Antimicrobial and Wound Healing Potential of Canthium coromandelicum
Leaf Extract-A Preliminary Study
S. Chandra Mohan,
The objective of this study was to evaluate the wound healing potential of
the ethonolic extract of Canthium coromandelicum. The study was done
on male albino rats using excision model. The ethanolic extract, treated wounds
were found to epithelize faster and the rate of wound contraction was significantly
increased as compared to control and standard wounds (p<0.001and p<0.01).
From the results, the ethanolic extract of Canthium coromandelicum had
greater wound healing activity than the standard ointment Cipladine. The enhanced
wound healing activity of alcoholic extract may be due to free radical scavenging
action and the antibacterial property of the phyto constituents (viz., tannins
and flavonoids) present in it which either due to their individual or additive
effect fastens the process of wound healing. Presence of flavonoids and tannins
in alcohol extract was also confirmed by preliminary phytochemical investigation.
to cite this article:
S. Chandra Mohan, K. Sasikala and T. Anand, 2014. Antimicrobial and Wound Healing Potential of Canthium coromandelicum
Leaf Extract-A Preliminary Study. Research Journal of Phytochemistry, 8: 35-41.
Received: November 10, 2013;
Accepted: December 22, 2013;
Published: March 29, 2014
Our skin acts as a protective layer from surroundings. The opening or break
of skin leads to wounds. Wound healing is complex process that proceeds in three
major path viz. inflammation, cellular proliferation and remolding (Glynn,
1981; Clark, 1996). Healing needs the combined efforts
of different tissues and cell lineages (Martin, 1997).
Wound healing involves blood clotting, fibrin formation and inflammation. Healing
is not complete until the disrupted surfaces are firmly knit by collagen (Buffoni
et al., 1993). The basic aim of wound healing is to reduce tissue
damage and proper environment to restore the affected wound area (Pierce
and Mustoe, 1995). The mechanism of wound repairing means reconstruction
of damaged tissues (Phillips et al., 1991). Wound
repairing drugs are still limited in drug industry (Udupa
et al., 1995). However, the major problem of management of wound
healing is the cost effect of therapy and its side effects. (Suh
et al., 1998). The Reactive Oxygen Species (ROS) are deleterious
to wound healing process due to the harmful effects on cells and tissues. Absorbable
synthetic biomaterials are considered to be degraded via ROS (Aliyev
et al., 2004). Inflammation results in a coordinated influx of neutrophils
at the wound site. Wound related non-phagocytic cells also generate free radicals
by involving non-phagocytic NAD(P)H oxidase mechanism (Griendling
et al., 2000). Thus, the wound site is rich in free radicals and
the presence of free radicals will result in oxidative stress leading to lipid
peroxidation, DNA breakage. Evidence for the role of oxidants in the pathogenesis
of many diseases suggests that antioxidants may be of therapeutic use in these
conditions. Topical applications of compounds with free-radical-scavenging properties
in have shown to improve significantly wound healing and protect tissues from
oxidative damage (Thiem and Grosslinka, 2004). Medicinal
preparations of plant origin are widely used for the treatment of various diseases
of skin. This treatment is especially effective in the case of chronic disorders,
since phytotherapy can be carried out over a long period of time without risk
of inducing side effects. Most part of the phyto components are non toxic and
provide highly effective treatment of many diseases. Many ayurvedic herbal plants
have a important role in the process of wound healing. Plants heal the wounds
in natural way. The healing process can be monitored by evaluating the rate
of contract of the wound. In this present study, Canthium coromandelicum
leaves were evaluated for wound healing activity.
MATERIALS AND METHODS
Plant materials: The plants were authenticated by Dr. S. Singaravadivel,
Senior Scientist, Head of the Department, Department of Microbilogy, Indian
Institute of Crop Processing Technology, Thanjavur, Tamil Nadu, India. The plant
Canthium coromandelicum leaves were dried and extracted with ethanol
using soxhlet apparatus and prepared as paste.
Phytochemical analysis: Chemical tests were carried out on the aqueous
extract and on the powdered specimens using standard procedures to identify
the constituents as described by Mohan et al. (2012a).
Microorganisms: Staphylococcus aureus (Gram positive), Escherichia
coli (Gram negative) and Bacillus subtilis (Gram positive) and Candida
albicans were the microorganisms used and they were obtained from the Microbiology
Laboratory of the Thanjavur Medical College Hospital, Thanjavur. These microorganisms
were identified and confirmed by Microbiologists, Department of Microbiology,
Thanjavur Medical College, Thanjavur.
Antimicrobial assay: Antibiogram was done by disc diffusion method (NCCLS,
1993; Awoyinka et al., 2007) using plant
extracts. Petri plates were prepared by pouring 30 mL of NA/PDA medium for bacteria/fungi.
The test organism was inoculated on solidified agar plate with the help of micropipette
and spread and allowed to dry for 10 min. The surfaces of media were inoculated
with bacteria/fungi from a broth culture. A sterile cotton swab is dipped into
a standardized bacterial/fungi test suspension and used to evenly inoculate
the entire surface of the Nutrient agar/PDA plate. Briefly, inoculums containing
Staphylococcus aureus, Escherichia coli and Bacillus subtilis
were spread on Nutrient agar plates for bacteria and Candida albicans
was spread on potato dextrose agar for fungus strains. Using sterile forceps,
the sterile filter papers (6 mm diameter) containing the crude extracts (50,
100 and 150 μL) were laid down on the surface of inoculated agar plate.
The plates were incubated at 37°C for 24 h for the bacteria and at room
temperature (30±1°C) for 24-48 h for yeasts strains. Each sample
was tested in triplicate.
Measurement of zone of inhibition: The antimicrobial potential of test
compounds were determined on the basis of mean diameter of zone of inhibition
around the disc in millimeters. The zones of inhibition of the tested microorganisms
by the extracts were measured using a millimeter scale.
Animals: Male albino rats of Wistar strain approximately weighing 180-200
g were used in this study. Male albino rats were purchased from Indian Institute
of Science, Bangalore. The animals were housed in spacious polypropylene cages
bedded with rice husk. The animal room was well ventilated and maintained under
standard experimental conditions (Temperature 27±2°C and 12 h light/dark
cycle) throughout the experimental period. All the animals were fed with standard
pellet diet and water were provided ad libitum. Before one week to the
experiment, animals were accustomed to new climate and surroundings. The animal
feed composition is crude protein (22.3%), crude oil (4.01%), crude fiber (4.02%),
ash (8.02%) and sand silica (1.02%).
Method of ointment preparation: In this preparation 1 g of suitable
extract is mixed with 10 g of ointment base (10%). Then it is stirred well until
homogenous base is obtained.
Wound healing experiments: The wound site was prepared following the
excision wound model (Opara, 1999). The animals were
anaesthetized with diethyl ether and the hairs on the skin of the back, shaved
with sterilized razor blades. The dorsal fur was shaved and a full thickness
of the excision wound of 1.5 cm in width and 0.2 cm depth was done. After complete
wounding, wound was washed and cleaned out with normal saline solution and the
medicated cream was applied.
Experimental protocol: Animals bearing partial thickness wound were
distributed into various groups such as Group I served as control, Group II
served as extract and Group III served as standard treated groups. Each group
had six animals. Standard drug povidone iodine (Commercially called as CIPLADINE)
was selected as standard drug for the comparison of wound healing actions in
Wound treatment: The wounds of the animals were treated topically using
the extracts. Group 2 was treated with the plant extract of Canthium coromandelicum.
Group 3 (positive group) was treated with the standard drug purchased from medical
store. It is a broad spectrum antibiotic used in the treatment and prevention
of local infections of the teat, hoof and skin diseases in animals. While group
I which was serve as the negative control group was left untreated.
Wound measurement: The measurement of the wound areas was taken from
the day of the excision of the wound and every two or three days interval until
the epithelization of the wound was completed. The area of the wound contraction
was measured in different treated and control group on 3rd, 6th ,9th and 12th
day. Wound contraction which contributes to wound closure was studied by tracing
of the site. A meter ruler was placed over the wound and the estimate taken
to produce a scale calculation. The percentage of wound closure was calculated
as follows using the initial and final area drawn on glass slides during the
experiments (Wall et al., 2002):
Where n is a No. of days (3rd, 6th ,9th and 12th day).
RESULTS AND DISCUSSION
In the present study the phytochemical screening (Table 1),
antimicrobial activity and the rate of wound contraction by excision wound model
was studied. The diameter sizes in mm of the zone of inhibition are shown in
the Table 2. The area of wound healing expressed in percentages
given in the Table 3.
||Phytochemical screening of Canthium coromandelicum
||Antimicrobial activity of Canthium coromandelicum extract
|Values were expressed as Mean±SD
||Percentage of wound healing activity of Canthium coromandelicum
|Values were expressed as Mean±SD for six rats in each
group, *Significantly different from control (p<0.001). #
Significantly different from standard (p<0.01)
||Percentage of wound healing activity of Canthium coromandelicum
The graphical representation of area of wound healing present in Fig.
1 and the photographical representation in Fig. 2(a-f).
The percentage of wound contraction includes by recording the changes in wound
area at fixed intervals of time, viz., 3rd, 6th ,9th and 12th day after treated
with extract. However, on 12th post wounding day, Group I animal
showed 78.07% of healing which may be due to self immunity of animal whereas
the standard treated group (Group II) showed 98.97% healing and the extract
treated group (Group III) showed 90.46% healing. When obtained result compared
with standard and control, the activity of the extract was found to be significant
(p<0.01; p<0.001). Wound healing normally involves an initial inflammatory
phase followed by fibroblast proliferation, formation of collagen fibers and
shrinking, occurring concurrently but independent of one another. Several plants
are having wound healing potential. Phytoconstituents such as flavonoids, glycosides
and tannins are reported to have wound healing property (Raina
et al., 2008; Venkatanarayana et al.,
2010). Wound healing effect is also attributed to free radical scavenging
activity of flavonoids.
||Wound healing activity of Canthium coromandelicum,
(a) Control-0th day (b) Control-12th day, (c) Plant extract-0th day, (d)
Plant extract treated rat-12th day, (e) Standard drug 0th day and (f) Standard
drug treated rat 12th day
Lipid peroxidation refers to the oxidative degradation of lipids, the final
products of lipid peroxidation may produce cancer (Mohan
et al., 2012b). The process of lipid peroxidation is important in
burns, wounds and skin ulcers. It is believed that the increase in strength
of collagen fibers by any drug that stops lipid peroxidation. Flavonoids, glycosides
and tannins are known to promote wound healing process mainly by their astringent
and antimicrobial property (Vinothapooshan and Sundar,
2010; Venkatanarayana et al., 2010).
Preliminary phytochemical analysis of the leaves of Canthium coromandelicum
revealed presence of flavonoids, tannins, phenolic compounds and glycosides.
Presence of flavonoids and tannins in extracts of leaves of Canthium coromandelicum
may be responsible for its wound healing activity. The present study demonstrated
that Canthium coromandelicum leaves extract was capable of promoting
wound healing activity. Enhanced wound contractions suggest that Canthium
coromandelicum leaves extract has potential in the management of wound healing
when compared to standard.
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