WOUND HEALING ACTIVTY OF TRICHODESMA INDICUM LINN: IN- VIVO
EVALUATION IN RAT USING INCISION WOUND MODEL. K .KANNADHASAN. *
1.
International Journal of Pharmacy and Integrated Life Sciences “Where improvisation meets innovation”
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ISSN: 2320 - 0782
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WOUND HEALING ACTIVTY OF TRICHODESMA INDICUM LINN: IN- VIVO EVALUATION IN RAT USING INCISION WOUND MODEL K .KANNADHASAN*1, R.RADHA1 & N.JAYASHREE1 1
Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai - 600003.
ABSTRACT The herbal cream formulated with Trichodesma indicum Linn were tested for wound healing property on experimentally induced incision wound models in rats. Wistar rats has been divided in 4 groups each comprising of 6 rats .Group I served as control , Group II – Std ,Group III Pet. Ether, Group IV – Ethanol treated with herbal applied on topically. From the result obtained the Ethanol herbal cream
cream
treated with
incision wound models showed significant increase tensile strength was observed. Ethanol extract showed increase hydroxy proline content when compared to standard drug .Histological evaluation showed there were a marked in more collagen formation and more granulation tissue formation in ethanol herbal cream treated groups than control and reference groups. The result of the present study substantiate the traditional claims that the
use
of
herbal cream prepared
from plant possesses
significant wound healing
promoting activity and provides scientific evidence to the ethno- medicinal property of plants in the wound healing of wounds. KEYWORDS: Herbal cream, Wound healing, Incision wound model, Hydroxyproline, Tensile strength. Article received on: 31/03/2013
Article accepted on: 28/04/2013
Corresponding Author: K Kannadhasan Address Email
Vol: 1(6) May 2013
: Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai – 600003, Tamil Nadu, India. :
[email protected]
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Volume 1 – Issue 6
Kannadhasan et. Al. , INTRODUCTION Wounds and skin damage is a key feature
ovate pits on the membranous axis when
of both the dermal pharmaceutical and
falling. Traditionally it is used as an
skin care products industries, the only
emollient and Poultice2
difference between them is the level of
The Trichodesma indicum root
damage. A wound occurs when the
ethanolic extract
integrity of any tissue is compromised
treat
(e.g. skin breaks, muscle tears, burns, or a
(Parianayagam et al 2011).3The plant is
bone fractures). A wound may be caused
useful in vitiated conditions of Vata and
as a result of a fall, or a surgical
Kapha, diarrhoea, dysentery
procedure; or by an infectious disease; or
diseases (Perianyagam et al 2005).4
by an underlying condition. When healing takes place in a direction away normal course. It
world
population
traditional medicines
depends
upon
for various
skin
such
study 7
T.
Indicum
exhibited
anti-
In this study, we investigated the possible
formulation
prepared from Trichodesm indicum Linn
herbal
cream
using
Trichodesma indicum Linn .Test for its
Boraganiaceae.
wound healing
It is morphologically annually erect, much
property
on incision
wound rat models. The wound healing
branched and is 15-45cm in height. Leaves
activity of herbal cream was assessed from
obtuse
physical, biochemical and histological
or sub acute, clotted above with stiff
parameters. The literature survey revealed
are bluish-white or nearly
that less work is done on
white when fully ripe. Leaving five deep
Vol: 1(6) May 2013
of T.
inflammatory (Perianayagam et al., 2006),
herbal cream .The herbal cream has been
hair. Flowers
ester
(Srikanth et al., 2002).6 In our previous
investigation was
linear or lanceolate-oblong,
ethyl
suppressant activity in Swiss albino mice
that are
study the wound healing properties of a
are
acid
Indicum has shown significant cough
produced by allopathic treatments. The
leaves belongs to family
non-steroidal compounds
methanol extract of whole plant
1
aim of the present
and skin
from leaves (Hasan et al., 1982)5 . The
scientific community to
overcome the side effects
as
24-hexacosadienoic
plants for wound healing has received by
anti-pyretic
Hexacosane, Ethylhexacosanoate and 21,
diseases. Recently ,the traditional use of
attention
and
have been reported from the plant,
in non
healing ,under healing .More than 80% of the
analgesic
Some of the chemical constituents
from its
may result
have been reported to
leaves
Trichodesma indicum Linn.
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Volume 1 – Issue 6
Kannadhasan et. Al. , more activity .
selected for cream preparation.
MATERIAL AND METHODS COLLECTION
OF
This two extracts were
PLANT HERABL CREAM PREPARATION
MATERIAL:
8-
11
The Plant materials were collected from
The herbal cream was prepared by using
Kayatharu (village) in Tirunelveli district,
following procedure:
in Tamilnadu. Leaves were dried in
1. White bees wax, Cetostearyl alcohol,
sunshade for a week. The plant was identified
and
authenticated
PEG 4000 were melted together in a
by
separate beaker and then add Tween
V.Chelladurai, Research officer-Botany
20, maintain the temperature at 75°C.
(Scientist-C), Central Council for Research
2. Required amount
in Ayurveda & Siddha. Govt.of India
dissolved in water phase in a separate
to get powder using mechanical grinder. sieved
powder
was
used
pet.ether or
ethanol extract (5%) was weighed &
(Rtd). The shade dried leaves were milled
The
of
beaker, to which add the glycerol and
for
methyl
evaluation and extraction purpose.
paraben,
maintain
the
temperature at 75°C. 3. When both the phase reached the
PREPARATION AND SELECTION
temperature, the aqueous phase was
OF EXTRACT:
added gradually drop by drop into oil
The fresh leaves of Trichodesma indicum Linn were used
phase with continuous stirring, to
for preparation of the
which lavender oil was added while it
cream .The plant material were shade dried
is hot.
and passed through 40 mesh sieves and
4. Kept it in a room temperature.
stored in closed vessels for further use. EXPERIMENTAL ANIMALS:
The dried powder leaves (200g) were extracted with Pet ether, ethyl acetate and Ethanol by using soxhlet method then cold
Wistar rats either sex (175-200g) were
maceration by using water. The yield of
used for the study. They were maintained
the extracts respectively 16%, 14%, 16%,
at the Experimental Animals laboratory,
& 14.5. Extracts were evaluated for
Madras Medical College, Chennai. They
antimicrobial activity by in vitro-method.
were individually housed. Maintained in
Based on the antimicrobial activity,the
clean polypropylene cages and fed with
ethanol and pet.ether extracts possessed
Commercially pellet diet
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and water ad
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Kannadhasan et. Al. ,
were removed on 8th post
libitum .Clearance from the Institutional
sutures
Animal Ethical committee was obtained
wound day and continued the applied of
for carrying
the cream .Animals were sacrificed on
out the
experiment (
3/243)CPSEA)
the 10th day. The portion of
EXPERIMENTAL DESIGN:
where the wound was created was
The Animals divided in 4 Groups and 6
removed after sacrificing the animal
animals in each group.
.One portion of the skin is to be used
GROUP I GROUP
the skin
- Cream base
for evaluation of tensile strength and
- Standard
the other portion will be taken for
II
(5% povidone iodine ointment)
Histo-pathological
GROUP III
Hydroxyl proline estimation.
- Test (5%
Evolution
and
herbal cream) (Pet.ether extract) DETERMINATION GROUP IV
OF
TENSILE
STRENGTH: 13
- Test (5%
herbal cream) (Ethanol extract) PROCEDURE: INCISION WOUND MODEL: 12
The
device for measuring wound
breaking strength was assembled in
PROCEDURE:
our
laboratory. The main component of the The rats were anaesthetized and
during
1ml
of
creation of
Intravenous
prior
to
device is a stand
which transfers force from the sample
ketamine
to a fsg15n1a (fig.1) .As the sensor computer
of the
intelligent
animals was shaved with cliper.
A
a moving arm
wound, with
hydrochloride (10mg/kg). The dorsal fur
electric
with
an
longitudinal
interface
we
module
Advantech co.,
used
Adam
an
4011 (
Cincinnati, oh). To
paravertebral incision of 6 cm long was
achieve
made through the skin and cuetaneous
used a servomechanism with a power
tissue
on
supply ± 3v and
After
the incision, the
the
sutured
0.5cm
surgical
threads
needle
back
as
described.
parted skin was
apart.
Using
a
the
range
wound was left
piezoelectric
sensor
and
to the breaking point of the sample . Software
drug was
recording
Vol: 1(6) May 2013
the range of output
covering continual tensile force increase
undressed; the control, test and standard applied
tensile force , we
force from 0 to 30 N, compatible with
(NO.000) and curved
(NO.11). The
vertical
once daily. The
83
for
data and
processing ,
analysis
was
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Volume 1 – Issue 6
Kannadhasan et. Al. , implemented in mat lab .On 10th days after
surgery, six
groups ketamine
animals
in
4. After hydrolysis by autoclaving the solution was filtered to remove cell debris.
each
were sacrificed by examine hydrochloride
5. Required volume was pipette separate tube.
injection. And
the skin wounds prevent post mortem transformation
.the
sutures
6. To each tube 1ml of 0.01M CuSo4, 1ml 2. 5N NaOH & 1 ml 6% H2O2 solution was added.
were
removed and using a template the skin area with wound was adjusted to
to
an 7. Shake vigorously for every 5 minutes, on water bath at 80°C for 5 minutes. (Reason: Heating & Shaking will destroy excess of peroxide since traces of peroxide will decrease color formation.)
optimal 3 x 2 cm ( length of incision = 2cm ) strip to obtain uniform samples .The samples were placed between the two clamps of the tensiometer .testing force was applied perpendicular to the
8. Tubes were chilled in ice water bath.
direction of the incisions. The maximal
9. Then to the above solution 4 ml of H2SO4 was added with vigorous shaking.
tensile strength was registered for each sample. All
data registered on computer
10. p-dimethylamino benzaldehyde (1 ml) was added to above solution to obtain orange color which was measured at 555nm in UV spectrometer.
in each sample.
11. Same procedure was repeated for determination of absorbance of known concentration of hydroxyproline which was compared with the absorbance of hydrolyzed tissue hydroxyproline. HISTOPATHOLOGICAL STUDIES:
DETERMINATION OF HYDROXYL PROLINE CONTENT14.
16
PROCEDURE:
The granulated
harvested from
incision wound model were subjected for
1. At the end of the 10th day, collagen tissue of incision wound was removed.
the histological study .These sample were then separately fixed in 10% formalin
2. Removed tissue was dried at 108° C for 16 hours & weighed
, dehydrated through grated alcohol series ,cleared in xylene and embedded in
3. Collagen tissue was placed in volumetric flask and 6N H2SO4 was added and autoclaved for 3 hours at 50 pounds pressure after sealing. Vol: 1(6) May 2013
tissue
paraffin wax ( melting point 56°C) . Serial sections of 5µm were cut and stained with haematoxylin 84
and
eosin
(H&E).The
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Volume 1 – Issue 6
Kannadhasan et. Al. , sections
were
examined
under
light
microscope and photomicrographs were
Table.1. Tensile strength :
taken.
GROUPS
Tensile Strength(g/cm)
RESULTS AND DISCUSSION Control(Cream
Incision Wound Models:
90.55±0.0872
Base) Std
(Povidone 102.55±0.8751
Ointment) (Pet.Ether
Extract 112.41± 0.0881*
cream) Ethanol
Extract 155.35±0.1217*
Cream
n = 6 animals in each group. The treated groups are compared by Student t test with the standard group. *P < 0.001 200
Tensile strength
150 100
tensile strength
50 0
Fig.no.3. Chart of Tensile Strength .
Fig.no.2 Incision wound before suturing Incision wound after suturing
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Kannadhasan et. Al. , Table.2. Hydroxyproline content.
HISTOPATHOLOGICAL STUDIES:
Concentration of Hydroxyproline (μg / 100mg Collagen Tissue) 72.65±0.57
S.No
Groups
1
Control ( cream base)
2
Std (Povidone iodine ointment)
85.5 ± 0 .12
3
(5%Pet.Ether Extract Cream)
90.4 ±0.53*
4
(5% Ethanol extract cream)
112.65 ± 0.63*
A
n = 6 animals in each group. The treated groups are compared by Student t-test with the standard group. *P < 0.0001
hydroxy proline
B
150 100 50 0
hydroxy proline
Fig.no.4 Chart of Hydroxyproline content.
C
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Kannadhasan et. Al. ,
as a standard and 5% pet ether extract cream and control..
Histopathological studies :
and
eosin
In
haematoxylin
stained
sections, wound
from the 5% w/w ethanol extract cream of T.I group showed more Granulation D
Tissues formation and Collagenization
Fig.no5.A-control group, B- standard group, C-5% pet.ether extract cream, D5% ethanol extract cream.
(Fig. 5.(D)) which was similar to the
Discussion
(Fig.5.(B)) .when compared to control
Tensile
Strength Content
standard
(Fig.5.(A)).
of healed
wound
increase
15.35±0.1217) compared to
(povidone
Poor
collagenization
and
granulation tissue formation were obtained in rats treated
model
with
5% Pet.
Ether
extract of T.I (Fig.5.(C))
5% ethanol extract of TI treated animal shows
control
iodine ) 10 days after wound creation
skin Incision Wound Model: In incision
positive
in tensile strength ( respectively
CONCLUSIONS
when
The present studies revealed that
pet. ether, control (12.41±
0.0881),( 9.55±0.0872).The mean tensile
plant
sources
strength was also significant in animal
cream possess
treated with standard drug(10.55±0.8751
healing
)
Present
used
in
the
the herbal
significant wound
promoting
activity. The
finding provides scientific
evidence to the ethanomedicinal property Hydroxyl proline content in healed skin
of
incision wound model:
.However, it needs further evaluation in
plants in
the
healing of wound
clinical settings before consideration for Based on the reports it is concluded that the
the of wounds.
cream formulated with 5% ethanol
extracts showed a increase in hydroxy proline content
when compared to
povidone iodine ointment (5%) was used Vol: 1(6) May 2013
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Volume 1 – Issue 6
Kannadhasan et. Al. , 7.
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