Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
235
COMPARATIVE STUDY BETWEEN THE ROLE OF THE
GRAVIOLA AND ETHANOLIC EXTRACT OF Eucalyptus
camaldulensis ON HEPATIC REGENERATION IN RABBITS
MODEL
Firas Hussain al-baw. Zainab J. Malik and A. J. AL- Khamas
Department of Pathology, college of Veterinary Medicine, Al- Qasim
Green,Babylon,Iraq.
Keywards:- graviola, eucalyptus, hepatoctomy.
Corresponding Author: [email protected]
ABSTRACT
The aim of this study was to investigate the effect of graveola and extract of
eucalyptus productions on liver regeneration. 16 mature males rabbit were used. The
animals were divided into two equal groups. The animals were anesthetized by
ketamine and xylazine as general anesthesia. The animals were anesthetized by
atropine sulphate (1mg /Kg B.W) intramuscularly as premedication and 15 minutes
later a mixture of xylazine hydrochloride (20mg /Kg B.W) and Ketamine
hydrochloride (40 mg /Kg B.W) intramuscularly. After anesthetized the rabbits and
preparation the site of operation was done in the upper part of abdomen, then Surgical
incision was done and exposure of fourth lobe of the live. The liver handling by
thump forceps and the stitches was done on the lobe. Three knots were tied and
dissecting scissors were used to cut the tied lobe just distal to the suture. Then the
abdominal wall was approximated with a running 3-0 Polyglactin suture, and the
skin was closed with a running 2-0 polyamide suture. And after hepatoctomy divided
the animals to two equal groups; first group give the graveola orally and second group
give the extracted eucalyptus orally too for 15 days . The clinical parameters included
temperature, respiratory , and heart rates, were within acceptable limits post
operatively in all animals in two groups. The intra-abdominal adhesions in different
degrees of evaluations and occurred among liver, different organs and abdominal wall
that more frequency in graviola group when compared with another treated
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
236
group.Histopathological Examinations was done by taken the liver biopsies on 15th
day postoperatively. The histopathological results of this study revealed that the liver
regeneration similar maturity in two groups.
INTRODUCTION
Graviola f(Annona muricata) is a miraculous d evergreen tree found in rainforest of
w America j. This 9small tree 9is well1known in herbal4medicine and7 has several
names: Soursop,pPaw, 6Guanabana. (1). Graviola acts2 as 7an antidepressant due
5to6 the novel alkaloids 4found 9in 3it is seeds and roots7. However, one 4should 4be
very careful8 while 3undergoing the treatment with 2this 0plant, because these
alkaloids 9may be toxic to y the nervous1 system6. (2)
Eucalyptus is one4 of three similar 6genera that are commonly referred to as
´eucalypts ´, the others being Corymbia and5 Angophora . many species, though by
no means all, are known as gum 3trees because they exude9 copious kino from6 any
brak. (3)
MATERIALS AND METHODS
Materials
Equipment Company Origin
Centrifuge CYAN Belgium
Light Microscope CYAN Belgium
Sensitive Balance Sartorius Germany
Soxhlet Atico India
Aspirating pipettes(5ml) Suzhou dukanging medical China
Embbedin processor Leica USA
Eppendrof tubes Sigma UK
Micropipette Eppendrof Germany
Micotom Leica USA
Non- heparin tube Suzhou dukanging medical China
Slides Suzhou dukanging medical China
Surgical sate Suzhou dukanging medical China
Syringe Suzhou dukanging medical China
Test tubes Suzhou dukanging medical China
Tissue processor Leica USA
Water bath Kottermann Germany
Suture Materials
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
237
Methods
Preparation of phosphate buffered saline (PBS) at PH 8
That prepared5 by dissolving the following8 chemicals in 1 L of distilled 6water
Substance:-
Sodium chloride 8gm
Potassium chloride 0.2gm
Potassium dihydrogen phosphate 0.2gm
Sodium dihydrogen phosphate 1.15gm
Distilled water 100ml
Plant leaves collection:-
The Eucalyptus leaves5 were5 collected during4 the period 7that extended4 from
the k park of veterinary h medicine college of Al- Qassim Green University6, h so we
washed the leaves to6 get rid d of dusts and then8 dried inside the hot air oven (100°
C) and8 then grinded to fine powder3 and saved in plastic bags6. At the end stored
inside the refrigerator until use. Preparation of the ethanolic extract8 (eucalyptus
camaldulensis):- twenty gms were taken f from powdered leaves and e extracted with
ethanol 70% by soxhlet apparatus9 for 24hr. and then the extract was taken and3
placed in a petri dish I inside5 the oven at 40° C for 48hr. and5 stored at 4°C until
used (4)
Experimental design
16 adult male rabbits at age (10- 12) months, and weighted between ( 1.5- 3) kg.
they were adapted at the animals house for 2 weeks before starting the experiment.
They divided to two groups and all groups had partial hepatoctomy from the third
lobe
1. The first group (8 animals):
After doing partial hepatoctomy give the animals orally the Graviola for 15 days
2. The second group (8 animals):
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
238
After doing partial hepatoctomy give the animals orally the ethanol extract of
eucalyptus camaldulensis leaves (200mg/ kg b.w.) for 15 days
Surgical procedure
Surgical Procedures: g Hepatic resection was p performed as described
previously by (5). The rabbits were anesthetized by 0.8 mg/kg intramuscular
Acepromazine, 0.05 mg/kg b Buprenorphine and 40 mg/kg of Ketamine then a
midline laparotomy4 w was performed. Surgical n incision was done in the
upper part of abdomen, and exposure of third lobe of the liver (Fig. 1), The
liver handling6 by thump forceps (Fig. 2), then the lobe partial resected gently
(2*1.5 cm) with a silk 3-0 suture tie was placed underneath it and positioned as
proximal to the origin of the lobe as possible. The two3 e ends of the suture
were tied6 over the lobe of liver at its base7 near the inferior vena cava (Fig.3).
Three knots were tied and dissecting scissors were used to cut the tied lobe just
distal to the suture. Then the abdominal wall was reapproximated with a running
3-0 Polyglactin suture, and the skin was closed with a running 2-0 polyamide
suture. (6and 7).
Figure (1): The midline laparotomy was performed. Surgical
incision was done in the upper part of abdomen and exposure of
fourth lobe of the live.
Figure (2): The exposure of third lobe of the liver and handling by thump forceps
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
239
Figure (3): The two ends of the suture were tied over the top
of the lobe at its base near the inferior vena cava then
surgical removed done.
RESULTS AND DISCUSSION
Clinical examination
The results of the physical and clinical examination for temperature, respiratory
rate, heart rate, defecation and urination during first week post operation revealed
that, slight elevated in temperature, respiratory rate and heart rate with seen normal
defecation and urination in all animals. Significant convergence in results between the
control group since the second days postoperation and treated group but early
disappeared in treated group when compared with control group at 3rd and 5th days
postoperation respectively, that may be due to increase the blood flow in operative
area. Beside that increase dilatation of blood vessels with increase permeability of
capillaries was agreed with other workers (8) whom mentioned that there were no
significant changes recorded in these clinical parameters before and after surgical
operation.
The animals activity which was represented by animal posture, motion, and alert
to the surrounding) and also the appetite in present study, were not altered. However
there were four animals in control group have normal appetite but limited activity
during three days postoperatively, this is may be attributed to visceral pain or
adhesion formation. The visceral pain during the first hours after surgical intervention
or pain associated with adhesion formation postoperatively reported in previous
researches (9,10).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Adhesions:
The intra-abdominal adhesions more frequency in treated group by graviola
than in treated group by eucalyptus camaldulensis leaves in different degrees of
evaluations and occurred among liver, different organs and abdominal wall.
Quantitative evaluation (MSAS) (Table-1-) showed that graviola group had large
number (3 animals) of adhesion at grade 2, while the treated group by eucalyptus
Table -1- Multiple sites adhesions schemes (MSAS).
Grade
4
Grade
3
Grade
2
Grade
1
Grade
0
Graviola group 1 3 3 1 0
5 1 2 0 0
eucalyptus
camaldulensis
Number of animals for each grade
Table 1
Groups
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
241
camaldulensis had large number (5 animals) of adhesions at grade 0, (1 animals)
at grade1 and (2animals) at grade 2 therefore the entrance to the abdominal cavity and
easy of visualization to the abdominal organs.
Qualitative evaluation (ATTS) (Table-2-) showed that graviola group had
grade2 adhesions at (5 animals). While treated group had grade 0 adhesions at (8
animals). Therefore the adhesions in treated group had little effect and easy to
perform adhesiolysis comparing with control group Adhesions formation is part of
normal healing process (Betsabee et al., 2017).
One of the most potent stimuli for initiation of an inflammatory response and thus adhesions formation is
surgical trauma. Routine surgical procedures involve various degree of tissue handling that initials tissue abrasion,
desiccation, ischemia, bleeding, infection and exposure to foreign materials, any of these factors can initiate
inflammatory responses which eventually lead to adhesion formation (Liakakos et al., 2001).
Table-2- Adhesive tissue tenacity schemes ( ATTS ).
Table 2 Number of animals for each grade
Groups Grade
4
Grade
3
Grade
2
Grade
1
Grade
0
Graviola group 2 2 4 0 0
3 2 1 0 0
eucalyptus
camaldulensis
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
242
Figure (4 )Macroscopic view shows grade 2 adhesion (MSAS) among
liver, omentum and abdominal wall in graviola group (A) and eucalyptus
camaldulensis group(B).
Hsitopathological Findings:
The histopathological findings6 of the e liver after partial hepatectomy were as
following:
The histopathological examination6 in treated group by Graveola there was early
epithelization and 6d dilation the central vein
But the histopathological examination7 in treated group by extract of eucalyptus
camaldulensis leaves show 9the inflammatory cells with 4granulation area but in
another section show 3dilation the bile duct
When we were showing9 the histopathological results, we had3 a view the graveola
resulting a good healing in liver through 15th days this due to many properties of it, so
due to novel alkaloid found in it4 is seeds and roots it is act an antidepresent (2). As
well as; have7 beneficial elements including antiviral7, anti-parasite and antiinflammatory9
properties (2).
But in the histopathological results of extract6 of eucalyptus camaldulensis we were
show have5 a good healind too but lesser than8 the group of graveola in rather9, so it
was have anti- inflammatory4 properties and have capable5 to inhibitor of cytokines
and 4stimulated the p phagocytosis ( 13 and 14) and have6 lipopolysaccharide I in5
nature that very good6 f for healing (15)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig.1: Histological section in the liver of SKL group showed dilation with congested
central vein (black arrows), dilated sinusoids (green arrows) and inflammatory cells
infiltration (blue arrows). H&E stain. 10X.
Fig.2: Histological section in the liver of SKL group showed dilation with congested
central vein (black arrow), dilated sinusoids (green arrow) and inflammatory cells
infiltration (blue arrow). H&E stain. 40X.
Fig.3: Histological section in the liver of SKL group showed dilation with congested
hepatic artery (black arrow) and inflammatory cells infiltration around bile duct (blue
arrow). H&E stain. 10X.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
244
Fig.4: Histological section in the liver of SKL group showed dilation with congested
hepatic artery (black arrow) , inflammatory cells infiltration (blue arrows) and dilation
of bile duct as well surrounded by inflammatory cells (green arrows). H&E stain.
40X.
Fig.5: Histological section in the liver of SQR group showed dilation of central vein
(black arrow) and severe inflammatory cells infiltration in entire hepatic parenchyma
(blue arrows). H&E stain. 10X.
Fig.6: Histological section in the liver of SQR group showed dilation of central vein
(black arrow) , inflammatory cells infiltration (blue arrows) and vacuolation of
hepatocytes (green arrows) as well as dilation of sinusoids (red arrows). H&E stain.
40X.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
245
Fig.7: Histological section in the liver of SQR group showed inflammatory cells
infiltration (blue arrow) and vacuolation of some hepatocytes (green arrows) and
pyknotic hepatocytes (black arrow). H&E stain. 40X.
Fig.8: Histological section in the liver of SQR group showed inflammatory cells
infiltration (black arrow) and congestion of portal vein (green arrows). H&E stain.
40X.
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