Comparative evaluation of ethanolic extracts of Litsea monopetala and Schleichera oleosa bark for wound healing activity in rabbits
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Date
2023
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Birsa Agricultural University, Ranchi
Abstract
The present study was conducted in the Department of Veterinary Surgery &
Radiology, College of Veterinary Sciences & Animal Husbandry, Birsa Agricultural
University, Kanke, Ranchi. For that 15 adult rabbits of either sex weighing 1.5-2.5 kg were
randomly procured from rabbit Farm College of Veterinary Sciences & Animal Husbandry
after assessing good health condition and divided in three groups based on completely
Randomized Design. Health status of rabbits was managed for 15 days in a uniform
environment to ensure minimum clinical variation during the study. Before experiment,
barks of Litsea monopetala and Schleichera oleosa were collected from forest around
Ranchi district. Barks were separately shade dried, powdered and hot extraction process
was done by using ethanol solution in soxhlet apparatus. Extracts were stored separately
at cool place for clinical evaluation further. All the animal were divided into three groups
having 5 animals in each group, group 1 animals were treated with normal saline, group 2
animals treated with ethanolic extract of litsea monopetala (Meda) bark &group3 animals
treated with ethanolic extract of Schleicheraoleosa (Kusum) bark on day 0th, 3rd, 7th, 12th
& 20th day interval. In each animal’s aseptically wound was created on dorsum position of
the rabbits, for that two full thickness skin wounds of size measuring 1cm x 1cm were
created surgically at the proposed site on either side of the dorso-median plane. A total of
4 wounds were created with 4 cm distance between two wound of same side and between
the contra lateral wounds. The wounds were treated daily with the respective treatment
groups solution and dressed with micropore adhesive tap till complete healing. Blood was
Collected from ear vein on 0, 3, 7, 12 and 20th days for assessment of systemic infection
by evaluation of Total Leucocyte count (TLC) & Differential Leucocytic count (DLC). To
study the histomorphological and histochemical changes in healing tissue, biopsy sample
was collected from the junction of wound and intact skin from all the experimental wounds
in each of the three groups on 3rd, 7th, 12th and 20th days. Finally, all the biopsy tissues
were preserved in 10% neutral buffer formalin solution for further study. Clinical
parameters like pain sensitivity, wound colour, granulation and exudates were taken into
account at different time interval. To measure the contraction percentage of wound, initial
area of the wound and final area of the wound were measured at different time intervals.
To study the histomorphology of created wound of different treatment groups, the biopsy
sample section was stained with Hematoxylin & Eosin solution under resolution of 400x
to assess the leukocyte infiltration, fibroplasia, epithelization and neo-vascularization on
3rd, 7th, 12th and 20th days of experimentation. To account the histochemical changes for
collagen, elastin and reticulin formation on different treatment groups were stained with
Van Gieson’s stain (collagen), Weigert’sresorcin-fuchsin stain (elastin) and Robb smith’s
modification of foot’s silver impregnation stains (reticulin) solution at 400x resolution on
3rd, 7th, 12th, and 20th days of experimentation. All the data obtained were subjected to
statistically analyzed as per method described by Snedecor and Cochran (2004) by using
SPSS software 16.0. One Way ANOVA was used to analyze uniform parametric data
whereas, Mann Whitney U test and Kruskal Wallis test were used to analyze nonparametric
data.
Results of the experiment for clinical observation found that pain was observed in
all the animals during first two days of surgical wound creation which slowly diminished
by 3rd day in all the animals. Significant (P<0.05) reduction of pain is observed in group 2
on 3rd day as compared to control group animals. Non-significant pain reduction was
observed from the 7thday in animals of all the groups. No significant difference in pinkish
wound colour & granulation tissue growth observed among the groups during the entire
time interval. Similarly, non-significant amount of serous type exudates was observed in
control group, however no exudates were observed in group 2 & group 3. Morphometric
study showed significantly higher (P<0.05) wound contraction in group 2 as compared to
control group from 3rd day of onwards at different time intervals. Almost complete healing
was observed in group 2 by day 20th. Histomrphological studies of biopsy sample of wound
healing process showed significantly higher (P<0.05) fibroplasia on 3rd day in group 2 as
compared to control animals. Marked (P<0.05) epithelialization observed in 3rd and 7th
days on group 2 as compared to control group animals. However, epithelialization was non
significantly variable between group 2 & group 3 animals during the entire period of
observation. Non-significant difference (P>0.05) was observed in leucocyte infiltration
among the groups at different intervals during entire period of experimentation.
Histochemical studies related to collagen formation was observed in third day in all
animals. Formation of thick collagen was significantly higher in group 2 on 7th day as
compared to other group. Wavy collagen formation was better in group 2 followed by
group 3 and then control group. Elastin formation was evident since 3rdday in all the
treatment group. Elastinfiber formation was significantly lower in control group as
compared to group 2 and 3. However, mean ± SE score of elastin fiber at different intervals
did not show significant variationsbetween group 2 and 3. Reticulin formation was
observed on 3rd day of experiment in all the groups. The dense and diffuse reticulinfiber
formation was significantly higher in group 2 & group 3 as compared to control on day
7th.while values were non significantly differed (P>0.05) between group 2 & group 3. The
non-significant reticulinfiber formation was observed on 12th & 20th days of experiment in
all the groups. Haematological observation related to TLC and DLC to assess systemic
infection of different groups on different time interval were non-significant. However,
alternation in TLC and DLC was observed in all groups after treatment but values were
within normal physiological range.
On the basis of present findings following conclusions may be drawn:
CONCLUSION
• Pain sensitivity score was lower (P<0.05) on 3rd day of observation in group 2
(Meda) as compared to control (group 1) and group 2 (Kusum). However, pain
sensitivity was similar between the control and Kusum groups. Meda has shown
good analgesic property from the day 7th onwards till the end of observation.
• Changes in wound colour score was found (P<0.05) on 12th days onwards in
Medagroup however color was similar between control and Kusum groups.
• Meda group showed non-significant increase (P>0.05) in granulation as compared
to other group.
• Percentage of contraction was significantly higher (P<0.05) in Meda as compared
to control and Kusum group. However, contraction was non significantly higher in
Kusum group compared to control.
• Meda groups showed non-significantly higher score of Leucocyte infiltration
&neovascularization process as compared to control and kusum.
• Fibroplasia was significantly higher (P<0.05) in meda group on day 3rd as
compared to control & Kusum.
• Epithelialization was higher (P<0.05) in Meda groups on different time intervals
as compared to control and Kusum groups.
• Collagen &reticulin formation was significantly higher (P<0.05) only on 3rd day in
Meda and Kusum group as compared to control, whereas, elastin formation was
significantly higher (P<0.05) in Meda and Kusum group as compared to control
during entire period of study.
• There were transient &non significant changes in the TLC and DLC parameters in
all the groups but remain in normal physiological limits.
• Based on the results it is concluded that treatment with Litsea monopatela (Meda)
showed better anti-inflammatory and analgesic properties in terms of wound
healing in rabbits as compared to control and Schleichera oleosa (Kusum) treated
rabbits.