and antimicrobial potency are more than 5% (Table
These findings are in agreement with report about sulfur-containing compounds
of Iranian garlic (Baghalian et al., 2005).
Methyl sulfonyl methane, also identified as dimethyl sulfone, is a naturally
occurring sulfur compound found in a variety of foods, including onions
and garlic. Methyl sulfonyl methane is an important nutrient and is essential
for the maintenance of connective tissues, joint function, proper enzyme
activity and hormone balance. Sulfur is very important for the formation
of collagen and is a major component in the synthesis of cartilage and
connective tissues. Sulfur is essential part of keratin which is necessary
for the maintenance of healthy situation of the skin, hair and nails.
Additionally, it gives strength, shape and hardness to their protein tissues.
Methyl sulfonyl methane can also decrease scar tissue by changing the
cross linking process in collagen to allow tissue repair and healing to
take place (Naguib , 2002). Methyl sulfonyl methane may be responsible
to the enhancing effect of aqueous garlic extract on wound healing.
From view point of microbiological evaluation, results of this experiment
were showed on the day 21, the total number of microorganisms was lesser
(4 ± 0.5x104 cm-2) when garlic extract were
applied compared to the control wounds (2 ± 0.5x105 cm-2)
Ankri and Mirelman (1999) attributed the antibacterial activity to the
specific chemicals in garlic. The nature of these chemicals and the mechanisms
of their action are not fully understood. Allicin is one of the sulfur
components of garlic extract, this compound is known to be responsible
for the most of the antibacterial property of garlic.
It is the most abundant thiosulfinate found in garlic and is generated
when an enzyme alliinase reacts with its substrate alliin. Alliin was
found to be a stable precursor that was converted to allicin by the action
of an enzyme termed allinase which was also present in the cloves (Ellmore
and Feldberg 1994). Allinase is present in extraordinarily large amounts
in garlic cloves (10% of the total protein content) (Harris et al.,
Enzyme and substrate are located in different compartments of the clove,
the transformation of alliin into the biologically active allicin molecule
upon crushing of a garlic clove is extremely rapid, being completed in
seconds (Lawson, 1996).
The antibacterial effect of allicin is broad spectrum. In most cases
the 50% lethal dose concentrations were somewhat higher than those required
for some of the newer antibiotics. Interestingly, various bacterial strains
resistant to antibiotics such as methicillin resistant Staphylococcus
aureus as well as other multidrug-resistant enterotoxicogenic strains
of Escherichia coli, Enterococcus, Shigella dysenteriae,
S. flexneni and S. sonnei cells were all found to be
sensitive to allicin (Chowdhury et al., 1991; Gonzalez-Fandos
et al., 1994; Holzgartner et al., 1992; Shadkchan et
Garlic extracts are also effective against Helicobacter pylorus which
is suspected of causing gastric ulcers (Celiini et al., 1996).
In view of the fact that the present experiment is the first study on
the garlic as an accelerator of burn wound healing, our results are not
comparable with those of previous works.
Considering these explanations, topical application of garlic causes
significant enhancement in the rate of wound contraction and decreases
the total bacterial count of burn wound surface.
Present results suggest that aqueous garlic extract may be used to accelerate
the process of burn wound healing in the dog. However, it seems further
studies are required to clarify other possible mechanisms involved in
the would healing and to evaluate the effects of various doses of garlic
extract during different periods of time.