Wound
Healing and Anti-Bacterial Effects of Cassia auriculata Extract
Mathew George* and Lincy Joseph
Jaipur National University, Jaipur
ABSTRACT:
AIM: objective
of the study was to find out the wound healing
and antibacterial effects of Cassia auriculata extract in rats.
METHOD: For wound healing studies Hot
water burn wounds and Wax burn wound
Methods: used. For anti-bacterial studies cup-plate method used.
RESULTS: Indicated
that the extracts of Cassia auriculata possessed wound healing and antibacterial activities.
KEY
WORDS: Wound healing,
Anti-bacterial activity, Cassia
auriculata extract ,burn, Cup-plate.
INTRODUCTION:
Renewed interest
on biological activities
of medicinal plants
emerged in early 1980’s
as the Council
of Scientific and
Industrial research have published
the information on the screening
of biological activities of many
medicinal plants using
experimental models1.
Medicinal plants are an important therapeutic aid for various ailments.
Scientific experiments on the antimicrobial properties of plant components were
first documented in the late 19th century2. Recently the use of
herbal preparations in remedies
for various medical
conditions have been
rapidly increasing
especially in India. In India, from ancient times,
different parts of medicinal plants have been used to cure specific ailments.
Today, there is widespread interest in drugs derived from plants. This interest
primarily stems from the belief that green medicine is safe and dependable,
compared with costly synthetic drugs that have adverse effects.
A
pathogen that can cause life – threatening infections patients with
burns and wounds. The extracts obtained
from plants are usually made in to different formulation, ether as ointment or
as lotion applied to the skin for wound3. During the past decade
anaerobic bacteria especially non – poring ones, have found as important
causative organisms of wound infection. A chemotherapeutic agent may act by
destroying the organism (bactericidal) or by inhibiting its growth
(bacteriostatic) .
Cassia
auriculata (family- Leguminaceae.) is used as
anti-oxidant and analgesic drug . Natural antimicrobials can be derived from
plants, animal tissues, or microorganisms. In this present study, Cassia auriculata extract
were screened for wound healing and antibacterial activity in rats and
micro organism respectively.
MATERIALS AND METHODS:
Animals: (For wound healing activity)4
Healthy male wistar rats weighing between
150-200 g were used in the studies.
They were individually
housed and maintained on normal diet and
water ad libitum. Animals
were randomly distributed
into various groups
each containing around 10
animals. Burn wounds were
inflicted on overnight starved animals
under pentobarbitone (25
mg/kg) anesthesia. Test extract
given orally at a dose of 300 mg
/kg . Apart from the
drugs under investigation, no local/systemic chemotherapeutic covers
were provided to
animals. Animal showing
signs of infection
was excluded from
the study and
replaced with a
fresh animal.
Organisms (For anti-bacterial activity)5:
The antibacterial activity of Cassia
auriculata extract were
studied by cup-plate method with ciprofloxacin as reference standard using two
gram positive organisms- Staphylococcus
aureus and Bacillus subtilis and two gram negative organisms namely Escherichia coli and Pseudomonas auruginosa.
Plant material: Cassia auriculata plant
was collected freshly
from in and around
Erode district of Tamilnadu,
India. Leaves and flowers of plant dried under shade, made into coarse powder
by grinding. Plant was identified and authenticated
at the herbarium Tamilnadu Agricultural University, Coimbatore. Ref : ID 08/NCP/ 2006.
Preparation of plant
extract:
Aqueous Extract:
To 20 g of
each dried plant
powder form , 500 ml water were added and
contents of flask were
mixed thoroughly by gentle
shaking. Flasks were kept for
four days with frequent shaking. After
the completion of
maceration process the
filtrates were obtained and
water evaporated to
get the dried
extract.(evaporation by keeping flasks in electric mantle at 80 0 C).The
residual extract was dissolved in water and
used in the studies.
Alcoholic (ethanol) extract:
To 20 g of
each dried plant
powder form , 500 ml ethanol were added
and contents of
flask were mixed thoroughly
by gentle shaking. Flasks were
kept for four days with frequent shaking. After the
completion of maceration
process the filtrates
were obtained and
solvent evaporated to
get the dried
extract.(evaporation by keeping flasks in electric mantle at 80 0 C.
GROUPS AND TREATMENT:
For Wound healing
Burn wound models:
Method of infliction
of Burn:
a)Hot water burn wounds6:
The
methods of Farrial
et al (1994) was
modified to produce
the hot water burn wound. On
day ‘0’ the animals
were anaesthetized and
given a fur
clipping on dorsal
side. A 2 X 3 cm.
glass cylinder was
placed on the
shaven back of
a rat. Hot water ( 2 ml)
at 980 C was
placed in the
glass cylinder. Thirty
seconds later the
water was quickly
drained off and
the exposed area
was wiped off
the water to
see the whitish
marked area.
b)Wax burn wound4:
On zero day, under anesthesia, the dorsum of each
rat was shaved. A 2 X 2 c.m Metal cylinder was placed on the
shaven back of the animals. To this was
poured melted wax
at 800 C and
the wax was
allowed to solidify
into the metal cylinder. Eight minutes after
this (during this time
wax solidified completely), the metal
cylinder containing solidified
wax adhering to the
layers of skin
was gently removed
to inflict a
distinctly demarked burn wound.
Assesment of burn wound healing:
The animals were
inspected daily and
the healing was
assessed based on
physical parameters, namely,
wound contraction and
epithelization, as well as
histologically.
a. Wound contraction: was studied
by tracing the
raw wound area
on a transparent
polythene paper on
every alternate day upto
14th post wounding
day. These wound tracing were retraced on a graph
paper to assess the area.
The wound concentration was calculated as
percentage of original
wound size(300 mm2) for each
animal of a
group. From this
group mean on predetermined
days, viz., 2nd, 6th,10th and 14th day
was calculated for
final analysis of
the results.
b. Epethelization: Falling of eschar
leaving no raw
area was considered
as end point
of complete re epithelization and
the days required
for this was
taken as a
period of epithelization.
c. Histopathology7: on day 0,2 and 10
some of the animals
under each model
were sacrificed and
the wounds excised
together with the
surrounding skin. They were fixed in formalin and embedded in paraffin.
Histological evaluation was performed on the haematoxylin and eosin (HE) stained
paraffin section.
For Antibacterial activity:
The antibacterial activity of extract was studied by
cup – plate method8.
MATERIALS AND METHODS:
1.
Medium
have been prepared as described in Indian pharmacopoeia.
2.
Sterilized Petri dishes, pipettes, boiling tubes and beakers.
3.
8 to
24hrs. old growth cultures in nutrient broth
4.
Sterilized
test tubes
5.
Sterile
6mm cork borer.
6.
Sterile
inoculation loops
7.
Sterilized
fine pointed forceps
8.
Nutrient
agar
9.
Tuberculin
syringes.
Preparation of media:
Media mentioned in Indian pharmacopoeia was prepared by
dissolving bacteriological peptone (6g), pancreatic digest of casein (4g),
yeast extract (3g), beef extract (1.5g), dextrose (1.0g) and agar (15.0g) in
distilled water to produce one liter of medium.
The pH of the solution was adjusted to 6.5-6.6 by using
1M sodium hydroxide and 1m hydrochloric acid. Then it was sterilized for 30
minutes at 15lbs pressure.
The organisms used in the present study for evaluating
antibacterial activity of test compounds were obtained from laboratory stock.
On the day of testing, the organisms were sub-cultured into sterile nutrient
broth. After incubating the same for three hours, the growth thus obtained was
used as inoculums for the test.
Sterilization of media and
Glass wares:
The media used in present study,
nutrient agar and nutrient broth, were sterilized in conical flasks of suitable
capacity by autoclaving at 15 lbs pressure for about 20 minutes. The cork
borer, Petri dishes, test tubes and pipettes were sterilized in hot air oven at
160°c for an hour.
Preparation of solutions of
test compounds:
10mg of each test compound was
dissolved in 10ml of DMF (dimethyl formamide) in serially and suitably labeled
sterile test tubes, thus giving a final concentration of 100µg/0.1ml
Method of testing:
Cup-plate method:
This method depends on the diffusion of an antibiotic
from a cavity through the solidified agar layer in a Petri dish to an extent
such that growth of the added micro-organism is prevented entirely in a
circular area or zone around the cavity containing a solution of antibiotic.
A previously liquefied medium was inoculated
appropriate to the assay with the requisite quantity of the suspension of
micro-organisms between 40-50°c and the inoculated medium was poured in to
Petri dishes to give a depth of 3 to 4m.m .Care had been taken to see that the
layers of the medium were uniform in thickness by placing the Petri dishes on a
leveled surface.
The dishes thus prepared were stored in a manner so as
to ensure that no significant growth or death of the test organism occurs
before the dishes were used and the surface of the agar layer was dry at the
time of use. With the help of sterile
cork borer, three cups of diameter, each 6m.m were punched and the set agar in
each Petri dish was scooped out. Using sterile pipettes the standard and the sample
solutions (0.1ml) of known concentrations were fed into the bored cups. The
order of the solutions were as follows;
Cup-1: Standard (ciprofloxacin)
Cup-2: solvent control (DMF)
Cup3-: Test compound
The Petri dishes were left standing for one to four
hours at room temperature as a period of pre-incubation diffusion to minimize
the effects of variation in time among the applications of different solutions.
These were then incubated for 24 hrs at 37°c.
The zone of inhibition developed, if any was then
accurately measured and recorded. Each zone of inhibition recorded were average
of six measurements. Solvent control (DMF) was also tested for zone of
inhibition.
Index:
Concentration of
ciprofloxacin -10µg/0.1ml in DMF.
Concentration of
test compound-100µg/0.1ml in DMF.
Diameter of cup-6m.m
Quantity in each cup-0.1ml
RESULTS:
In wound healing studies:
The results of the study implies that extract C. auriculata accelerates significant healing
process . In control animals wound contraction was to the extent of 33%,
56%, 66%, and 87% by day 2, 6, 10 and 14 respectively. These animals took 14.25
± 0.45 days for reepithelization. Cassia
auriculata, administered orally shortened the period of epithelization
significantly (p<0.01) by 3 days. Besides, it also promoted the wound
contraction throughout (Table 1).
Histological examination performed on the ten-day old
wounds showed a steady and progressive wound healing in control animals (Table
2 and 3). The dermis proliferated almost to reach normal level. Eschar was
getting separated off leaving space for epidermis to grow and complete
reepithelzation. Moderate amount of collagen and numerous inflammatory cells
could be seen in corium. However wounds in cassia auriculata extract treated
animals showed signs of advanced healing such as complete restoration of
epidermis, well organized high amount of collagen in dermis, and absence of
inflammatory cells in fully grown dermis. A
reduction of lipid
peroxidase of wounds
may reduce the
further loss of
tissue in wound
area and may thus
promote healing.
In anti-bacterial studies:
Antibacterial
activities of C. auriculata extracts
possess significant antibacterial
activity was compared with
10 mcg of standard
drugs, ciprofloxacin against
the organisms like E. coli, P. aeruginosa , S. aureus and B. subtilis ( table 4).
DISCUSSIONS:
Wound healing
effect:
A pathogen that
can cause life – threatening infections in
patients with burns and wounds9.. The extracts obtained from plants are usually
made in to different formulation.. Various biological and metabolic alterations
occur in wound infections10. These include the degradation of
adenosine triphosphate, significant of polyunsaturated fatty acid in the red
cell membrane, elevation of the activity of serum enzyme and fall in the level
of vitamin. These changes have been
associated with the formation of the lipld peroxidation product namely malon
dialdehyde (MDA) as a consequence of the wound infection. More over, there is
experimental evidence documenting super oxide radical (O2) involvement in the
pathogenesis of wound11.
The selective toxic action on the infecting organism is
the key to beneficial actions of antibiotic. These drugs can hit at least is
targets in bacteria.
§ The cell wall
§ The cytoplasmic membrane
§ The Ribosome
§ The RNA molecules involved in transcription
of genetic information
Antimicrobial can bind to ribosome and may interfere
with peptide chain formation in bacteria or with the transcription mechanisms12
.The spread of drug resistant pathogens is one of the most serious threats to
successful treatment of microbial diseases. The ultimate goal is to offer
appropriate and efficient antimicrobial drugs to the patient .The use of plant
extracts and phyto chemicals, both with known antimicrobial properties, can be
of great significance intherapeutic treatments. These products are known by
their active substances, forexample, the flavanoids, tannins, phenolic
compounds.13
Anti bacterial effect:
No obvious
difference in susceptibility was found between gram-negative and gram-positive
bacteria. There was no inhibition of growth with the vehicle control (10% DMF).
Data express:
Plant extracts have great potential as antimicrobial
/anti bacterial compounds against microorganisms. Thus, they can be used in the
treatment of infectious diseases caused by resistant microbes.
These plant extracts were also compared with standard
antibiotic. Aqueous extracts showed less activity than ethanol extracts
possibly because i) the same active substances were present in water extracts,
but in low concentrations ii) active substances were soluble in organic
solvents and, therefore, not present in water extracts as also suggested by de
Boer et al14.The antibacterial action of the extracts is more
pronounced on Gram positive than on Gram negative bacteria, and these findings
correlate to the observations of previous screenings15,16
of medicinal plants for antibacterial activity.
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