evaluation in rat using incision wound model

International Journal of Pharmacy and Integrated Life Sciences “Where improvisation meets innovation”

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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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of


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

Vol: 1(6) May 2013

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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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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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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

Vol: 1(6) May 2013

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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

87

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Kannadhasan et. Al. , 7.

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