ijlpr 2021; doi 10.22376/ijpbs/lpr.2021.11.2.

P98-106

International Journal of Life science and Pharma Research

Pharmacology for Drug Discovery

Research Article

GC-MS Analysis, Gastroprotective and In Silico Docking Studies of Phytoconstituents

from Ixora Javanica Flowers

M Ganga Raju* , Anusha K, N V L Suvarchala Reddy V, Gayathri S and Nikitha K

Department of Pharmacology, Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad 500090, Telangana, India.

Professor and Head, Department of Pharmacology, Gokaraju Rangaraju College of Pharmacy, Hyderabad, Telangana, India.

ABSTRACT: Ixora javanica belongs to the family Rubiaceae having phytoconstituents that are useful in the treatment of various diseases. In
our study, the methanolic extract of Ixora javanica flowers was screened for its antiulcer activity. The extract was analyzed using GC-MS to
identify significant active constituents, and these constituents were subjected to molecular docking to study their affinity towards the H+/K+
ATPase enzyme. The dry powder of the Ixora javanica flowers was extracted by methanol as the solvent for the soxhlation technique. The
extract was then subjected to the GC-MS examination. The in-vivo antiulcer activity was screened using ethanol, indomethacin in pylorus
ligation induced gastric ulcer models. The pharmacological evaluation of the extract was carried out using 200 and 400 mg/kg bd. wt.
Omeprazole (20 mg/kg, bd.wt, p.o) was used as the standard. Ulcer scores were calculated. Glide 5.6 (Schrodinger Inc.) was used for
generating docking simulation studies. The various phytochemical constituents identified from GC-MS study were formononetin, ferulic acid,
quinic acid, palmitic acid, oleanolic acid and maslinic acid in higher amounts. The extract exhibited a substantial reduction in the ulcer scores in
ethanol, indomethacin, and pylorus ligation prompted gastric ulcer prototypes. A decrease in the ulcers might be due to the presence of
phytochemical constituents like terpenoids, flavonoids, phenolic acids, glycosides, alkaloids, steroids, saponins, and tannins present in Ixora
javanica flowers, which clearly showed that the extract possesses gastroprotective activity. Molecular docking studies confirmed the H+/K+
ATPase inhibitory effect. Among the identified constituents, formononetin, ferulic acid, and rutin have shown the highest docking scores when
compared to other compounds.

Keywords: Anti-ulcer, Docking, H+/K+ ATPase, GC-MS, Ixora javanica, Omeprazole.

*Corresponding Author Recieved On 26 August 2020

M. Ganga Raju , Department of Pharmacology, Gokaraju Revised On 16 October 2020

Rangaraju College of Pharmacy, Bachupally, Hyderabad Accepted On 22 October 2020
500090, Telangana, India. Published On 03 March 2021

Funding This research did not receive any specific grant from any funding agencies in the public, commercial or not for profit sectors.

Citation M Ganga Raju, Anusha K, N V L Suvarchala Reddy V, Gayathri S, Nikitha K , Gc-Ms Analysis, Gastroprotective and In Silico
Docking Studies of Phytoconstituents from Ixora Javanica Flowers.(2021).Int. J. Life Sci. Pharma Res.11(2), P98-106
http://dx.doi.org/10.22376/ijpbs/lpr.2021.11.2.P98-106

This article is under the CC BY- NC-ND Licence (https://creativecommons.org/licenses/by-nc-nd/4.0)

Copyright @ International Journal of Life Science and Pharma Research, available at www.ijlpr.com

Int J Life Sci Pharma Res., Volume11., No 2 (March) 2021, pp P98-106

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1. INTRODUCTION 2.2 Phytochemical screening

Gastric ulcers are an important disease of the digestive The preliminary phytochemical screening was performed
system and affect 5–10% of the adult population, becoming a with the methanolic extract of Ixora javanica flowers (MEIJ)
global problem due to their higher morbidity and mortality, for the detection of various phytochemicals.9
as well as their medical, social and economic
impact.1 Population upsurge, the scanty supply of drugs, the 2.3 Identification of phytochemical constituents using
expensive cost of treatments, side effects of several synthetic GC-MS
drugs, and development of resistance to presently used drugs
for transmittable diseases have led to the augmented The GC-MS analysis was carried out by Shimadzu,
emphasis on the use of plant materials as a source of GCMSQP2010 model instrument coupled with mass
remedies for a varied variety of human illness.2 Helicobacter spectroscopy as the detector. The ZB-5MS Column with
pylori, genetic factors, alcoholic beverages and the use of non- dimensions 30m×0.32mm×0.25µm was used for analysis. The
steroidal anti-inflammatory drugs (NSAIDs) are the main oven temperature was adjusted to 50 ̊C and the solvent cut
contributing factors for gastric ulceration. Antacids, time 5 min. The column flow is 1.5 mL. The inlet
anticholinergics, proton pump inhibitors and histamine H2- temperature was kept at 250 ̊C, and the source temperature
receptor antagonists are drugs currently used for the of 210 ̊C and an interface temperature of 260 ̊C.
treatment however, they cannot be tolerated in the long-
term because of their safety profile.3 Thus, medicinal plants 2.4 Animals
have become attractive options for the development of
newer agents due to their lower side effects.4 Ixora Wistar albino rats of both sexes weighing up to 200-250 g
javanica, belonging to the family Rubiaceae, is traditionally were used. The animals were accommodated in enclosures
beneficial for numerous infirmities like hepatic disorder, under standard conditions. All the experimental protocols
cancer, microbial infection, antioxidant, pain, edema etc and were duly approved by the Institutional Animal Ethics
has been predictable for several therapeutic properties.5 The Committee (Protocol Apvl No: 1175/PO/Re/S/08/ CPCSEA).
flowers and leaves of Ixora javanica are used as a sedative
stomachic tonic, intestinal antiseptic and as astringent. The 2.5 Acute toxicity Studies
flower parts of this plant are used to heal sores and chronic
ulcers. Peptic ulcer disease had been a significant cause of Acute toxicity training was conceded out on Wistar albino
morbidity and mortality for almost more than a century. The mice by the oral route at a dose of 2000 mg/kg of the
term peptic ulcer refers to an acid peptic injury of the methanolic extract of Ixora javanica flowers as per the
digestive tract, resulting in mucosal break reaching the OECD- guidelines 425.10
submucosa. Peptic ulcers are usually located in the stomach
or proximal duodenum, but they can also be found in the 2.6 Experimental models
esophagus or Meckel's diverticulum. Traditionally, a 2.6.1 Ethanol-induced gastric ulcer model
hypersecretory acidic environment, together with dietary
factors or stress was thought to cause most peptic ulcer After 48 h fasting, rats were divided into 4 groups of 6
diseases. However, the discovery of Helicobacter animals (each group) and treated orally with distilled water
pylori infection and the ubiquitous use of Non- Steroidal Anti- (10 ml/kg), extract (200 and 400 mg/kg), and omeprazole (20
Inflammatory Drugs (NSAIDs) in the mid-20th century have mg/kg). Sixty minutes after this procedure, every animal
changed this perception.6 Recently, herbal compounds have received ethanol (1 ml/200 g). One hour later, the rats were
played an important role in the discovery and development euthanized, stomachs were removed, opened along the
of modern drugs against ulcers and other diseases due to greater curvature, and the ulcer score was determined.11
their potentially improved safety and efficacy over
conventional treatments.7 Its flowers are traditionally used 2.6.2 Indomethacin induced gastric ulcer model
for chronic ulcers.8 There have been no reports on antiulcer
activity of Ixora javanica in the current literature. Therefore, Rats were fasted for 48 h and treated orally with vehicle
the present study assessed the antiulcer activity of the (distilled water, 10 ml/kg), extract (200 and 400 mg/kg), and
methanolic extract of Ixora javanica and their isolated omeprazole (20 mg/kg). One hour after the treatment, 60
compounds affinity with H+/K+ ATPase inhibitory effect so mg/kg of indomethacin was administered orally to all the
that their binding interactions can be studied, with the aim of groups. Four hours later, the animals were euthanized,
developing a safe and effective drug for treating gastric ulcers. stomachs were removed, opened along the greater
curvature, and the ulcer score was determined.11
2. MATERIALS AND METHODS
2.6.3 Pylorus ligation induced gastric ulcer model
2.1 Plant material
Rats were fasted for 36 h and divided into 4 groups of 6
The flowers of Ixora javanica were collected and air-dried animals in each and treated orally with distilled water (10
under shade, powdered mechanically and stored in an airtight mL/kg), extract (200 and 400 mg/kg), and omeprazole (20
container. The powder was extracted using a soxhlet mg/kg). The pylorus was ligated under light thiopental
sodium anaesthesia with care taken not to cause bleeding or
apparatus and ethanol as solvent, dried, and stored in a
to occlude blood vessels. Six hours after ligation, the animals
refrigerator for further use. The plant part was authenticated were sacrificed by an overdose of thiopental sodium, and the
(IJ 27022018) by a botanist at the New Government Degree stomach part was isolated, contents were collected,
College, Kukatpally, Hyderabad, India. measured for volume, and subjected to analyse the acidity
against 0.1 N NaOH to pH 8 using a pH meter. The total

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acid output was calculated. Each stomach was examined for 2.6.5 Determination of total acidity
lesions.12,13
A fraction of 1mL gastric juice diluted with 1mL of distilled
2.6.4 Determination of pH water was taken into a 50 mL conical flask, and two drops of
phenolphthalein indicator was added to it and titrated with
A fraction of 1mL gastric juice was diluted with 1mL of 0.01N NaOH until a permanent pink colour was observed.
distilled water, and the pH of the solution was measured The volume of 0.01N NaOH consumed was noted.12,13 The
using pH meter.12,13 total acidity is expressed as mEq/L by the following formula:

Acidity = (Vol. of NaOH × N × 100)/0.1 mEq/L

2.6.6 Determination of Free acidity 2.6.7 Determination of Gastric volume

The Topfer's reagent was used. A fraction of gastric juice After sacrificing the animal, the stomach is dissected out,
was titrated with 0.01N NaOH until the canary yellow gastric juice is collected, drained into tubes & was
centrifuged at 1000 rpm for 10 min, and volume is noted.14
colour was observed. The volume of 0.01N NaOH
consumed was noted. The free acidity was calculated by the 2.6.8 Percentage protection
same formula for the determination of total acidity.
Percentage protection was calculated by using the formula:

% protection = ([Ulcer Score of control – Ulcer Score of the test]/ Ulcer Score of control) X 100

2.6.9 Histopathology
3. STATISTICAL ANALYSIS
For histopathology assessment, the dissected stomach tissues
were fixed in a 10% buffered formalin solution. Sections were Data were expressed as mean ± S.E.M. Comparisons
deparaffinised and were stained with haematoxylin and between means of different groups were analyzed by
eosin.12 ANOVA followed by the Dunnett’s test. The Graph Pad
Prism software package, version 8 (Graph Pad Software, Inc.,
2.7 Docking studies San Diego, CA, USA), was used to perform all statistical
investigations.
Docking simulations predicted the binding orientation of drug
candidates to their protein targets. Glide 5.6 (Schrodinger 4. RESULTS AND DISCUSSION
Inc.) was used for generating docking simulation studies.
2.1 Phytochemical screening
Docking simulations were performed in Dell precision T-
1500 workstation Intel (R) Core (TM) i7 CPU 860 @GHz; The preliminary phytochemical investigation for the
12.0 GB Ram, 1 TB Hard disk. Protein-ligand interactions methanolic extract of Ixora javanica flowers showed the
were visualized using Maestro 9.1. Proton pump inhibitors presence of flavonoids, tannins, steroids, glycosides,
(PPIs) block the gastric H+/K+ ATPase, inhibiting gastric acid carbohydrates, alkaloids, saponins, and terpenoids.
secretion. The crystallized x-ray structure of H+/K+ ATPase
(PDB ID: 5A5N) was retrieved from the RCSB protein bank. 4.2 Identification of phytochemical constituents using
GC-MS
Protein-ligand interactions were stimulated though flexible
glide-ligand docking with XP extra precision mode. The best- The crude methanolic extract of the Ixora javanica flower was
docked structures were chosen using the glide score subjected to GC-MS analyzer. Nearly 40 bio compounds
function. The more negative the glide score, the more were identified from extract, namely formononetin, ferulic
favourable the binding. Additionally, the docked ligand poses acid, quinic acid, palmitic acid, oleanolic acid, maslinic acid,
were visualized, and the different ligand-receptor interactions rutin, serine, supraene, malonic acid, 2-t-butyl-4-methyl-5-
were studied.15 In the present study, six compounds, namely oxo (1,3) dioxolane carboxylic acid, erythro 4- hydroxy
arginine lactone in high amounts. Fig 1 shows the GC-MS
formononetin, ferulic acid, rutin, oleanolic acid, ursolic acid,
chromatogram of the extract.
and maslinic acid, are docked against H+/K+ ATPase (PDB
ID: 5A5N).

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Fig 1: GC-MS Chromatogram of MEIJ

4.3 Acute toxicity studies Table 1. The extract at a dose of 200 mg/kg bd. wt., 400
mg/kg bd. wt. and standard drug omeprazole at 20 mg/kg bd.
Methanolic extract of I. javanica flowers was tested on female wt. showed significant inhibition of ulcers by 44%, 65.05%,
Wistar Albino mice up to a dose of 2000 mg/kg bd.wt., p.o. and 87.78%, respectively.
Methanolic extract of I. javanica flowers did not exhibit any
signs of toxicity and mortality up to 2000 mg/kg. bd.wt. All 4.4.1 Macroscopic appearance of the gastric mucosa in
animals were safe even after 14 days of observation. The Ethanol-induced ulcer models
pharmacological evaluation was done using 200 and 400
mg/kg bd. wt. In figure 2, disease control 4-5 bands of thick and dark red
erosions with 0.5-1 mm in width were observed. In MEIJ, 200
4.4 Ethanol-induced gastric ulcer model mg/kg bd.wt 1-2 bands of thick and dark red erosions and
erythema were observed. In MEIJ, 400 mg/kg bd.wt 3-4 bands
Methanolic extract of I. javanica flower extracts has shown a of light red erosions and erythema were observed. In
significant reduction in ulcer scores at 200 mg/kg., bd.wt Omeprazole, 20 mg/kg bd.wt no lesions, but slight erythema
(p<0.001), 400 mg/kg., bd.wt (p<0.001) and standard was observed. A significant reduction in ulcers was observed
omeprazole 20 mg/kg., bd. wt.) (p<0.001) when compared to in the test extract and the standard groups when compared
the disease control group. The results were tabulated in to the disease control group.

Fig 2: Macroscopic appearance of the gastric mucosa in Ethanol induced ulcer model

4.4.2 Histopathology studies of ethanol-induced gastric hyperplasia was observed. In MEIJ 400 mg/kg., bd. wt. group,
ulcerated rat stomach scant inflammatory cells, and gastric mucosal thickness
appeared to be normal, no pits were observed. In the
Rat's stomach in the disease control group, gastric mucosal standard omeprazole (20 mg/kg bd.wt.) group, mucosal
hyperplasia, gastric pits, and inflammation were observed. In thickness appeared to be normal, no inflammation and no
MEIJ 200 mg/kg., bd. wt. group, gastric mucosa scant pits were observed (Figure 3).
inflammatory cells appeared. No gastric pits but slight

Fig 3: Histopathological study of ethanol induced gastric ulcerated rat stomach

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Ethanol is an ulcerogenic agent that induces gastric mucosa ethanol-induced ulcers. Terpenoids, flavonoids, tannins,
by promoting disturbances of mucosal microcirculation, glycosides, and alkaloids present in MEIJ could be the reason
ischemia, and appearance of free radicals, endothelin release, for its antiulcer activity in ethanol-induced ulcers.
degranulation of mast cell and inhibition of prostaglandins and
decrease of gastric mucus production.16 Oleanolic acid 4.5 Indomethacin induced gastric ulcers
(terpenoid) treatment caused a significant increase in PGE2
Methanolic extract of I. javanica flowers has shown a
content in the gastric epithelial cells, which resulted in a
significant reduction in ulcer scores at 200 mg/kg., bd.wt
decrease in ulcers.17 Ursolic acid (terpenoid) decreases in the (p<0.001), 400 mg/kg., bd.wt (p<0.001) and standard
LPO level while the increase in SOD, CAT, and GSH levels. omeprazole 20 mg/kg bd. wt. (p<0.001) when compared to
Phenols exhibit antioxidant properties by the virtue of the disease control group. Results were shown in Table 1.
scavenging free radicals by breaking radical chain reactions, The extract at a dose of 200 mg/kg bd. wt., 400 mg/kg bd. wt.
attenuating peroxides level, and triggering antioxidant and standard drug omeprazole at 20 mg/kg bd. wt. showed
defense enzyme systems contributing to the antiulcer effect. significant inhibition of ulcers by 47.78%, 63.05%, and 82.63%,
respectively.
Tannins have astringent effects which stimulate protein
precipitating and vasoconstriction resulting in information of 4.5.1 Macroscopic appearance of the gastric mucosa in
impenetrable protective barriers preventing gastric ulcer by indomethacin-induced ulcer model
reducing the number of ulcer score.18 Formononetin
(flavonoid) decreases the abruptly increased MDA Figure 4 depicts the macroscopic appearance of the gastric
concentration due to exposure to ethanol.19 Glycosides mucosa in the indomethacin-induced ulcer model. In disease
possess potent antioxidant properties by decreasing lipid control (group I), grade 5 lesions, along with haemorrhage,
were observed. In group II (MEIJ, 200 mg/kg bd. wt), 2-3
peroxidation and increasing antioxidant level.20 Alkaloids have
erosions with <5 mm in length and 0.5-1 width erythema
gastro protective effects by stimulating nitric oxide were observed. In group III (MEIJ, 400 mg/kg bd. wt) pits in
manufacture, acting by modifying the levels of pro- the gastric mucosa were observed. Group IV (Omeprazole,
inflammatory cytokines (IL-8), and reducing the action of 20 mg/kg bd. wt) no lesions, but small petechiae were
myeloperoxidase (MPO) and lipid peroxidation, signifying a observed. A significant reduction in ulcers was observed in
potential antioxidant activity.21 MEIJ significantly reduced the the test extract and the standard groups when compared to
the disease control group.
ulcer index and afforded significant protection against

Fig 4: Macroscopic appearance of the gastric mucosa in indomethacin induced ulcer model

NSAIDs induce gastric ulcers by inhibiting prostaglandin indomethacin-induced ulcers. Flavonoids, saponins, steroids,
synthesis via the cyclooxygenase pathway. In the stomach, and alkaloids present in the extract might be responsible for
prostaglandins protect against mucosal injury by stimulating the antiulcer activity.
bicarbonate and mucus secretion, maintaining mucosal blood
flow, and regulating mucosal cell turnover and repair.22 4.6 Pylorus ligation induced gastric ulcers
Flavonoids inhibited the activity of PG metabolizing enzyme
15-hydroxy-PG- dehydrogenase and elevated the PGE2 Methanolic extract of I. javanica flower extracts has shown a
content of the gastric mucosa in Wistar Albino rats subjected significant reduction in ulcer scores at 200 mg/kg., bd.wt
to absolute ethanol-induced gastric mucosal damage.23 (p<0.001), 400 mg/kg., bd.wt (p<0.001) and standard
Saponins have ulcer protective effect by the formation of omeprazole 20 mg/kg bd. wt. (p<0.001) when compared to
protective mucus, which shields the mucosa from acid the disease control group. The results were tabulated in
damage by selectively inhibiting prostaglandins. Steroids have Table 1. The extract at a dose of 200 mg/kg bd. wt., 400
caused the inhibition of gastric acid secretion or boosting the mg/kg bd. wt. and standard drug omeprazole at 20 mg/kg bd.
mucosal defense mechanism by increasing mucus production, wt. showed significant inhibition of ulcers by 45.35%, 61.74%,
stabilizing the surface epithelial cells, or interfering with the and 81.96%, respectively.
PGs synthesis.20 Alkaloids reduce the levels of gastric juice
and free/total pepsin, blocked the activity of H+/K+ ATPase, 4.6.1 Macroscopic appearance of the gastric mucosa in
normalized gastrin levels, and reduced levels of Ca2+ in the pylorus induced ulcer model
parietal cells; furthermore, it enhanced gastric mucosa
defense mechanisms by increasing the levels of prostaglandin In the disease control group, 1-3 erosions with 0.5-1 mm and
E2 (PGE2) and mucin.21 MEIJ significantly reduced the ulcer slight haemorrhage were observed. In MEIJ, 200 mg/kg bd.wt
index and afforded significant protection against group, small erosions with <5 mm in length, and erythema

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were observed. In MEIJ (400 mg/kg bd.wt.) group, petechiae reduction in ulcers was observed in the test extract and the
were observed and omeprazole, 20 mg/kg bd.wt., group, standard groups when compared to the disease control
erythema was observed, as shown in figure 5. A significant group.

Fig 5: Macroscopic appearance of the gastric mucosa in pylorus induced ulcer model

Pylorus ligation causes accumulation of acid and pepsin, the mucosa from toxins and other irritants.27 Saponins have
which leads to the auto digestion of gastric mucosa and also been stated to retain the antiulcer property, possibly
ulceration.24 Ferulic acid (phenol) administration of ferulic due to its surfactant-like properties.28 MEIJ has shown a
acid to pylorus ligated animals resulted in a decrease in ulcer significant reduction in the gastric volume, free and total
index and volume of gastric contents. Total acidity and free acidity, and an increase in the pH, thus, proving its antiulcer
acidity was reduced upon administration of ferulic acid along activity. The phenolic acids, terpenoids, tannins, and saponins
with decrement of lipid peroxidation.25 Ursolic acid present in the MEIJ could be responsible for the reduction of
(Terpenoid) treated Wistar Albino rats showed a significant ulcers in the pylorus ligated rats. The compounds interacted
increase in gastric pH and gastric juice volume and total acid- with the active site of H+/K+ ATPase was found to inhibit
pepsin output. Gallic acid (Phenolic acid) caused a decrease in H+/K+ ATPase activity. Formononetin, ferulic acid, and rutin
gastric juice volume, total acidity, free acidity, and pepsin have shown the highest docking scores when compared to
concentration. Pepsin concentration significantly decreased other compounds. More negative, the glide score is better
after treatment with gallic acid. Gallic acid decreased the than the binding affinity. Hydrogen bonding is an exchange
plasma protein leakage from gastric mucosa by strengthening reaction where the hydrogen bond donors and acceptors of
the mucosal barrier. The strengthening of mucosal defense is the free protein and ligand break their hydrogen bonds with
further exemplified by a decrease in cell exfoliation, as seen water and form new ones in the protein-ligand complex.29
from the reduction in DNA content of the gastric juice.26 More negative the score, stronger is the hydrogen bonding.
Tannins possess as an antiulcer agent by its astringency Rutin and oleanolic acid have the highest hydrogen bonding
property and vasoconstriction effects. Due to the score when compared to other compounds. Ferulic acid,
precipitation of micro proteins on the ulcer site, a protective formononetin, and ursolic acid have the highest lipophilicity
layer was formed, which hinders gut secretions and protects score when compared to other compounds.

Table 1: Effect of MEIJ on various groups in different models of rats

Ethanol induced Indomethacin induced Pylorus ligation
Groups
Ulcer Score % Protection Ulcer Score % Protection Ulcer Score % Protection
Disease control 4.75±0.11 - 7.66±0.21 - 3.66±0.21 -
*,$ *,$ *,$
MEIJ (200 mg/kg) 2.66±0.1 44% 4.00±0.3 47.78% 2.00±0.18 45.35%
MEIJ (400 mg/kg) 1.66±0.1*,$ 65.05% 2.83±0.21*,$ 63.05% 1.40±0.08*,$$ 61.74%
Omeprazole
0.58±0.08* 87.78% 1.33±0.01* 82.63% 0.66±0.1* 81.96%
(20 mg/kg)

Values are expressed as mean ± SEM, (n=6). Statistical analysis was performed by using ANOVA followed by Dunnett’s test.
Results were expressed as (*= p <0.001) vs disease control group and ($= p <0.001, $$= p <0.05) vs standard group.

4.7 Ulcer healing study (p<0.001) and standard omeprazole (20 mg/kg bd. wt.)
(p<0.001). The MEIJ has shown a significant increase in the
Methanolic extract of I. javanica flowers has shown a pH at 200 mg/kg bd. wt. (p <0.05), 400 mg/kg bd. wt.
significant reduction in the gastric volume, free and total (p<0.05) and standard omeprazole (20 mg/kg bd. wt.) (p
acidity at 200 mg/kg bd. wt. (p <0.001), 400 mg/kg bd. wt. <0.001), as shown in Table 2.

Table 2: Effect of MEIJ on gastric contents

Group Gastric volume pH Free acidity Total acidity
Normal control 0.366±0.09 1.86±0.08 58±0.7 46±0.5
Disease control 2±0.1 1.21±0.05 85.5±0.4 76.166±0.7
#,*,$ #,**,$
MEIJ (200 mg/kg) 1.466±0.06 3.1±0.04 77.167±0.4 #,*,$ 67±0.5 #,*,$
*,$ #,**,$$
MEIJ (400 mg/kg) 1.01±0.06 3.6±0.08 68.667±0.8 #,*,$ 58.666±0.8 #,*,$

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Omeprazole
0.633±0.04 #,* 4.66±0.06 #,* 60.83±0.6 #,* 50.33±0.49 #,*
(20 mg/kg)

Values are expressed as mean ± SEM, (n=6). Statistical analysis was performed by using ANOVA followed by Dunnett’s test. Results were expressed
as (#= p <0.001) vs normal control group (*= p <0.001, **= p <0.05) vs disease control group and ($= p <0.001, $$= p <0.05) vs standard group.

4.8 Molecular Docking show that formononetin is having highest glide score (-8.37)
amongst the test compounds followed by ferulic acid (-8.30),
Docking studies of MEIJ and standard omeprazole were rutin (-7.34), oleanolic acid (-5.53), ursolic acid (-4.30),
observed against H+/K+ ATPase protein (PDB ID: 5A5N). maslinic acid (-3.60). Standard compound omeprazole has a
The parameters analyzed in the study include glide score, glide score of -8.63. The hydrogen bonding interactions of
hydrogen bonding, and lipophilicity (Table 3). The results these compounds were shown in Figure 6.

Table 3: Docking results of MEIJ against H+/K+ ATPase protein (PDB ID: 5A5N)
S. No Name of the compound Glide score Hydrogen bond Lipophilicity
1 Omeprazole -8.63 -1.61 -0.13
2 Formononetin -8.37 -0.70 -3.32
3 Ferulic acid -8.30 -0.56 -3.31
4 Rutin -7.34 -3.54 -1.50
5 Oleanolic acid -5.53 -1.66 -0.71
6 Ursolic acid -4.30 0 -2.31
7 Maslinic acid -3.60 -0.96 -1.17

Fig 6. Docking interaction studies of isolated compounds from MEIJ with the PDB- 5A5N

In the figure 6, docking interaction studies were represented bond interaction with Val 1008, Phe 1009, Val 1013, Glu
having the following interactions. A: Hydrogen bonding 1017, Val 1018, Tyr 1021, Ala 1060, Ile 1074. C: Hydrogen
interaction of omeprazole with PDB- 5A5N. Omeprazole bonding interaction of ferulic acid with PDB- 5A5N. Ferulic
(total score- 8.63) demonstrated hydrogen bonding acid (total score -8.30) demonstrated hydrogen bonding
interactions with Asn 1064 and lipophilic bond interactions interaction with Met 1029 and lipophilic bond interaction
with Ile 1074, Val 1018, Tyr 1063, Val 1013, Val 1021. B: with Phe 1009, Val 1013, Tyr 1021, Ala 1060, Tyr 1063, Ile
Hydrogen bonding interaction of formononetin with PDB- 1074. D: Hydrogen bonding interaction of rutin with PDB-
5A5N. Formononetin (total score – 8.37) demonstrated 5A5N. Rutin (total score -7.34) demonstrated hydrogen
hydrogen bonding interaction with Tyr 1063 and lipophilic bonding interaction with Arg 1077, Asn 1064 and lipophilic

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bond interaction with Val 1008. Val 1013, Ala 1060, Ile 1074. thus, proving its antiulcer activity. Molecular docking studies
E: Hydrogen bonding interaction of oleanolic acid with PDB- confirmed the H+/K+ ATPase inhibitory effect of the
5A5N. Oleanolic acid (total score -5.53) demonstrated compounds obtained from GC-MS study. Further studies are
hydrogen bonding interaction with Arg 1007, Lys 1011 and required to focus on the isolation of specific phytochemicals
lipophilic bond interaction with Asp 1030, Val 1008, Thr and elucidating the mechanism of action.
1010, Phe 1009, Pro 1012, Val 1018, Val 1013. F: Hydrogen
bonding interaction of ursolic acid with PDB- 5A5N. Ursolic 6. AUTHORS CONTRIBUTION STATEMENT
acid (total score -4.30) demonstrated lipophilic bond
interaction with Val 1008, Pro 1012, Val 1013, Val 1018, Ile The authors Dr M. Ganga Raju, Anusha, Gayathri and Nikitha
1074. G: Hydrogen bonding interaction of maslinic acid with conceived the present idea. Anusha, Gayathri, and Nikitha
PDB- 5A5N. Maslinic acid (total score -3.60) demonstrated designed and performed the experiments. Dr M. Ganga Raju
hydrogen bonding interaction with Val 1008 and lipophilic and Dr. NVL Suvarchala analyzed the data and wrote the
bond interaction with Val 1008, Val 1013, Val 1018. paper with input from all authors.

5. CONCLUSION 7. ACKNOWLEDGEMENTS

The plant extract was prepared using methanol, and the The authors are grateful to the principal and management of
phytochemical screening revealed the presence of the Gokaraju Rangaraju College of pharmacy, for the
phytoconstituents such as terpenoids, flavonoids, tannins, constant support and encouragement during the course of
steroids, alkaloids, saponins, glycosides, and carbohydrates. the work.
The GC-MS analysis of extract showed the presence of 40
bio compounds. Acute toxicity studies have revealed that 8. CONFLICT OF INTEREST
methanolic extract of Ixora javanica flowers was found to be
safe up to 2000 mg/kg bd.wt. The methanolic extract of Ixora Conflict of interest declared none.
javanica flowers has shown a significant reduction in ulcers;

9. REFERENCES

1. Ji CX, Fan DS, Li W, Guo L, Liang ZL, Xu RM, Zhang 10. Test No. 425: acute oral toxicity: up-and-down
JJ. Evaluation of the anti-ulcerogenic activity of the procedure. OECD guidelines for the testing of
antidepressants duloxetine, amitriptyline, fluoxetine chemicals. Section 4; 2008.
and mirtazapine in different models of experimental doi: 10.1787/9789264071049-en.
gastric ulcer in rats. Eur J Pharmacol. 2012 Sep; 691(1- 11. Sofidiya MO, Agunbiade FO, Koorbanally NA,
3):46-51. Sowemimo A, Soesan D, Familusi T. Antiulcer activity
doi: 10.1016/j.ejphar.2012.06.041, PMID 22789173. of the ethanolic extract and ethyl acetate fraction of
2. Khan MA. Introduction and importance of medicinal the leaves of Markhamia tomentosa in rats. J
plants and herbs | national health portal of India Ethnopharmacol. 2014;157:1-6.
[internet]; updated 2016 May 20. National Health doi: 10.1016/j.jep.2014.09.012, PMID 25240588.
Portal India. 12. Nawale SR, Priyanka N, Das S, Ganga Raju M. Data of
3. Santin JR, Lemos M, Klein Júnior LCK, Niero R, de in vivo screening of antiulcer activity for methanolic
Andrade SF. Antiulcer effects of Achyrocline satureoides extract of Vernonia elaeagnifolia DC. Data Brief. 2019
(Lam.) DC (Asteraceae) (Marcela), a folk medicine Mar; 23:103753. doi: 10.1016/j.dib.2019.103753.
plant, in different experimental models. J 13. Dordević S, Petrović S, Dobrić S, Milenković M,
Ethnopharmacol. 2010 Jul;130(2):334-9. Vucićević D, Zizić S, Kukić J. Antimicrobial, anti-
doi: 10.1016/j.jep.2010.05.014, PMID 20546870. inflammatory, anti-ulcer and antioxidant activities of
4. Sumbul S, Ahmad MA, Mohd A, Mohd A. Role of Carlina acanthifolia root essential oil. J Ethnopharmacol.
phenolic compounds in peptic ulcer: an overview. J 2007 Feb;109(3):458-63.
Pharm Bioallied Sci. 2011;3(3):361-7. doi: 10.1016/j.jep.2006.08.021, PMID 17011148.
doi: 10.4103/0975-7406.84437. PMID 21966156. 14. Sakat SS, Tupe P, Juvekar A. Gastroprotective effect of
5. Kharat AR, Nambiar VV, Tarkasband YS, Pujari RR. A Oxalis corniculata (whole plant) on experimentally
review on phytochemical and pharmacological activity induced gastric ulceration in Wistar rats. Ind J Pharm
of genus Ixora. Int J Res Pharm Chem. 2013 Jul– Sci. 2012 Jan–Feb;74(1):48-53.
Sep;3(3):628-35. doi: 10.4103/0250-474X.102543, PMID 23204622.
6. Lanas A, Chan FKL. Peptic ulcer disease. Lancet. 2017 15. Karim N, Khan I, Abdelhalim A, Abdel-Halim H,
Feb;390(10094):613-24. doi: 10.1016/S0140- Hanrahan JR. Molecular docking and antiamnesic
6736(16)32404-7, PMID 28242110. effects of nepitrin isolated from Rosmarinus officinalis
7. Ranjan KN, Shreechandan P, Priyadarshini P, Sampad on scopolamine-induced memory impairment in mice.
SS. Herbal drugs in treatment of Peptic Ulcer. J BIO Biomed Pharmacother. 2017 Dec;96:700-9.
Innov. 2017 May; 6(3):499-508. doi: 10.1016/j.biopha.2017.09.121, PMID 29040957.
8. Dontha S, Kamurthy H, Manthripragada B. 16. Nwidu LL, Nwafor P, Vilegas W. Antiulcer Effects of
Phytochemical and pharmacological profile of Ixora: a ethyl acetate Fraction of Carpolobia lutea Leaf. J App
review. Int J Pharm Sci Res;567-84:2015Feb; 6(2). Pharm Sci. 2012;2(8):233-42.
doi: 10.13040/IJPSR.0975-8232.6(2).567-84. doi: 10.7324/JAPS.2012.2841.
9. Khandelwal K. Practical pharmacognosy. 2nd ed. 17. Sánchez M, Theoduloz C, Schmeda-Hirschmann G,
MAH: Niral Prakashan; 2008. p. 149-56. Razmilic I, Yáñez T, Rodríguez JA. Gastroprotective
and ulcer-healing activity of oleanolic acid derivatives:

P- 105
ijlpr 2020; doi 10.22376/ijpbs/lpr.2021.11.2.P98-106 Pharmacology

in vitro–in vivo relationships. Life Sci. 2006 24. Kaur M, Singh A, Kumar B. Comparative antidiarrheal
Apr;79(14):1349-56.doi: 10.1016/j.lfs.2006.03.044. and antiulcer effect of the aqueous and ethanolic stem
18. Pandey D, Joshi A, Hemalatha S. Anti-ulcer study of bark extracts of Tinospora cordifolia in rats. J Adv
standardized ethanol root extract of Aganosma Pharm Technol Res. 2014 Jul–Sep;5(3):122-8. doi:
dichotoma and isolated ursolic acid. Int J Pharm Pharm 10.4103/2231-4040.137417, PMID 25126533.
Sci. 2017 Feb;9(4):172. 25. Umre R, Ganeshpurkar A, Ganeshpurkar A, Pandey S,
doi: 10.22159/ijpps.2017v9i4.16957. Pandey V, Shrivastava A, Dubey N. In vitro, in vivo and
19. Alauddin, Chaturvedi S, Azmi L, Shukla I, Naseem Z, in silico antiulcer activity of ferulic acid. Future J Pharm
Rao C, Agarwal N. Gastroprotective effect of
Sci. 2018;4(2):248-53. doi: 10.1016/j.fjps.2018.08.001.
formononetin against ethanol-induced gastric
ulceration in rats via augmentation of cytoprotective 26. Asokkumar K, Sen S, Umamaheswari M,
markers and curtailing apoptotic gene expression. Sivashanmugam AT, Subhadradevi V. Synergistic effect
Pharmacogn Mag. 2018 Jun;14(59):605-12. of the combination of gallic acid and famotidine in
doi: 10.4103/pm.pm_205_18. protection of rat gastric mucosa. Pharmacol Rep. 2014
20. Ignatius V, Narayanan M, Subramanian V, Periyasamy Aug;66(4):594-9.
BM. Antiulcer Activity of Indigenous Plant Operculina doi: 10.1016/j.pharep.2014.01.006, PMID 24948059
turpethum Linn. Evid Based Complement Alternat 27. Sahoo SK, Sahoo HB, Priyadarshini D, Soundarya G,
Med. 2013 Jan;2013:272134. Kumar ChK, Rani KU. Antiulcer activity of ethanolic
doi: 10.1155/2013/272134. PMID 23476683. extract of Salvadora indica (W.) leaves on albino rats. J
21. do Nascimento RF, de Sales IR, de Oliveira Formiga R, Clin Diagn Res. 2016 Sep;10(9):FF07-FF10.
Barbosa-Filho JM, Sobral MV, Tavares JF, Diniz Mde F, doi: 10.7860/JCDR/2016/20384.8470, PMID 27790462.
Batista LM. Activity of alkaloids on peptic ulcer: what’s 28. Onwukwe OS, Azubike NC, Eluke BC, Anulika OO,
new? Molecules. 2015 Jan;20(1):929-50. Cornelius OO, Ikechukwu CJ, Peter AU. Evaluation of
doi: 10.3390/molecules20010929, PMID 25580688. the antiulcer properties of aqueous and methanol
22. Thabrew M, Arawwawala LDAM. An Overview of in extracts of Vitex doniana on indomethacin induced
vivo and in vitro models that can be used for evaluating gastric ulcers in albino rats. Pharmacol Online. 2018
anti-gastric ulcer potential of medicinal plants. Austin Apr;1:68-74.
Biol. 2016 Jun;1(2):1007. 29. Zhao H, Huang D. Hydrogen bonding penalty upon
23. Parmar NS, Parmar S. Anti-ulcer potential of ligand binding. PLOS ONE. 2011 Jun;6(6):e19923.
flavonoids. Ind J Physiol Pharmacol. 1998;42(3):343-51. doi: 10.1371/journal.pone.0019923, PMID 21698148.

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