Screening of In-Vitro Antioxidant and Antiulcer Activity of Millingtonia hortensis Linn Flowers
Rashmi Rajeghorpade1*, Varsha Shende2,
Vinod Pawar2, Sohan Patel1, Satyajit Sahoo1
1. Dept. of Quality Assurance, Pioneer Pharmacy College (235), Vadodara-390019,
India
2. Dept. of Pharmacology, SVPM’s College of Pharmacy, Malegaon
(Bk), Baramati, 413115, MS, India.
Correspondence: rashmmerajeghorpadey@gmail.com
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Article Information
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Abstract
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Research Article
Received: 01/08/2024
Accepted: 17/08/2024
Published:31/08/2024
Keywords
Millingtonia
hortensis, Antiulcer Activity,
in-vitro
Antioxidant Activity,
Pylorus
Ligation,
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Investigations
were conducted on the polyphenolic content, in vitro antioxidant activity,
and antiulcer properties of the methanolic extract of Millingtonia hortensis Linn flowers MEMHF. Reducing power,
hydroxyl radical activity, and superoxide anion scavenging, while the
antiulcer activity was evaluated using rat pylorus ligation. As the quantity
of extract rose (5-100μg/ml), the antioxidant activity also increased. The
total flavonoid and phenol concentrations of MEMHF were determined to be
31.69 mg/ml GAE and 163.73 mg/ml QE per 1000 mg of extract, respectively.
Pretreatment with MEMHF at doses of 250 and 500 mg/kg resulted in a
substantial reduction in stomach volume, total acidity, free acidity, and
ulcer index. Additionally, it raised pH in comparison to the control group,
indicating antiulcer action. At the very least, some of the reported
antioxidant and antiulcer action may be attributable to the presence of
phenols and flavonoids.
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INTRODUCTION
Though very little is known about their mode of action, herbal
medications made from plant extracts are being used more and more to treat a
wide range of clinical disorders. The pharmacological analysis of many plants
utilized in the traditional Indian medical system is gaining popularity. [1].
The part that damage mediated by free radicals plays in the genesis of
human illnesses has garnered more attention in recent years. Humans' levels of
oxidants and antioxidants are kept in balance throughout regular metabolism,
which is crucial for maintaining ideal physiological conditions. A number of
chronic diseases, including heart disease, some types of cancer, and peptic
ulcers, have been linked to an imbalance that can result in an overproduction
of oxidants under specific circumstances. This imbalance can cause oxidative
damage to body cells and molecules. [2, 3]. Tissue damage is caused by
oxygen-derived free radicals, which include hydrogen peroxide, superoxide
anions, and hydroxyl radicals. These radicals are cytotoxic. Because they start
biomolecular oxidation, which results in cell death and oxidative stress, an
excessive quantity of ROS is dangerous. Furthermore, oxidative stress results
in unintentional enzyme activation and oxidative system damage. [3].
One of
the main gastrointestinal diseases, peptic ulcer disease (PUD), is brought on
by an imbalance between defensive (mucin, prostaglandin, and bicarbonate) and
offensive (acid, pepsin, and Helicobacter pylori) components. Thus, the main
treatment strategy for peptic ulcer disease has been to reduce the generation
of stomach acid. Proton pump inhibitors (PPIs) and H2 receptor antagonists are
two anti-ulcer medications that are available for the treatment of PUD;
however, clinical examination of these medications has shown adverse effects,
recurrence rates, and drug interactions. This has served as justification for
the creation of new antiulcer medications, and as a result, studies looking for
novel compounds have expanded to include medicinal plants that may provide more
protection and reduce the likelihood of relapses. [4].
Millingtonia
hortensis L. (Family: Bignoniaceae) is a widely dispersed and cultivated plant
that is sometimes referred to as the "Indian cork tree" and is also
known as "Akas neem" [5]. Thai traditional medicine uses the dried
flowers of Millingtonia hortensis L. as an asthma treatment. The
pharmacological actions of Millingtonia hortensis have been found to include
antifungal, antiproliferative, antimutagenic, antibacterial, and larvicidal
properties. Although this herb has some established medical uses, its
anti-ulcer activity has not yet been reported. [6].
Antioxidants are essential for scavenging and suppressing free
radicals, which protects people against infection and degenerative illnesses.
As far as we are aware, there are no scientific studies on its ability to treat
ulcers. Consequently, the goal of this study was to demonstrate the antiulcer
and antioxidant
properties.
MATERIALS AND METHODS
Plant Collection
and Identification:
The basic plant material of Millingtonia hortensis Linn flowers used
for the investigation was collected from Medicinal garden of S.V.P.M’s College
of Pharmacy, Malegaon (Bk) in the month of November-December. The plant
material was identified and authenticated by R.B. Deshmukh (Botanist),
Department of Botany, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar,
Baramati, Maharashtra, India.
Preparation
of Extract
Using a Soxhelet Extractor, the dried
flowers were coarsely pulverized and defatted with petroleum ether at 60–80˚C.
After that, methanol extraction was performed on the resulting marc at 60–65˚C.
A simple distillation process was used to concentrate the extract. The
semisolid extract was refrigerated below 10˚C in an airtight container. The
extract yield as a percentage was 41.06%.
Preliminary
Phytochemical Screening
In order to identify any
phytoconstituents present, a preliminary phytochemical screening was performed
on MEMHF using the conventional techniques outlined in K. R. Khandelwal's
Practical Pharmacognosy. [7].
Animals
Albino rats of the Wistar
strain (weighing 120–150 g) and albino mice (20–30 g) from the SVPM's College
of Pharmacy animal house were used in the pharmacological investigations. Six
animals in a group were kept in polypropylene cages with typical housing parameters:
a room temperature of 27 ± 3˚C, relative humidity of 60 ±10%, and a 12-hour
photo period. Before a week, all of the animals were allowed to become used to
the trial room setting. They were also fed regular mouse pellets from M/s
Prashant Traders, Pune, and were given access to tap water whenever they wanted
it. Normal standards of cleanliness were upheld. [8].
In accordance with CPCSEA rules, institutional animal
ethics committee (IAEC) of SVPM's College of Pharmacy, Malegaon (Bk), Baramati
(Reg. No. 1214/ac/08/CPCSEA) granted ethical permission for conducting animal
studies.
Acute Toxicity Study
Based on the acute toxic
class technique (OECD Guideline No. 423), the acute oral toxicity of MEMHF was
investigated in female albino mice (20–30 g) that were randomly selected and
kept under normal circumstances. Before the dose, the animals fasted for three
to four hours while having access to unlimited water. There was a one- to
two-hour eating restriction following the extract dosage. Animals were watched
separately for a total of 14 days, at least once during the first 30 minutes
following dose, and sometimes throughout the first 24 hours. [9].
In-vitro Antioxidant
Activity
Reducing
power
Oyaizu's approach was
used to measure the reducing power of MEMHF. A reference standard of sodium
metabisulphate (25 μg/ml) and MEMHF at varying concentrations (5-100 μg/ml)
were employed. [10] After that, 1 ml of
the sample was combined with 2.5 ml of K3[Fe(CN)6] (1%), and 2.5 ml of
phosphate buffer (0.2 M, pH 6.6). For twenty minutes, the mixture was incubated
at 50˚C. Following the incubation period, 2.5 ml of 10% trichloroacetic acid
was added to the mixture, and it was centrifuged for 10 minutes at 3000 rpm.
The absorbance (OD) was measured at 510 nm after the top layer of the solution
(2.5 ml) was combined with 2.5 ml of distilled water and 0.5 ml of FeCl3
(0.1%). Three separate people carried out the experiment.Higher absorbance of
the reaction mixture indicates greater reducing power.
Antioxidant
K3[Fe(CN)6] + FeCl3 K4[Fe(CN)6].3H2O
+ FeCl2
The reducing power (%) was
evaluated by using the formula-
[Absorbance of control- Absorbance of test]
Reducing power (%) = ×100
Absorbance of control
Superoxide anion scavenging activity
In this
experiment, 0.1 ml of sample solution with varying doses of MEMHF (5-100 μg/ml)
or ascorbic acid (25μg/ml) as a standard in water was combined with 1 ml of NBT
solution (156 μM NBT in 100 mM phosphate buffer, pH 7.4) and 1 ml of NADH
solution (468 μM in 100 mM phosphate buffer, pH 7.4). 100 μl of Phenazine
Methosulphate Solution (60 μM PMS in 100 mM Phosphate Buffer, pH 7.4) was added
to the mixture to start the reaction. Using a spectrophotometer set to 510 nm,
the absorbance was measured following five minutes of incubation at 25˚C. [11].
Decrease in absorbance of the reaction
mixture resulted in superoxide anion scavenging activity of the test samples.
Superoxide
ion scavenging/inhibition (%) was calculated by using the formula-
[Absorbance of control- Absorbance of test]
Scavenging (%) = ×100
Absorbance of control
Hydroxyl radical scavenging activity
The Hathwell
Gutteridge 2-deoxyribose degradation test has been used to quantify the
production of hydroxyl radicals [12]. 1 mM deoxyribose and 0.2 mM
phenylhydrazine hydrochloride were produced in 50 mM phosphate buffer (pH 7.4).
0.4 ml of sample solutions of MEMHF and Standard were collected, together with
0.6 ml of 1 mM Deoxyribose. 1.6 ml of reaction solution was created by adding
0.2 ml of phosphate buffer. 0.4 ml of 0.2 mM phenylhydrazine was added after 10
minutes of incubation. One hour of incubation was followed by the addition of
one milliliter of 2.8% TCA and one percent (w/v) thiobarbituric acid to the
reaction mixture, which was then heated for ten minutes in a boiling water
bath. After cooling the tubes, the absorbance was measured at 510 nm. Decrease in absorbance indicates the
increase in the hydroxyl radical scavenging activity. The % scavenging effect
is calculated as
[Absorbance of control- Absorbance of test]
Scavenging (%) = ×100
Absorbance of control
Total
Flavonoid Content
Quercetine was used as a
standard in the Aluminum chloride (AlCl3) technique to calculate the total
flavonoid content. After adding 0.25 ml of the MEMHF extract to 1.25 ml of
double-distilled water, 75 μlit of 5% NaNO2 was added. AlCl3 (0.15 ml, 10%) was
added after 5 minutes at room temperature. The reaction mixture was then
treated with 0.5 ml of 1 mM NaOH after a further 6 minutes of RT incubation.
Lastly, 275 μlit of double-distilled water were added to the reaction mixture
to dilute it. After another 20 minutes of RT incubation, the absorbance at 510
nm (Schimadzu UV-Vis 1700) was measured. Every test was run in triplets. The
standard value of quercetine was used to compute the flavonoid content. [13].
Total
Phenolic Content
The Folin-Ciocalteu
reagent was used to determine the phenols. Gallic acid (Standard Phenolic
Compound) or MEMHF (0.5 mg of 10 ml) extract was diluted and combined with
distilled water, aqueous Na2NO3, and Folin Ciocalteu reagent (5 ml, 1:10
diluted). After the solutions were let to remain for 15 minutes, the total
phenol content was measured using calorimetry (Schimadzu UV-Vis 1700) at 510
nm. Milligrams of GAE per g dry weight of extract were used to express the
concentration of all phenolic components in the extract. [13].
Evaluation
of Antiulcer Activity
Wistar Albino rats of either sex
weighing about 150-200g (pregnancy was excluded) were divided into four groups
of five each
Group 1st - Control
Group 2nd -
Standard
Group 3rd - MEMHF (250 mg/kg
body weight)
Group 4th - MEMHF (500mg/kg
body weight)
In order to
prevent coprophagy, the animals were housed in separate cages and fasted for 24
hours before having their stomachs ligated (PL) with water permitted. A minor
midline incision below the xiphoid process was made to expose the abdomen under
mild ether anesthesia. The pyloric section of the stomach was then gently
pulled out and ligated to prevent tension to the pylorus or injury to its blood
supply. With great care, the stomach is restored, and the abdominal wall's
disrupted sutures were repaired. The medications are taken orally one hour
before the pyloric ligation procedure. During the recovery phase, they are
denied food and liquids, and four hours after the procedure, they are killed.
The stomach is cut open, the contents are emptied into a tube, and the pH and
free and total acidity are measured. After that, the stomach is cut open along its
larger curvature so that any ulcers may be seen. The size and severity of
ulcers determine the degree of ulceration, which is rated from zero to five.
Place the stomach onto the slide glass The following arbitory scoring system
was used to grade the incidence or severity of lesion-
Score 10 –
Duodenal epithelium
Score 20 –
Petichial / Flank Haemorrhages
Score 30 – One or
two ulcers
Score 40 –
Multiple ulcers
Score 50 –
Perforated ulcers
Mean ulcers score for each animal is
expressed as ulcer index.
For five minutes,
centrifuge the stomach contents at 1000 rpm. Take note of the loudness. Take 1
milliliter of the supernant liquid and use pure water to dilute it to 10
milliliters. Using a pH meter, note the solution's pH. Using Topfer's reagent,
titrate the solution against 0.01N NaOH. When the solution takes on an orange
hue, titrate until the end point. Take note of the NaOH volume, which
represents the free acidity. Titrate the mixture more until the hue turns pink
again. [14].
Note the total
volume of NaOH which corresponds to the total acidity (mEq/i/100mg) can be
expressed as
Acidity
= Vol. of NaOH x Normality x 100 mEq/I/100g
0.1
Compare the
gastric volume, acidity and ulcer index of control animal and the animal
treated with ranitidine.
Statistical
Analysis
Every data point is expressed as the
mean ± standard error mean and subjected to one-way Analysis of Variance
(ANOVA). Graph Pad Prism 6 software is then used to compare the test and
control groups. The P value of < 0.001 was considered to be statistically
significant.
RESULTS AND DISCUSSION
Preliminary
phytochemical screening
Preliminary
phytochemical screening of MEMHF showed the presence of Flavonoids, Saponin
glycoside, Steroids, Tannic and Phenolic compounds.
Acute
toxicity study
The MEMHF was
studied for acute toxicity study at dose of 2000mg/kg i.p. in Swiss mice. The
extract was found devoid of mortality of animals. Hence 2500mg/kg was
considered as LD50 cut off value. So the doses selected for extract
as per the OECD guideline no. 423 (Annexure -2d) fixed dose method are
mentioned below
250 mg/kg (1/10th
of 2500mg/kg)
500mg/kg (1/5th of 2500mg/kg)
In vitro Antioxidant Activity
The MEMHF at 5-100 μg concentration
showed significant Antioxidant activity. The results are tabulated in table
1-4. Sodium metabisulphate used as Standard.
Reducing
Power
It was observed that MEMHF have
demonstrated concentration dependent increase in the % of absorbance, indicating
reducing power property. All the tested concentrations of MEMHF exhibited
greater absorbance than control and results found statistically significant.
100 μg of both extracts showed greater reducing power i.e. 193.65%. However,
sodium metabisulphate 25 μg (standard) showed reducing property (90.43%).The
results are summarized in Table 1.
Superoxide
Anion Scavenging Activity
All the concentrations of MEMHF
showed significant scavenging effect. MEMHF at100μg exhibited 57.80 %
inhibition. Sodium metabisulphate 25 μg showed 53.63 % scavenging activity. The
results are summarized in Table 2.
Hydroxyl
Radical Scavenging Activity
Phenyl hydrazine in solution
generates OH• radicals as measured by 2-deoxyribose degradation. It was
found that MEMHF at graded concentrations (5-100 μg) scavenged OH• radicals and
inhibit the production of TBA reactive material significantly. The results are
tabulated in Table 3.
Total Flavonoid and Total Phenolic Content
Results of total Flavonoid and total
Phenolic content was summarized in Table 4.
Antisecretory
and Antiulcer activity
The MEMHF at 250 and 500 mg/kg caused
comparable ulcer protection and also decreased the acid volume in pretreated
rats. The results are tabulated in table 5 and graph of different parameters are
showed in Fig 1 to 5.
Effect
on Stomach Histology
Histopathological profile of pyloric
ligated stomach of control group showed hemorrhages, congestion, leucocytic
infiltration, edema and necrosis. Pretreatment with MEMHF (250 and 500 mg/kg)
and Ranitidine (50 mg/kg) resulted comparatively less destruction than control.
Stomach Cyto-architecture is showed in Fig 6.
Table 1: Results of
the Reducing power of MEMHF
|
Groups/Treatment
|
Absorbance at 510 nm
|
% Reducing property
|
|
Control
Control + Standard 25 μg
Control + MEMHF 5 μg Control +
MEMHF 10 μg
Control + MEMHF 25 μg Control +
MEMHF 50 μg
Control + MEMHF 100 μg
|
0.11
± 0.008
0.22 ±
0.02***
0.16 ±
0.003
0.18 ±
0.003
0.19 ±
0.005***
0.24 ±
0.005***
0.34 ±
0.005***
|
-
90.43
36.88
53.63
68.18
110.23
193.65
|
Values are the mean ±
S.E.M., ( n=3)
Significance ***
P<0.001, **P<0.01, *P<0.05, compared to control
Table 2: Results Superoxide Anion Scavenging Activity of extract of MEMHF
|
Groups
|
Absorbance
At 510 nm
|
% Scavenging
Activity
|
|
Control
Control + Standard 25 μg
Control + MEMHF 5 μg
Control + MEMHF 10 μg
Control + MEMHF 25 μg
Control + MEMHF 50 μg
Control + MEMHF 100 μg
|
0.535 ±
0.012
0.195 ±
0.034***
0.480 ±
0.003
0.426 ±
0.008***
0.351 ±
0.009***
0.317 ±
0.005***
0.226 ±
0.003***
|
-
63.53
10.34
20.38
34.37
40.64
57.80
|
Values are the mean ±
S.E.M., (n=3)
Significance ***
P<0.001, *P<0.01, *P<0.05, compared to control.
Table 3: Results of Hydroxyl Radical Scavenging Activity of MEMHF
|
Group
|
Absorbance
At 510 nm
(After 1 hr)
|
Percentage
Inhibition
|
Absorbance
At 510 nm
(After 4 hr)
|
Percentage
Inhibition
|
|
Control
Control + Standard 25 μg
Control + MEMHF 5 μg
Control + MEMHF 10 μg
Control + MEMHF 25 μg
Control + MEMHF 50 μg
Control +
MEMHF 100 μg
|
0.34
± 0.02
0.11
± 0.01***
0.34
± 0.006
0.31
± 0.007
0.23
± 0.006***
0.18
± 0.004***
0.11
± 0.005***
|
-
66.65
7.28
8.96
35.76
48.18
68.73
|
0.36
0.13
± 0.018***
0.34
± 0.004
0.28
± 0.006*
0.22
± 0.006***
0.16
± 0.005***
0.12
± 0.005***
|
-
64.12
6.42
76.84
38.55
65.82
65.39
|
Values are the mean ±
S.E.M., (n=3)
Significance ***
P<0.001, *P<0.01, *P<0.05, compared to control.
Table 4: Results of Total Flavonoid and Total Phenolic content of MEMHF
|
Extract
|
Total Flavonoid Content
(mg/ml Quercetine equi. per 1000 mg Extract)
|
Total Phenolic Content
(mg/ml Gallic acid
equi. per 1000 mg Extract)
|
|
MEMHF
|
163.73 ± 0.120
|
31.69 ± 0.084
|
Table 5: Results of Antiulcer activity of MEMHF
|
Group/Treatment
mg/kg
|
Acid volume
(ml/100gm,b.w)
|
pH
|
Free acidity
(mEq/l/100mg)
|
Total acidity
(mEq/l/100mg)
|
Mean ulcer index
|
|
Control
MEMHF
250mg/kg,
p.o
MEMHF
500mg/kg,
p.o
Std Ranitidine
50mg/kg,
p.o
|
3.51±1.158
2.35±1.263***
2.25±1.603***
1.91±0.105***
|
2±1.702
4±1.250***
4.5±1.175***
4.67±0.964***
|
53±1.812
39.33±3.378***
23.5±4.266***
15.75±1.566***
|
67.5±1.153
56.66±3.506***
34.5±4.196***
28±2.353***
|
50
35
32.5
28.24
|
(Values are mean ±SEM,
for % animals)
In control group, the
production of ulcer and increase in acid volume was observed. Pretreatment with
MEMHF at 500mg/kg showed marked protection in ulcers which was significant
protection in ulcers which was significant when compared with control. Millingtonia hortensis (500mg/kg) showed
maximum inhibition of gastric acid, free acid and total acid. Also lower dose
could able to show the observable mucosal protection.
|
|
Hemorrhages and congestion
|
|
|
Ulceration
|
|
|
Healing
|
|
|
Leucocytic
infiltration
|
|
|
Necrosis
|
|
|
Edema
|
Fig.6. Stomach
Cyto-architecture. (a) Stomach architecture of Normal control (b) Stomach architecture after Pylorus Ligation (c) Stomach architecture of PL+MEMHF 250
mg/kg (d) Stomach architecture of PL+MEMHF 500
mg/kg.
DISCUSSION
Numerous antioxidant methods and
modifications have been proposed to evaluate antioxidant activity and to
explain how antioxidants function. Of these, reducing power, superoxide anion
scavenging activity, and hydroxyl radical scavenging activity are the most
commonly used for the evaluation of antioxidant activities of extracts.
It was reported that oxidative
stress, which occurs when free radical formation exceeds body’s ability to
protect itself, forms the biological basis of different chronic conditions.
Based on the data obtained from this study, MEMHF exhibits free radical
indicator or scavenging activity as well as a primary antioxidant that reacts
with free radicals, which may limit free radical damaging effect [15].
Peptic ulcer results due to
overproduction of gastric acid or decrease in gastric mucosal production.
Pylorus Ligation-induced ulcers occur because of an increase in acid-pepsin
accumulation due to pylorus obstruction and subsequent mucosal digestion.
Further the role of free radicals is also reported in the induction of ulcers
[16, 17].
The present study reveals that MEMHF
treated groups showed a significant (P < 0.001) increase in gastric juice
pH, reduces the gastric volume, free acidity, total acidity and improved cyto-architecture
of stomach.
The most important mechanism of
action responsible for the anti-ulcer activity of MEMHF is due to presence of
flavonoids and polyphenolic compounds. Flavonoids have antioxidant properties,
which involves free radical scavenging, transition metal ions chelation,
inhibition of oxidizing enzymes, increase of proteic and nonproteic
antioxidants and reduction of lipid peroxidation [18, 19, 20].
CONCLUSION
The result of our study
proved that crude extract of Millingtonia hortensis possess antioxidant and
antiulcer activity against different experimental models. Hence it can be
postulated that presence of polyphenols in Millingtonia hortensis may correlate
for the present activities.
ACKNOWLEDGEMENT
The authors
are grateful to management of S.V.P.M’s College of Pharmacy, Malegaon (Bk),
Baramati, for providing the necessary facilities through the Principal, College
of Pharmacy, Malegaon (Bk), Baramati, Dist-Pune, Maharashtra, India.
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