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955| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1
Research Article
Evaluation of Antiulcer Activity of Extract of Calycophyllum
Spruceanum (Benth) Bark
VENKATAIAH GUDISE1*
, DR. I VEENA RANI2
, P. LAXMI SUMA3
, R. SHREYA GUPTA4
, R. BHAVYA SRI5
Department of Pharmacology, SSJ College of Pharmacy, VN Pally, Gandipet, Hyderabad, TS-500075,
India
Email: venkatpharma22@gmail.com
Received: 16.10.19, Revised: 17.11.19, Accepted: 22.12.19
ABSTRACT
Objective The objective of the study was to evaluate the antiulcer activity of ethanolic extract of Calycophyllum
spruceanum bark.
Methodology The ethanol-HCL and pylorus ligation induced models was selected using swiss albino mice and
the animals were fasted overnight and then grouped as negative control (0.9% normal saline), standard
(omeprazole 30mg/kg), test 1 (CSBE 100mg/kg) and test 2 (CSBE 200mg/kg). The ulcer score, ulcer index and
percent ulcer inhibition were determined in ethanol/HCL model. The volume of gastric secretion, pH, total
acidity, ulcer index and percent of ulcer inhibition was determined in pylorus ligation model. The ulcerogenic
abrasions in the mucosal membrane were observed under a microscope (10X) and scored manually.
Results The preliminary phytochemical screening indicated the presence of sterols, alkaloids, glycosides,
flavonoids, saponins and coumarins. The bark extract was tolerable at dose of 2000mg/kg. The selected doses
of the extracts of 100, 200mg/kg was shown the significant and dose dependent antiulcer activity in ethanol/HCL
and pylorus-ligation models as 11.08 %, 32.59% and 28.29%, 37.03% respectively. The dose of 200mg/kg of CSBE
was shown the superlative activity ((#
p˂0.001)) in both models.
Conclusion: It was concluded that, the ethanolic extract of Calycophyllum spruceanum bark has a significant
antiulcer activity. This activity might be due to presence of supportive phytochemical like flavonoids, alkaloids,
steroids. and glycosides. Hence, further evaluation is needed to isolate and identify the specific constituents
responsible for the activity.
Keywords Calycophyllum spuceanum; Ethanol-HCL; Pylorus-ligation; Antiulcer activity.
INTRODUCTION
The peptic ulcer is a common disease globally with
10 to 19% was recognised and hospitalisation is
ranging about 10 to 17% and highly prevalent
0.12 to 1.5% yearly1
and affects 10 % of the global
population with various causes and about 15000
deaths per year1-3
. The ulcer is defined as
degeneration and necrosis of the GI mucosa
exposed to gastric secretions or chemicals
(induced) or H. Pylori bacteria or drugs like
NSAIDS and most commonly found in duodenum
(duodenal ulcers) or stomach (gastric ulcers) in
ratio of 4:1 as acute or chronic characterised by
indigestion, nausea and vomiting, abdominal
discomfort and burning sensation. The ulceration
leads to development of various complications like
haemorrhage, perforation and malignant if not
treated4
. These ulcers can be established due to
imbalance between aggressiveness of gastric
content like acid, pepsin, H. pylori and bile salts
and gastroprotective mechanism of mucus,
prostaglandins and bicarbonates5
. The recent
approach to the peptic ulcer was accomplished by
GI acid secretion inhibition, increasing of gastric
secretion, preventing the necrosis, apoptosis and
proliferation of epithelial cells for ulcer healing
property. The currently used medicinal products
are PPI’s, histamine antagonists, PG analogues,
antacids, cytoprotective agents and antibiotics for
H. pylori bacterial infections, but most of these
drugs are producing various adverse effects on
prolonged usage and drug-food or drug-food
interactions. Hence, the phyto-derived compounds
are gradually increasing for various pathological
problems6
. Hence, it is necessary to screen for
developing the novel phytochemicals for the
treatment of various diseases including ulcers.
The stem bark of Calycophyllum spruceanum
(Benth) as a common name, capirona belongs to
the family of rubiaceae and alycophyllum genus
with habitat of 30m height found in south america
and india and reported the presence of various
constituents like seco-iridoids, 8-O-
tigloyldiderroid, loganin, secoxyganin, loganetin
ISSN 0975-2366
DOI:https://doi.org/10.31838/ijpr/2020.12.01.070
Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum
(Benth) Bark
956| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1
and diderroside7
. There are few studies was
reported as anti-fungal activity, mucosal infections,
anti-aging, antioxidant, wound healing,
antidiabetic activity8-11
.
MATERIALS AND METHODS
Materials
Chemicals and drugs
Omeprazole was obtained as gift sample from Dr.
Reddys laboratories, Hyderabad, India., ethanol
from merk Pvt, Ltd., plant material was obtained
from botanist, Dr. Madhavachetty, SV University,
tirupathi, India. The voucher no. 2101.
Methods
Preparation of extracts
The ethanolic extract was prepared using Soxhlet
apparatus. The bark plant was collected, cleaned
and washed with distilled water and dried under
shade. The bark was made into course powder
and further sieved to get fine powder for the
methanolic extraction. The powdered bark
(150gm) was kept for soxhlation using 1000 ml of
the solvent (ethanol) at 60-700
c until the solvent
colour faded away. then the extract was filtered,
concentrated under reduced pressure (400
c) using
hot-air-oven, then calculated the percent yield and
stored in the air tight container under 4ºC in
refrigerator until use12
.
Phytochemical screening
The preliminary phytochemical screening of the
extract was performed according to standard
procedures13
and the results shown in results
section.
Experimental animals
Swiss albino mice (20-25g), maintained under
standard conditions (27 ± 2°C; relative humidity
60 ± 5 %, light dark cycle of 12 hrs.) and fed with
standard pellet diet and water ad libitum were used
for present study. The protocol was approved by
IAEC of SSJ College of Pharmacy,
Vattinagulapally, Gandipet mondal, Rangareddy
dist, Hyderabad, Telangana state, India. The Reg.
no: 1488/PO/Re/S/11/CPCSBEA/03/2018.
Determination of MTD
The maximum tolerated dose (MTD) was carried
accordance to the OCED guidelines14
. Five
animals were kept for overnight fasting prior to
drug administration and one animal was treated
with 2000mg/kg as a single dose of
Calycophyllum spruceanum bark extract via oral
route and the food was withheld for further 3–4
hours, four hours later remaining four animals was
received with limit test dose and the mice were
individually observed individually at least once
during the first 30 minutes after dosing,
periodically during the first 24 hours (with special
attention given during the first 4 hours), and daily
thereafter, for a total of 14 days, for behavioural,
neurological, and autonomic profiles (skin and fur,
eyes and mucous membranes, and also
respiratory, circulatory, autonomic and central
nervous systems, and somato motor activity and
behaviour pattern. Attention should be directed to
observations of tremors, convulsions, salivation,
diarrhoea, lethargy, sleep and coma) and for any
lethality, morbidity & Mortality.
ANTIULCER ACTIVITY
Treatment schedule
Overnight fasted animals grouped and treated as
follows: Negative control (0.9% saline), Standard
(omeprazole 30mg/kg), Test 1 (CSBE 100mg/kg)
and Test 2 (CSBE 200mg/kg).
Ethanol-HCl -induced model
All groups were induced gastric ulceration by orally
administration of 0.2 ml of a solution containing
60% ethanol and 0.3M HCl. Then the animals
were sacrificed by anaesthesia using diethyl ether
1h after the administration of ethanol & HCL, and
the stomachs were removed and inflated by
injection of with 0.9% normal saline solution (1mL).
The ulcerated stomachs were fixed in 5% formalin
for 30 min and opened along the greater curvature
to expose the gastric mucosal layer. The
ulcerogenic abrasions in the mucosal membrane
of the glandular region were observed under a
microscope (10X) and scored manually. The
scoring of ulcerations was patterned as follows:
normal gastric mucosa was scored as 0, pinpoint
ulcers were scored 0.5, one or two small
ulcerations were given 1.0 and ulcers with
diameters greater than 3 mm with heavy bleeding
were given a score 2.0. The ulcer index (UI) was
calculated from the sum of the scores of all lesions
for each stomach, and the mean ulcer index (mean
UI) was calculated for each group. The results
shown in table 6.3.1 and 6.3.2, Figure 6.3.1,
6.3.2 and 6.3.3 of the results chapter. Percent
ulcer inhibition of the samples was determined
using the following equation15,16
.
Pylorus-ligation model
All the grouped animals were selected and divided
into groups and allowed for fasting for 12hrs with
access to drinking water ad libitum and treated as
stated above model, then the stomach was
exposed by doing midline abdominal incision (1cm
long) just below the sternum was made under
diethyl ether anaesthesia. Then then pylorus was
ligated by taking care that neither damage to
blood vessels nor traction on the pylorus occurs.
The abdominal wall was closed by applying
sutures. Then the test compounds were
Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum
(Benth) Bark
957| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1
administered orally. 6hrs later, all the animals
were sacrificed by ether anaesthesia. The
abdomen was opened and a ligature was placed
around oesophagus close to diaphragm and
complete stomach was removed, the contents was
drained into centrifuge tubes. The greater
curvature of the stomach was opened and pinned
on dissection trays for scoring the ulceration17
. The
number of ulcers was notes as follows:
0= Normal coloured stomach; 0.5= Red colour;
1= Spot ulcer; 1.5= Haemorrhagic streak; 2=
Deep ulcer; 3=Perforation
The ulcer index was calculated by using following
formula:
The percent inhibition was calculated by using the
following formula.
Statistical analysis
The statistical analysis of the study results was done
by GraphPad Prism, version 8.0, using ANOVA
followed by Dennett’s Multiple comparison. The
data was represented as mean±SD. The statistical
significance considered was p< 0.05, when
compared with negative control group.
RESULTS AND DISCUSSION
Preliminary Phytochemical screening
The ethanolic extract of Calycophyllum
spruceanum shown the presence of bark Phenols,
flavonoids, alkaloids, steroids. and glycosides,
fixed oils, saponins and coumarins.
Maximum Tolerated Dose
The Calycophyllum spruceanum bark extract was
not shown any signs of morbidity and mortality at
a dose of 2000mg/kg body weight for 14 days of
observation. Hence the biological evaluation was
carried out at doses of 100 and 200 mg/kg doses
of extract.
Effect of CSBE in HCL & Ethanol model
The gastric ulcers can develop due to the
aggressive property of HCL when exposed to the
lumen part of the stomach18-20
. The pyloric-ligation
induced ulceration might be due to auto-digestion
and accumulation of HCL then leads to breakdown
of mucosa barrier and leads to formation of
lesions with or without haemorrhagic condition.
There are many commercial medicines like
ranitidine, omeprazole was using for treatment of
both ulcers21
.
The ethanol also damages the gastric mucosa and
alters the permeability and radical formation which
further develops necrosis of the mucosal layer18,22
.
The present study was indicated that, the extract
was shown the significant antiulcer activity.
The study results established that, the ethanolic
extract was shown the significant potential against
induced ulcerations in swiss albino mice. The
pinpoint ulcer was highly increased due to HCL
and ethanol in negative control group, whereas the
ulcers was significantly reduced in omeprazole
(32mg/kg) treated group (#
p˂0.001), 100mg/kg
and 200mg/kg of CSBE (#
p˂0.001) respectively.
The small ulcers were highly increased because of
HCL and ethanol in the negative control group.
Whereas the small ulcers were significantly
reduced in omeprazole (32mg/kg) treated group
(#
p˂0.001) respectively. when compared to
negative control the ulcer was reduced in
100mg/kg of CSBE treated group (*p˂0.05) and
also the ulcers were decreased in 200mg/kg of
CSBE treated group (#
p˂0.001) respectively.
The ulcer with ≥ 3mm with bleeding was increased
in negative control group due to the presence of
HCL & ethanol. Ulcer ≥ 3mm bleeding was
significantly decreased in omeprazole (32mg/kg)
treated group (#
p˂0.001) respectively and
100mg/kg of CSBE treated group ($
p˂0.01) &
300mg/kg of CSBE treated group (#
p˂0.001)
respectively.
Ulcer index was highly increased in negative
control because of the negative control group of
animals were present with the HCL & ethanol and
significantly decreased in omeprazole (32mg/kg)
treated group (#
p˂0.001), 100mg/kg of CSBE
treated group (#
p˂0.001) and 200mg/kg of CSBE
treated group (#
p˂0.001). In this study % ulcer
inhibition was noticed as 15.22%, 11.08% and
32.59% of omeprazole (32mg/kg), CBSE
(100mg/kg) and CBSE (100mg/kg) respectively.
High % ulcer inhibition in 200mg/kg of CSBE
treated group. All the results were shown in table
1, figure 1. The structural alteration in stomach
was shown in figure 2.
Table 1: Mean date of antiulcer activity of CSBE in Ethanol & HCL induced model
Group and Treatment
(mg/kg.p.o)
Ulcer score (mean±SD)
Ulcer Index
(mean±SD)
%
UI
Pinpoint Ulcer Small Ulcers
Ulcer ≥ 3mm
with bleeding
Negative Control
(0.9% NS)
5.00±0.894 3.50±1.22 2.67±0.516 9.93±0.071 -
Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum
(Benth) Bark
958| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1
STD
Omeprazole (30mg)
1.83±0.752#
1.17±0.752#
1.17±0.753#
6.51±0.378#
15.22
Test 1
CSBE (100mg)
1.67±0.516#
2.17±0.753*
1.50±0.548s
8.52±0.376#
11.08
Test 2
CSBE (200mg)
1.50±0.547#
1.33±0.516#
1.17±0.408#
5.17±0.298#
32.59
Values are expressed as mean±SD (n=6); *p˂0.05, $
p˂0.01, #
p˂0.001 compared to negative control
group; CSBE: Calycophyllum spruceanum bark extract.
Figure 1: Graphical representation: effect of CSBE extract in ethanol & HCL induced model
Figure 2. Structural alteration in ethanol & HCL induced model
Effect of CSBE in Pylorus-ligation model:
The pH is significantly neutral in the omeprazole
(32mg/kg) treated groups (#
p˂0.001, 100mg/kg
and 200mg/kg of CSBE treated group (#
p˂0.001)
when compared to the negative control. High dose
extract was shown more potent.
The total acidity is highly increased in the negative
control treated group due to the pylorus ligation
and omeprazole (32mg/kg) treated groups are
significantly (#
p˂0.001) reduced the total acidity,
the test 100mg/kg and 200mg/kg of CSBE were
decrease the total acidity with non-significantly.
The ulcer index was significantly (#
p˂0.001)
decreased in the omeprazole (32mg/kg) treating
group, the test groups of CBSE (100mg/kg and
200mg/kg) treating group when compare to
negative control.
The percentage ulcer inhibition was noted as
36.98%, 28.29% and 37.03% of omeprazole
(32mg/kg), CSBE (100mg/kg) and CSBE
(200mg/kg) and all the results were shown in table
2 and figure 2. Numerous studies indicated that,
the phytochemical constituents like phenols and
flavonoids are accountable for gastro-protective
activity23
and the above compounds are having the
ability to trigger the mucus formation and
protection against reactive oxygen species and
other mucosal irritants24
. Therefore, the antiulcer
activity of CSBE ethanolic extract might be
presence of such supportive phytochemicals.
Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum
(Benth) Bark
959| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1
Table 2: Mean date of antiulcer activity of CSBE in pylorus ligation-induced ulcer
Group and Treatment
(mg/kg.p.o)
Volume of
gastric secretion
(mL)
pH
Total acidity
(mEq/L)
Ulcer index
(10/X)
%
UI
Negative Control
(0.9% NS)
1.80±0.217 3.333±0.1751
74.17±2.63
9
7.67±1.03 -
STD
Omeprazole (30mg)
0.6167±0.1835
# 6.167±0.1211# 60.33±3.01
1#
4.83±0.753
# 36.98
Test 1
CSBE (100mg)
1.667±0.1633# 5.100±0.08944
#
71.50±3.88
6
5.50±0.548
# 28.29
Test 2
CSBE (200mg)
0.6333±0.1751
# 6.033±0.3559# 70.52±3.51
3
4.83±0.753
# 37.03
Values are expressed as mean±SD (n=6); *p˂0.05, $
p˂0.01, #
p˂0.001 compared to negative control
group; CSBE: Calycophyllum spruceanum bark extract.
Figure 2: Graphical representation of the results in pylorus-ligation model (mean±SD).
CONCLUSION
Based on the study results, we concluded that, the
ethanolic extract of Calycophyllum spruceanum
bark has a significant antiulcer activity may be due
to its ability to ability to trigger the mucus formation
and protection against reactive oxygen species and
other mucosal irritants. This activity might be due
to presence of supportive phytochemical like
flavonoids, alkaloids, steroids. and glycosides.
Hence, further fractional and structural elucidation
is needed to find specific compounds responsible
for this activity.
ACKNOWLEDGEMENT
Authors are very much thankful to Dr. K. Vanitha
Prakash, Principal, SSJ College of Pharmacy for
providing the research facilities and technical
support.
ABBREVIATIONS
HCL : Hydrochloric acid
CBSE : Calycophyllum spruceanum
bark extract
H. pylori : Helicobacter pylori
GI : Gastrointestinal
PPI’s : Prostaglandin inhibitors
PG : Prostaglandin
IAEC : Institutional animal ethics
committee
MTD : Maximum tolerated dose
OECD : Organisation for economic
control and cooperation
UI : Ulcer index
% UI : Percent ulcer inhibition
SD : Standard deviation
STD : Standard
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  • 1. 955| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1 Research Article Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum (Benth) Bark VENKATAIAH GUDISE1* , DR. I VEENA RANI2 , P. LAXMI SUMA3 , R. SHREYA GUPTA4 , R. BHAVYA SRI5 Department of Pharmacology, SSJ College of Pharmacy, VN Pally, Gandipet, Hyderabad, TS-500075, India Email: venkatpharma22@gmail.com Received: 16.10.19, Revised: 17.11.19, Accepted: 22.12.19 ABSTRACT Objective The objective of the study was to evaluate the antiulcer activity of ethanolic extract of Calycophyllum spruceanum bark. Methodology The ethanol-HCL and pylorus ligation induced models was selected using swiss albino mice and the animals were fasted overnight and then grouped as negative control (0.9% normal saline), standard (omeprazole 30mg/kg), test 1 (CSBE 100mg/kg) and test 2 (CSBE 200mg/kg). The ulcer score, ulcer index and percent ulcer inhibition were determined in ethanol/HCL model. The volume of gastric secretion, pH, total acidity, ulcer index and percent of ulcer inhibition was determined in pylorus ligation model. The ulcerogenic abrasions in the mucosal membrane were observed under a microscope (10X) and scored manually. Results The preliminary phytochemical screening indicated the presence of sterols, alkaloids, glycosides, flavonoids, saponins and coumarins. The bark extract was tolerable at dose of 2000mg/kg. The selected doses of the extracts of 100, 200mg/kg was shown the significant and dose dependent antiulcer activity in ethanol/HCL and pylorus-ligation models as 11.08 %, 32.59% and 28.29%, 37.03% respectively. The dose of 200mg/kg of CSBE was shown the superlative activity ((# p˂0.001)) in both models. Conclusion: It was concluded that, the ethanolic extract of Calycophyllum spruceanum bark has a significant antiulcer activity. This activity might be due to presence of supportive phytochemical like flavonoids, alkaloids, steroids. and glycosides. Hence, further evaluation is needed to isolate and identify the specific constituents responsible for the activity. Keywords Calycophyllum spuceanum; Ethanol-HCL; Pylorus-ligation; Antiulcer activity. INTRODUCTION The peptic ulcer is a common disease globally with 10 to 19% was recognised and hospitalisation is ranging about 10 to 17% and highly prevalent 0.12 to 1.5% yearly1 and affects 10 % of the global population with various causes and about 15000 deaths per year1-3 . The ulcer is defined as degeneration and necrosis of the GI mucosa exposed to gastric secretions or chemicals (induced) or H. Pylori bacteria or drugs like NSAIDS and most commonly found in duodenum (duodenal ulcers) or stomach (gastric ulcers) in ratio of 4:1 as acute or chronic characterised by indigestion, nausea and vomiting, abdominal discomfort and burning sensation. The ulceration leads to development of various complications like haemorrhage, perforation and malignant if not treated4 . These ulcers can be established due to imbalance between aggressiveness of gastric content like acid, pepsin, H. pylori and bile salts and gastroprotective mechanism of mucus, prostaglandins and bicarbonates5 . The recent approach to the peptic ulcer was accomplished by GI acid secretion inhibition, increasing of gastric secretion, preventing the necrosis, apoptosis and proliferation of epithelial cells for ulcer healing property. The currently used medicinal products are PPI’s, histamine antagonists, PG analogues, antacids, cytoprotective agents and antibiotics for H. pylori bacterial infections, but most of these drugs are producing various adverse effects on prolonged usage and drug-food or drug-food interactions. Hence, the phyto-derived compounds are gradually increasing for various pathological problems6 . Hence, it is necessary to screen for developing the novel phytochemicals for the treatment of various diseases including ulcers. The stem bark of Calycophyllum spruceanum (Benth) as a common name, capirona belongs to the family of rubiaceae and alycophyllum genus with habitat of 30m height found in south america and india and reported the presence of various constituents like seco-iridoids, 8-O- tigloyldiderroid, loganin, secoxyganin, loganetin ISSN 0975-2366 DOI:https://doi.org/10.31838/ijpr/2020.12.01.070
  • 2. Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum (Benth) Bark 956| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1 and diderroside7 . There are few studies was reported as anti-fungal activity, mucosal infections, anti-aging, antioxidant, wound healing, antidiabetic activity8-11 . MATERIALS AND METHODS Materials Chemicals and drugs Omeprazole was obtained as gift sample from Dr. Reddys laboratories, Hyderabad, India., ethanol from merk Pvt, Ltd., plant material was obtained from botanist, Dr. Madhavachetty, SV University, tirupathi, India. The voucher no. 2101. Methods Preparation of extracts The ethanolic extract was prepared using Soxhlet apparatus. The bark plant was collected, cleaned and washed with distilled water and dried under shade. The bark was made into course powder and further sieved to get fine powder for the methanolic extraction. The powdered bark (150gm) was kept for soxhlation using 1000 ml of the solvent (ethanol) at 60-700 c until the solvent colour faded away. then the extract was filtered, concentrated under reduced pressure (400 c) using hot-air-oven, then calculated the percent yield and stored in the air tight container under 4ºC in refrigerator until use12 . Phytochemical screening The preliminary phytochemical screening of the extract was performed according to standard procedures13 and the results shown in results section. Experimental animals Swiss albino mice (20-25g), maintained under standard conditions (27 ± 2°C; relative humidity 60 ± 5 %, light dark cycle of 12 hrs.) and fed with standard pellet diet and water ad libitum were used for present study. The protocol was approved by IAEC of SSJ College of Pharmacy, Vattinagulapally, Gandipet mondal, Rangareddy dist, Hyderabad, Telangana state, India. The Reg. no: 1488/PO/Re/S/11/CPCSBEA/03/2018. Determination of MTD The maximum tolerated dose (MTD) was carried accordance to the OCED guidelines14 . Five animals were kept for overnight fasting prior to drug administration and one animal was treated with 2000mg/kg as a single dose of Calycophyllum spruceanum bark extract via oral route and the food was withheld for further 3–4 hours, four hours later remaining four animals was received with limit test dose and the mice were individually observed individually at least once during the first 30 minutes after dosing, periodically during the first 24 hours (with special attention given during the first 4 hours), and daily thereafter, for a total of 14 days, for behavioural, neurological, and autonomic profiles (skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somato motor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma) and for any lethality, morbidity & Mortality. ANTIULCER ACTIVITY Treatment schedule Overnight fasted animals grouped and treated as follows: Negative control (0.9% saline), Standard (omeprazole 30mg/kg), Test 1 (CSBE 100mg/kg) and Test 2 (CSBE 200mg/kg). Ethanol-HCl -induced model All groups were induced gastric ulceration by orally administration of 0.2 ml of a solution containing 60% ethanol and 0.3M HCl. Then the animals were sacrificed by anaesthesia using diethyl ether 1h after the administration of ethanol & HCL, and the stomachs were removed and inflated by injection of with 0.9% normal saline solution (1mL). The ulcerated stomachs were fixed in 5% formalin for 30 min and opened along the greater curvature to expose the gastric mucosal layer. The ulcerogenic abrasions in the mucosal membrane of the glandular region were observed under a microscope (10X) and scored manually. The scoring of ulcerations was patterned as follows: normal gastric mucosa was scored as 0, pinpoint ulcers were scored 0.5, one or two small ulcerations were given 1.0 and ulcers with diameters greater than 3 mm with heavy bleeding were given a score 2.0. The ulcer index (UI) was calculated from the sum of the scores of all lesions for each stomach, and the mean ulcer index (mean UI) was calculated for each group. The results shown in table 6.3.1 and 6.3.2, Figure 6.3.1, 6.3.2 and 6.3.3 of the results chapter. Percent ulcer inhibition of the samples was determined using the following equation15,16 . Pylorus-ligation model All the grouped animals were selected and divided into groups and allowed for fasting for 12hrs with access to drinking water ad libitum and treated as stated above model, then the stomach was exposed by doing midline abdominal incision (1cm long) just below the sternum was made under diethyl ether anaesthesia. Then then pylorus was ligated by taking care that neither damage to blood vessels nor traction on the pylorus occurs. The abdominal wall was closed by applying sutures. Then the test compounds were
  • 3. Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum (Benth) Bark 957| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1 administered orally. 6hrs later, all the animals were sacrificed by ether anaesthesia. The abdomen was opened and a ligature was placed around oesophagus close to diaphragm and complete stomach was removed, the contents was drained into centrifuge tubes. The greater curvature of the stomach was opened and pinned on dissection trays for scoring the ulceration17 . The number of ulcers was notes as follows: 0= Normal coloured stomach; 0.5= Red colour; 1= Spot ulcer; 1.5= Haemorrhagic streak; 2= Deep ulcer; 3=Perforation The ulcer index was calculated by using following formula: The percent inhibition was calculated by using the following formula. Statistical analysis The statistical analysis of the study results was done by GraphPad Prism, version 8.0, using ANOVA followed by Dennett’s Multiple comparison. The data was represented as mean±SD. The statistical significance considered was p< 0.05, when compared with negative control group. RESULTS AND DISCUSSION Preliminary Phytochemical screening The ethanolic extract of Calycophyllum spruceanum shown the presence of bark Phenols, flavonoids, alkaloids, steroids. and glycosides, fixed oils, saponins and coumarins. Maximum Tolerated Dose The Calycophyllum spruceanum bark extract was not shown any signs of morbidity and mortality at a dose of 2000mg/kg body weight for 14 days of observation. Hence the biological evaluation was carried out at doses of 100 and 200 mg/kg doses of extract. Effect of CSBE in HCL & Ethanol model The gastric ulcers can develop due to the aggressive property of HCL when exposed to the lumen part of the stomach18-20 . The pyloric-ligation induced ulceration might be due to auto-digestion and accumulation of HCL then leads to breakdown of mucosa barrier and leads to formation of lesions with or without haemorrhagic condition. There are many commercial medicines like ranitidine, omeprazole was using for treatment of both ulcers21 . The ethanol also damages the gastric mucosa and alters the permeability and radical formation which further develops necrosis of the mucosal layer18,22 . The present study was indicated that, the extract was shown the significant antiulcer activity. The study results established that, the ethanolic extract was shown the significant potential against induced ulcerations in swiss albino mice. The pinpoint ulcer was highly increased due to HCL and ethanol in negative control group, whereas the ulcers was significantly reduced in omeprazole (32mg/kg) treated group (# p˂0.001), 100mg/kg and 200mg/kg of CSBE (# p˂0.001) respectively. The small ulcers were highly increased because of HCL and ethanol in the negative control group. Whereas the small ulcers were significantly reduced in omeprazole (32mg/kg) treated group (# p˂0.001) respectively. when compared to negative control the ulcer was reduced in 100mg/kg of CSBE treated group (*p˂0.05) and also the ulcers were decreased in 200mg/kg of CSBE treated group (# p˂0.001) respectively. The ulcer with ≥ 3mm with bleeding was increased in negative control group due to the presence of HCL & ethanol. Ulcer ≥ 3mm bleeding was significantly decreased in omeprazole (32mg/kg) treated group (# p˂0.001) respectively and 100mg/kg of CSBE treated group ($ p˂0.01) & 300mg/kg of CSBE treated group (# p˂0.001) respectively. Ulcer index was highly increased in negative control because of the negative control group of animals were present with the HCL & ethanol and significantly decreased in omeprazole (32mg/kg) treated group (# p˂0.001), 100mg/kg of CSBE treated group (# p˂0.001) and 200mg/kg of CSBE treated group (# p˂0.001). In this study % ulcer inhibition was noticed as 15.22%, 11.08% and 32.59% of omeprazole (32mg/kg), CBSE (100mg/kg) and CBSE (100mg/kg) respectively. High % ulcer inhibition in 200mg/kg of CSBE treated group. All the results were shown in table 1, figure 1. The structural alteration in stomach was shown in figure 2. Table 1: Mean date of antiulcer activity of CSBE in Ethanol & HCL induced model Group and Treatment (mg/kg.p.o) Ulcer score (mean±SD) Ulcer Index (mean±SD) % UI Pinpoint Ulcer Small Ulcers Ulcer ≥ 3mm with bleeding Negative Control (0.9% NS) 5.00±0.894 3.50±1.22 2.67±0.516 9.93±0.071 -
  • 4. Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum (Benth) Bark 958| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1 STD Omeprazole (30mg) 1.83±0.752# 1.17±0.752# 1.17±0.753# 6.51±0.378# 15.22 Test 1 CSBE (100mg) 1.67±0.516# 2.17±0.753* 1.50±0.548s 8.52±0.376# 11.08 Test 2 CSBE (200mg) 1.50±0.547# 1.33±0.516# 1.17±0.408# 5.17±0.298# 32.59 Values are expressed as mean±SD (n=6); *p˂0.05, $ p˂0.01, # p˂0.001 compared to negative control group; CSBE: Calycophyllum spruceanum bark extract. Figure 1: Graphical representation: effect of CSBE extract in ethanol & HCL induced model Figure 2. Structural alteration in ethanol & HCL induced model Effect of CSBE in Pylorus-ligation model: The pH is significantly neutral in the omeprazole (32mg/kg) treated groups (# p˂0.001, 100mg/kg and 200mg/kg of CSBE treated group (# p˂0.001) when compared to the negative control. High dose extract was shown more potent. The total acidity is highly increased in the negative control treated group due to the pylorus ligation and omeprazole (32mg/kg) treated groups are significantly (# p˂0.001) reduced the total acidity, the test 100mg/kg and 200mg/kg of CSBE were decrease the total acidity with non-significantly. The ulcer index was significantly (# p˂0.001) decreased in the omeprazole (32mg/kg) treating group, the test groups of CBSE (100mg/kg and 200mg/kg) treating group when compare to negative control. The percentage ulcer inhibition was noted as 36.98%, 28.29% and 37.03% of omeprazole (32mg/kg), CSBE (100mg/kg) and CSBE (200mg/kg) and all the results were shown in table 2 and figure 2. Numerous studies indicated that, the phytochemical constituents like phenols and flavonoids are accountable for gastro-protective activity23 and the above compounds are having the ability to trigger the mucus formation and protection against reactive oxygen species and other mucosal irritants24 . Therefore, the antiulcer activity of CSBE ethanolic extract might be presence of such supportive phytochemicals.
  • 5. Venkataiah Gudise et al / Evaluation of Antiulcer Activity of Extract of Calycophyllum Spruceanum (Benth) Bark 959| International Journal of Pharmaceutical Research | Jan - March 2020 | Vol 12 | Issue 1 Table 2: Mean date of antiulcer activity of CSBE in pylorus ligation-induced ulcer Group and Treatment (mg/kg.p.o) Volume of gastric secretion (mL) pH Total acidity (mEq/L) Ulcer index (10/X) % UI Negative Control (0.9% NS) 1.80±0.217 3.333±0.1751 74.17±2.63 9 7.67±1.03 - STD Omeprazole (30mg) 0.6167±0.1835 # 6.167±0.1211# 60.33±3.01 1# 4.83±0.753 # 36.98 Test 1 CSBE (100mg) 1.667±0.1633# 5.100±0.08944 # 71.50±3.88 6 5.50±0.548 # 28.29 Test 2 CSBE (200mg) 0.6333±0.1751 # 6.033±0.3559# 70.52±3.51 3 4.83±0.753 # 37.03 Values are expressed as mean±SD (n=6); *p˂0.05, $ p˂0.01, # p˂0.001 compared to negative control group; CSBE: Calycophyllum spruceanum bark extract. Figure 2: Graphical representation of the results in pylorus-ligation model (mean±SD). CONCLUSION Based on the study results, we concluded that, the ethanolic extract of Calycophyllum spruceanum bark has a significant antiulcer activity may be due to its ability to ability to trigger the mucus formation and protection against reactive oxygen species and other mucosal irritants. This activity might be due to presence of supportive phytochemical like flavonoids, alkaloids, steroids. and glycosides. Hence, further fractional and structural elucidation is needed to find specific compounds responsible for this activity. ACKNOWLEDGEMENT Authors are very much thankful to Dr. K. Vanitha Prakash, Principal, SSJ College of Pharmacy for providing the research facilities and technical support. ABBREVIATIONS HCL : Hydrochloric acid CBSE : Calycophyllum spruceanum bark extract H. pylori : Helicobacter pylori GI : Gastrointestinal PPI’s : Prostaglandin inhibitors PG : Prostaglandin IAEC : Institutional animal ethics committee MTD : Maximum tolerated dose OECD : Organisation for economic control and cooperation UI : Ulcer index % UI : Percent ulcer inhibition SD : Standard deviation STD : Standard REFERENCES 1. G. Vimala and F. Gricilda Shoba, A Review on Antiulcer Activity of Few Indian Medicinal Plants, International Journal of Microbiology, 2014;2014. 2. Bharathi DP, Jegad E, Kavimani S, Antiulcer activity of aqueous extract of fruits of Momordica cymbalaria Hook f. in Wistar rats Pharmacognosy Research, 2010; 2(1):58-61.
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