Screening
For Wound Healing Activity of Alcoholic Extract of Ocimum kilimandscharicum Leaves
Sachin R Patil*1,Swati
Patil2, M B Patil1, Ravi Kumar1 and
Mahesh S Paschapur1
1K.L.E.S’s
College of Pharmacy, Ankola-581314, Karnataka, India.
2Principal KM Kundnani College of Pharmacy,
Cuffe Parade, Mumbai
ABSTRACT
Objective: To investigate
and rationalize the effects of Ocimum
kilimandscharicum leaves (Lamiaceae)
for their wound healing activity and probable mechanism underlying its
protective effects in albino wistar rats.
Methods: The wound healing activity was evaluated using three
different types of wound models viz; the excision, incision and dead space
wound model. A supportive study was also made on granuloma tissue to
estimate the level of hydroxy
proline. The healing tissues obtained on the 11th post wounding day
of the incision wound model were processed for histological study.
Results: The animals receiving the alcoholic extract of Ocimum kilimandscharicum leaves has
shown to possess significant increase in wound closure rate, skin breaking
strength, granuloma breaking strength, hydroxy proline content, granuloma dry
weight and decrease in mean scar area.
Conclusion: The prohealing potential of the Ocimum kilimandscharicum leaves extract may probably be as a result
of the presence of a mixture of phytoconstituents including flavonoids,
steroids, etc. and increased collagen deposition as well as better alignment
and maturation.
KEY
WORDS: Ocimum kilimandscharicum, alcoholic extract, wound healing, hydroxy
proline, phytochemicals.
INTRODUCTION
A wound is
disruptions of tissue integrity that results in damage and is typically
associated with lose of function. Wound healing can be defined as a complex
dynamic process that results in the restoration of anatomic continuity and
function. It is a finely orchestrated and overlapping sequence of events involving;
control of infection, resolution inflammation, angiogenesis and generation
functional connective matrix, contraction, resurfacing, differentiation and
remodeling1.
Injury of the skin induces repair mechanism
that restores its functions in protecting the individual against environmental
factors that might be harmful. Three different phases constitute the
physiologic process of wound-healing; (i) substrate phase, (ii) proliferative
phase and (iii) remodeling phase2-3. All these steps are
orchestrated in controlled manner by a variety of cytokines including growth
factor. Some of these growth factors like platelet derived growth factor (PDGF),
transforming growth factor β (TFG- β), fibroblast growth factor (FGF)
and epidermal growth factor (EGF) etc. have been identified in self healing
wounds4-5.
Table 1: Effect of alcoholic extract of Ocimum kilimandscharicum on healing of Excision wound
|
Wound Model |
Day |
Control |
Alc 200# |
Alc 400^ |
|
Wound closure (days) |
|
22.27±0.92 |
19.10±0.33** |
17.83±0.44*** |
|
Mean scar area(mm2) |
|
39.57±0.61 |
28.69±0.64*** |
25.69±0.66*** |
|
%wound contraction by day |
4th |
9.698±0.44 |
14.42±0.35*** |
19.19±0.41*** |
|
8th |
29.84±0.42 |
41.18±0.64*** |
51.43±0.84*** |
|
|
12th |
50.91±0.80 |
70.47±0.48*** |
81.49±0.81*** |
|
|
16th |
73.50±1.0 |
84.26±1.50*** |
97.70±0.71*** |
|
|
20th |
92.44±0.77 |
--- |
--- |
Alc 200#: Alcoholic extract
200mg/kg b.w., Alc 400^: Alcoholic extract 400mg/kg b.w.
(Values are
Mean ± SEM from 6 animals in each group), Data analyzed by One-way ANOVA
followed Dunnett’s test, P values: ***P<0.0001 considered as
significant when compared to control.
Table 2: Effect of alcoholic extract of Ocimum kilimandscharicum on healing of incision wound
|
Group(N) |
Breaking Strength (g) |
|
Control |
282.7±4.85 |
|
Alc 200# |
469.4±4.48*** |
|
Alc 400^ |
503.5±3.02*** |
Alc 200#: Alcoholic extract
200mg/kg b.w., Alc 400^: Alcoholic extract 400mg/kg b.w. (Values are Mean ± SEM from 6 animals in each
group), Data analyzed by One-way ANOVA followed Dunnett’s test, P
values: ***P<0.0001 considered as significant when compared to control.
Ocimum kilimandscharicum (Lamiaceae) is an aromatic undershrub with pubescent
quadrangular branchlets; found throughout India and also cultivated. The leaves
are acrid, thermogenic, aromatic, antibacterial, insecticidal, antiviral and
deodorant.
Scientifically, Ocimum kilimandscharicum has been
documented to possess insect repellant property6. It is employed as
indigenous medicine for a variety of ailments like cough, bronchitis, viral
infections, foul ulcers, anorexia and wounds7-8.
Keeping this in
view, wound-healing potential of alcoholic extract of leaves of Ocimum kilimandscharicum has been
investigated on different parameters of wound healing in wistar rats.
MATERIALS AND METHODS:
Plant Material:
The fresh leaves
of Ocimum kilimandscharicum were
collected in the month of July 2008 and authenticated from Dr. Bhandary,
Professor of Botany, Government Arts and Science College, Karwar, Karnataka.
Preparation of Extract:
Around 1kg of
leaves were separated and dried under shade for about a week and extracted by
hot percolation method using soxhlet apparatus. The shade-dried leaves were
pulverized to reduce to 60 mesh, powder then subjected to Soxhlet extraction
with 95% alcohol, the alcoholic extract was concentrated in a rotary evaporator
and dried in vacuum desiccator using sodium sulphite. The dried alcoholic
extract was suspended in distill water using 1% Tween 80 and then used for pharmacological
screening.
Preliminary
phytochemical screening of the extract gave positive results for proteins,
carbohydrates, volatile oils,
fatty acids,
flavonoids, tannins, saponins, sterols and triterpenoids.
Experimental Animals:
Swiss Albino Mice
(25-30g) and Wister Albino Rats (180-210g) of either sex were used in the
study. These were procured from Venkateshwara Enterprises, Bangalore. They were
maintained under standard husbandry conditions. The animals were given
standardized laboratory feed and water ad
libitum.
Acute Toxicity Studies:
Swiss albino mice
of either sex (18-22g b.w.) were used for acute oral toxicity study. The study
was carried out as per the guidelines set by OECD and up to 2000mg/kg b.w. of
extract there were no signs and symptoms seen when the animals monitored for
48h. Based on the results obtained from this study, the dose for wound healing
activity was fixed to be 200mg/kg b.w. and 400mg/kg b.w. for dose dependent
study.
Animals were
divided into three groups (n=6). They were starved for 12h prior to initiate
the experiment. Group I served as control, which received 1% Tween 80. Group II and III served as test, received
alcoholic extract at dose of 200mg/kg b.w. and 400mg/kg b.w. respectively.
.
Excision Wound Model:
Animals were
anaesthetized with ether and shaved on the part to be exposed. A circular wound
of 2.5cm was made on depilated dorsal thoracic region of rat. The oral dose was
given once a day to the animals. The observations were made for time period of
complete epithelization, mean scar area and also the percentage wound
contraction was made on 4th, 8th, 12th, 16th and 20th post wounding day9.
Incision Wound Model:
The Ehrlich and
Hunt method was adopted10. The animals were anaesthetized with ether
and shaved and wounded two para vertebral incision of 6mm were made through the
entire thickness of the skin, on the either side of the vertebral column with
the help of a sharp blade. The incision were sutured using silk thread, sutures
were removed on 8th post wounding day and tensile strength was determined on
10th post wounding day by adopting continuous constant water flow technique.
Dead Space Wound Model:
Dead space wound
was made by implantation of polypropylene tube (0.5cmX2.5cm), beneath the
dorsal para vertebral skin. On the 10th day the granuloma tissue
form on the dead space wound was dissected and tensile strength was determined.
The excess tissue was cut into two approximately equal halves. One of the
granuloma tissues was dried in an oven at 60° C and the dry weight was noted.
Acid hydrolyze of the dried tissue was used for determination of hydroxy
proline. Other part of the tissue was kept in 10% formalin solution for
histopathological studies to evaluate the effect of the extract on collagen formation11.
Histopathological
Study:
A section of
granuloma tissue from all three
group animals of the incision wound model was processed for histological study. The
amount of collagen was quantified using Vangeison stain.
Fig 1: Histological section of granulation tissue of the
control group showing more macrophages (a) and less collagen (b) deposition.
Fig 2: Histological section of granulation tissue of the Alc
200 treated group showing moderate collagen deposition (a).
Statistical
Analysis:
Results were
expressed as Mean ± SEM. The difference between experimental groups was
compared by One-way Analysis of Variance (ANOVA) followed by Dunnett’s test.
The results were considered statistically significant when P<0.0001.
RESULTS:
In the present
investigation, preliminary phytochemical analysis of alcoholic extract revealed
the presence of flavonoids, tannins, saponins, sterols, carbohydrates, proteins
and triterpenoids.
In all the models
studied, significantly improved wound-healing activity has been observed with
the Ocimum kilimandscharicum leaves extract at both the doses, compared to
that of the control group of animals. The results of excision wound model are
shown in Table No.1. The animals treated with alcoholic extract
showed a significant decrease in epithelization period as evidenced by shorter
period for fall of escher as compared to control. The drug extract also
facilitated the rate of wound contraction significantly at both the dose levels
(200mg/kg b.w. and 400mg/kg b.w.).
The result of
incision wound model is shown in Table No.2. The animals treated with alcoholic
extract showed significant increase in tensile strength on 10th post
wounding day at both the doses as compared to control.
The drug treated animals of dead space wound model showed
significant increase in dry granuloma weight, granuloma breaking strength and
in the level of hydroxyproline content (Table No.3) at both the dose levels.
Histopathological study revealed that there is increase in collagen and very
few macrophages deposition in the drug treated group (Fig. 2, 3) compared to
control (Fig.1).
Fig 3: Histological section of granulation tissue of the Alc
400 treated group showing more collagen (a) and less macrophages (b)
deposition.
DISCUSSION:
Wound healing
involves various phases. Initially involves acute inflammatory phase followed
by the synthesis of collagen and other extra cellular macromolecules, which are
later removed to form a scar12. Drugs, which influence one phase,
may not necessarily influence another. Hence, different models have been used
in our study to assess the effect of various phases, which run concurrently,
but independent of each other.
The biological
response regulating the body’s own cellular defense mechanisms contributes to
the wound and its repair. It has been observed that wound healing effects may
result due to collagen I expression up-regulation13 along with
increased tensile strength of the tissue14. Since collagen is
principal component of connective tissue, which provides a structural
frame-work, strength and milieu for tissue regeneration, it plays a major role
in wound healing process15. It has also been documented that
oxidative stress plays an important role in the pathophysiology of several
diseases including wound healing.
Many plant
extracts and medicinal herbs have shown potent antioxidant activity. In this
present study, phytochemical investigation revealed that ethanolic extract
contains high amount of tannins, saponins, flavonoids, sterols and other major
phytochemicals, which may be responsible for the antioxidant activity.
Table 3: Effect of alcoholic extract of Ocimum
kilimandscharicum on healing of dead space wound
|
Groups |
Breaking Strength (g) |
Granulation tissue Dry
Weight (mg/100g) |
L-Hydroxy
Proline (µg/100g) |
|
Control |
227.9±2.30 |
39.09±0.80 |
755.4±73.06 |
|
Alc 200# |
281.4±3.20*** |
48.24±0.93*** |
1828±96.56*** |
|
Alc 400^ |
309.7±2.99*** |
54.53±1.06*** |
2239±118.2*** |
Alc 200#: Alcoholic extract 200mg/kg
b.w., Alc 400^: Alcoholic extract 400mg/kg b.w.
(Values are
Mean ± SEM from 6 animals in each group), Data analyzed by One-way ANOVA
followed Dunnett’s test, P values:
***P<0.0001 considered as significant when compared to control.
The wound healing
potential of the Ocimum kilimandscharicum
leaves extract may probably be as a result of the presence of a mixture of this
phytoconstituents16-17. Thus from this study it is concluded that
the Ocimum kilimandscharicum leaves
extract has a
reproducible
wound healing potential and thereby justifies its use in folklore medicine in
India.
CONCLUSION:
Thus, it can be
concluded that the ethanolic extract of Ocimum
kilimandscharicum leaves possess
potent wound healing activities. The inhibitory activity of the extract
justified the use of the plant as a non-specific wound healing activity in folk
medicine. Further detailed investigations needs to be underway to determine the
exact phytoconstituents, which are responsible for the wound healing activity.
ACKNOWLEDGMENT:
The authors are thankful to Dr. M.B.Patil, Principal, K.L.E.S’s
College of Pharmacy, Ankola, Uttar Kannada district, Karnataka, India. for
providing all the facilities and support to carry out the research work.
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Received on 30.08.2009
Accepted on 10.09.2009
© A&V Publication all right reserved
Research J. Pharmacology and
Pharmacodynamics 2009; 1(2): 93-96