SCREENING OF METHANOLIC EXTRACT OF STERCULIA SCAPHIGERA WALL SEEDS FOR ULCERPROTECTIVE & ANTIOXIDANT ACTIVITY

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1332

Ogale et al. World Journal of Pharmacy and Pharmaceutical Sciences

SCREENING OF METHANOLIC EXTRACT OF STERCULIA

SCAPHIGERA WALL SEEDS FOR ULCERPROTECTIVE &

ANTIOXIDANT ACTIVITY

Sunita C. Ogale*1, Sanjay B. Kasture

1, Veena S. Kasture

2 , Roshan Tiwari

3,

Zaid Temrikar4

1Department of Pharmacology, VIVA Institute of Pharmacy, Virar (E),

Palghar, India

1Sanjivani College of Pharmaceutical Education and Research, Kopargaon, Dist.

Ahmednagar,India

ABSTRACT

In this study the antiulcer and antioxidant activity of methanolic extract

& methanolic fraction of Sterculia scaphigera wall seeds (MESS &

MFSS) was investigated. The methods used to induce ulcer were by

physical method i.e. by pyloric-ligation (4 hr.) and by chemical method

i.e. administering ethanol orally (40%,2ml/100gm, for 15 days, twice

daily). Pretreatment of rats with the methanolic extract (50 mg/kg body

weight,i.p.and p.o.) and methanolic fraction (25 and 50 mg/kg body

weight, p.o.) of Sterculia scaphigera markedly reduced pyloric-ligation

and ethanol - induced rise in volume of gastric acid secretion, total

gastric acidity, pH of gastric content and ulcer index which was

supported by the limited extent of histological damage.

KEY WORDS: Sterculia scaphigera, Antioxidant, Antiulcer, Ethanol sunita ogale.

INTRODUCTION

Sterculia scaphigera Wall is a tropical herb of the Sterculiaceae family, mainly distributed in

Vietnam, Thailand, Malaysia, Indonesia, and South China[1]

(Wang et al.,2003).In Chinese

system of medicine, this plant is commonly used for the treatment of phlegm and relieving

sore throat, and to relieve constipation[2]

(Xiao P. G., 2002). In India, seeds of this plant are

available in Herbal Pharmacies for treatment of constipation and hyperacidity. The water

WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS

SSJJIIFF IImmppaacctt FFaaccttoorr 22..778866

VVoolluummee 44,, IIssssuuee 0011,, 11333322--11334466.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357

*Correspondence for

Author

Sunita C. Ogale

Department of

Pharmacology, VIVA

Institute of Pharmacy,

Virar (E),

Palghar, India

Article Received on

13 Nov 2014,

Revised on 05 Dec 2014,

Accepted on 27 Dec 2014


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infusion of this herb can promote peristalsis of the intestines[3]

. However, there is no

systematic scientific study indicating these effects of Sterculia scaphigera. The seeds contain

alkaloids, sterculin, bassorin, tannins and some astringents[3]

(ENaturalHealthCenter.com

(e2121.com).

Since liver is the major drug metabolizing and detoxifying organ in the body, it is most

vulnerable to damage. Ethanol consumption is considered to be a risk factor in the

development of liver damage. Ethanol when administered chronically is known to potentiate

hepatotoxicity of carbon-tetrachloride (CCl4). Alcoholic liver disease (ALD) is the common

consequence of prolong and heavy alcohol intake. The fatal changes in the liver include fatty

liver, hepatitis and hepatic cirrhosis[4]

(Seitz et al,.2005). Reactive oxygen species (ROS) and

other free radicals believe to be the key mechanism of ALD[5]

(Lindros K.O.,1995). It is well

established that CCl4 is metabolized in the liver to highly reactive trichloromethyl radical

which initiate free radical-mediated lipid peroxidation of the cytoplasmic membrane

phospholipids and causes functional and morphological changes in the cell membrane leading

to accumulation of lipid-derived oxidants causing liver injury[6,7]

(Recknagel R.O.,1967;

Recknagel et al.,1989). It also induces hydropic degeneration, centrilobular necrosis, fatty

changes, cirrhosis and hepatoma[8]

(Smuckler et al.,1962). In the absence of reliable liver

protective drugs, herbs may play role in relieving liver disorders. Many plants having

antioxidant activity also demonstrate hepatoprotective activities[9]

(De et al.,1996).The

preliminary studies indicated that the methanolic extract and its methanol soluble fraction

possess antioxidant activity in the DPPH assay. Therefore the present study aims to

investigate the protective effect of methanolic extract and methanolic fraction of Sterculia

scaphigera on rat liver damage induced by ethanol-CCl4.

MATERIALS AND METHODS

Preparation of extract

The seeds of Sterculia scaphigera were purchased from Herbal Pharmacy at Nashik and

authenticated at the Agharkar Research Institute, Pune. The seeds (0.5 kg) were crushed to a

coarse powder and were defatted with petroleum ether (60-80OC) and then extracted with

methanol using cold extraction methods with occasional stirring for seven day. The extract

was dried in air (yield - 32g). The extract was stored in refrigerator and reconstituted in

distilled water just before use. The methanolic extract (8 g) was further fractionated with n-

hexane, ethyl aceate and methanol. The yield of methanolic fraction was approximately 1g.


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Drugs and Chemicals

1,1-diphenyl-2-picryl hydrazyl (DPPH) was purchased from Sigma-Aldrich, Mumbai.

Standard drug Ranitidine (GSK, India), Alcohol (Qualigens Fine Chemicals, Mumbai), were

used. All other reagents used were of analytical grade. All drug solutions were freshly

prepared before each experiment. Sterculia scaphigera extract & fraction were dissolved in

distilled water and administered orally.

Animals

Wistar Albino rats (150-200g) were obtained from Serum Institute, Pune. Animals were

housed five per cage under standard laboratory conditions of temperature (25 ± 20C) with

food and water and relative humidity of 45-55% and 12/12 light/dark cycle. The experiments

were carried out according to the guidelines of the Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), New Delhi, India, and the Institutional

Animal Ethical Committee (IAEC) approved protocol of this study.

Phytochemical screening: Phytochemical screening of methanolic extract and its methanolic

fraction of Sterculia scaphigera was carried out as described by Harborne[10]

(Harborne J.B

.,1973), Trease and Evans[11]

(Trease and Evans, 1989).

Acute toxicity: Rat were treated orally with 2 g/kg of MESS & MFSS using up and down

method (OECD guidelines) and mortality, if any, was observed for 24 hour.

Antioxidant activity: The antioxidant activity or the inhibition of the generation of free

radicals is important in the protection against ethanol-CCl4 induced hepatopathy[12]

(Castro et

al.,1974) The ability of test agent to scavenge free radical was determined by using 1,1-

diphenyl, 2-picrylhydrazyl (DPPH) assay as described by Hatano[13]

(Hatano.,1988) . The

known quantity of extract and its fraction were dissolved in methanol and the solutions were

serially diluted. The dilutions of the methanolic extract and its methanolic fraction were

added to solution of DPPH. The mixture was shaken and allowed to stand at room

temperature for 30 min and the absorbance was measured at 517nm using spectrophotometer.

L-ascorbic acid was used as reference standard. The % inhibition was calculated from the

following equation:

% inhibition = [(absorbance of control - absorbance of test sample)/absorbance of

control] x 100%.


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The antioxidant activity of each sample was expressed in terms of IC50 (micromolar

concentration required to inhibit DPPH radical formation by 50%), calculated from the

calibration curve.

Antiulcer activity

Pyloric ligation induced ulcer

Rats in a group of five were fasted 48 h prior to receiving dose of the vehicle, MESS

(50mg/kg,orally & i.p.), MFSS( 25 & 50 mg/kg,orally) and ranitidine (10 mg/kg,orally &

i.p.). Pyloric ligation was done by ligating the pyloric end of the stomach of rats 1 h after

drug administration[14]

(Shay et al., 1945). Rats were allowed to recover and stabilize in

individual cage and were deprived of water during post-operative period. After 4 h of

surgery, rats were sacrificed and gastric juice was collected and subjected to analysis. Gastric

contents were analyzed for total acidity by titrating against 0.01N NaOH using

phenolphthalein as indicator. The number of ulcers was noted and the severity recorded as

described by Kulkarni, 1999[15]

.

Alcohol induced ulcers in rats

Rats in groups of five each were treated orally with vehicle, ethanol (40%,2ml/100gm,p.o.),

ranitidine (10 mg/kg,orally) & MESS (50mg/kg,orally & i.p),MFSS( 25 & 50 mg/kg,orally)

for 15 days twice a day. On day 16th

, the animals were killed by an overdose of ether. The

abdomen was immediately opened to remove the stomach and ulcer index was calculated as

described above.

Histopathological study : Immediately after the sacrifice of the anesthetized animals, their

stomach tissues were removed and immediately fixed in 10% buffered formalin. These

tissues were processed and embedded in paraffin wax after being dehydrated in alcohol and

subsequently cleared with xylene. Five-micrometer thick serial histological sections were

obtained from the paraffin blocks and stained with hematoxylin and eosin.The sections were

examined under light microscope and photomicrographs were taken[16]

( Luna,1996).

Statistical analysis: All values are mean ± S.E.M. obtained from six animals. For statistical

analysis, One- Way ANOVA with Dunnet's Test was used to compare the groups. In all the

cases a difference was considered significant when p was <0.05.


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RESULTS

Phytochemical screening

Phytochemical screening of methanolic extract and methanolic fraction of Sterculia

scaphigera showed that it contained tannins, terpenoids, alkaloids, amino acids,

polysaccharides and flavonoids.

Acute toxicity

There was no mortality in rats treated with MESS & MFSS upto 2 g/kg, p.o.

Antioxidant activity

The IC50 of methanolic extract, its methanol soluble fraction and L-ascorbic acid were found

to be 200 μg/ml, 98 μg/ml, and 7 μg/ml respectively.

Antiulcer activity

Pyloric ligation induced ulcer

In pyloric ligation model,MESS (50 mg/kg,orally & i.p.), MFSS( 25 & 50 mg/kg,orally)

produced significant (p<0.05) reduction in ulcer index and also significantly reduced the

gastric volume, total acidity, and increased the pH of the gastric fluid, proving its

antisecretory activity when compared with control group. as compared to control. But there

was no significant decrease in volume. Ranitidine (10 mg/kg, orally & i.p.) also significantly

(P < 0.01) reduced ulcer index, gastric volume, total acidity, and increased the pH of the

gastric fluid, of pyloric-ligation induced gastric ulcers. Observations are given in Table 1.

Alcohol induced ulcers in rats

Administration of ethanol produced haemorrhagic gastric lesions in the gastric mucosa of the

control group.Administration of methanolic extract of Sterculia scaphigera and its

methanolic fraction reduced these lesions as evidenced by a significant (P < 0.01) reduction

in the ulcer index when compared with the group receiving ethanol alone. Ranitidine also

significantly (P < 0.01) reduced ulcer index of ethanol-induced gastric ulcers. Observations

are given in Table 2.

Group I --- Normal saline (Vehical control)

Group II ---Control group 40% ethanol

Group III ---Ranitidine (10 mg/kg , p.o.) + 40% ethanol

Group IV---MeOH extract of Sterculia scaphigera (50mg/kg, i.p.) + 40% ethanol


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Group V---MeOH extract of Sterculia scaphigera (50mg/kg,p.o.) + 40% ethanol

Group VI--- MeOH fraction of Sterculia scaphigera (25mg/kg,p.o.) + 40% ethanol

Group VII---MeOH fraction of Sterculia scaphigera (50mg/kg,p.o.) + 40% ethanol

Histopathology of stomach sections in pyloric-ligation induced ulcer in rats

Histopathological examinations when compared to the histoarchitecture of the stomach of

Group I (normal) animals, stomach of Group II rats (exposed to pyloric-ligation) revealed

extensive damage of mucosa, characterized by loss of gland architecture with erosion of the

epithelial layer and infiltration by inflammatory cells. In Group IV,V,VI&VII rats (exposed

to MESS & MFSS and pyloric-ligation),only minimal disruption of mucosa was observed

which is similar to that of Group III rats (exposed to ranitidine and pyloric-ligation ). (Fig.1)

Histopathology of stomach sections in ethanol induced ulcer in rats

Histopathological examinations when compared to the histoarchitecture of the stomach of

Group I (normal) animals,stomach of Group II rats (exposed to ethanol) revealed extensive

damage of mucosa, characterized by loss of mucus and chief cells. In Group IV,V,VI&VII

rats (exposed to MESS & MFSS and ethanol),only minimal disruption of mucosa was

observed which is similar to that of Group III rats (exposed to ranitidine and ethanol). (Fig.2)

Table 1. Effect of Sterculia scaphigera methanolic extract & methanolic fraction on

pyloric-ligation induced ulcers in rats

Treatment Groups

(Dose in mg/kg,i.p.)

Total gastric

content (ml) pH

ml of NaOH

reqd.

Total acidity

[mEq/L]

Ulcer Index

Control [pyloric-

ligation] 4.38±0.224 3.143±0.32 5.929±0.322 59.29±3.22 4.714±0.285

RTD(10) 3.5±0.178 * 4.8±0.184* 3.943±0.17* 39.43±1.68* 3.143±0.39*

RTD(10) p.o. 3.61±0.128* 4.64±0.179* 3.786±0.09* 37.86±0.88* 2.643±0.24*

MESS(50) 3.74±0.141* 5.14±0.142* 3.72±0.108* 37.29±1.08* 2.714±0.46*

MESS(50) p.o. 2.72±0.153* 5.0±0.154* 4.343±0.13* 43.43±1.25* 2.214±0.36*

MFSS(25) p.o. 3.32±0.130* 4.31±0.045* 4.314±0.05* 43.14±0.45* 2.857±0.18*

MFSS (50) p.o. 3.04±0.149* 3.97±0.035* 3.97±0.035* 39.7±0.35* 2.214±0.36*

F 6, 28 =

P =

12.38

<0.05

14.71

<0.05

25.10

<0.05

25.10

<0.05

7.43

<0.05

Values represent the mean of 5 rats ± SEM

* Statistically significant compared with control group (p <0.05)

RTD --- Ranitidine

MESS --- Methanolic Extract of Sterculia scaphigera

MFSS --- Methanolic Fraction of methanolic extract of Sterculia scaphigera


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Table 2: Effect of methanolic extract & methanolic fraction of Sterculia scaphigera on

ethanol induced ulcers in rats

Treatment Groups

(Dose in mg/kg,p.o.) Ulcer index

I 0.6±0.114

II 2.5 ± 0.316*

III 0.8 ± 0.122 #

IV 1.0 ± 0.158#

V 0.7 ± 0.122#

VI 1.2 ± 0.122#

VII 1.0 ± 0.223#

F ( 6, 28) =

P =

11.89

<0.05

Values represent the mean of 5 rats ± SEM

* Statistically significant compared with vehicle control group (p <0.05)

# Statistically significant compared with ethanol group (p <0.05)

Group II should be compared with Group I.

Groups III, IV, V, VI and VII should be compared with Group II

Fig.1 Results of Histology : (Ulcer induced by pyloric-ligation)

Fig. A Fig. B

Fig. C Fig. D


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Fig. E Fig. F

Fig. G Fig. H

Fig. I

Fig.1 Histopathological examination of Stomach sections in normal and different

treatment groups along with pyloric-ligation.

Fig. A-- The vehicle treated rats exhibited stomach section showing normal

gastric mucosa.

Fig B&C--Stomach sections of the rats with pyloric-ligation showed loss of gland

architecture with erosion of the epithelial layer and infiltration by

inflammatory cell.


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Fig.D&E--The rats treated with Ranitidine(10mg/kg,i.p.&p.o.resp.) along with

pyloric-ligation showed almost normal architecture of gastric mucosa.

Fig.F&G– The rats treated with methanolic extract of Sterculia scaphigera

(50mg/kg,i.p&p.o.resp.) along with pyloric-ligation showed almost normal architecture

of gastric mucosa.

Fig.H&I– The rats treated with methanolic fraction of Sterculia scaphigera

(25&50mg/kg,p.o.resp.) along with pyloric-ligation showed almost normal architecture

of gastric mucosa.

Fig.2 Results of Histology : (Ulcer induced by administration of ethanol)

Fig. A Fig. B

Fig. C Fig. D

Fig. E Fig. F


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Fig. G Fig. H

Fig.2. Histopathological examination of Stomach sections in normal and different

treatment groups along with administration of ethanol

Fig.A -- The vehicle treated rats exhibited stomach section showing normal

gastric mucosa.

Fig.B&C -- Stomach sections of the ulcer induced rats with ethanol showed loss

of mucus and chief cells.

Fig.D -- Stomach sections of rats treated with Ranitidine along with ethanol

Showed almost normal cellular architecture of gastric mucosa.

Fig.E&F -- Stomach sections of the rats treated with methanolic extract of

Sterculia scaphigera (50 mg/kg,i.p.& orally,resp.) along with ethanol showed almost

normal cellular architecture of gastric mucosa.

Fig.G&H -- Stomach sections of the rat treated with methanolic fraction of

Sterculia scaphigera (25& 50mg/kg,orally resp.) along with ethanol showed almost

normal cellular architecture of gastric mucosa.

DISCUSSION AND CONCLUSION

The results obtained in the present study show that the methanolic extract of Sterculia

scaphigera and its methanolic fraction possess antioxidants & antiulcer activity.It has been

demonstrated that many drugs and formulations having potent antioxidant action are

effective in healing experimentally induced gastric ulcers[17,18,19]

(Dhuley, 1999; George et

al., 1999; Goel and Sairam, 2002). Many plants exhibit efficient antioxidant properties owing

to their phenolic constituents. Most of tannins and flavonoids are phenolic compounds and

are responsible for antioxidant properties of many plants[20]

(Larson, 1988). The DPPH assay

is based on the ability of 1,1-diphenyl-2 picryl-hydrazyl a stable free radical, to decolorize in

the presence of antioxidants. The DPPH contains an odd electron which is responsible for

absorbance at 517 nm and also for visible deep purple color. When DPPH accepts an electron


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donated by an antioxidant compound, it is decolorized which can be quantitatively measured

from the change in absorbance. That is why with the increasing concentration of extract %

scavenging activity also increases.

Peptic ulcer is one of the major gastro-intestinal disorders, which occur due to an imbalance

between the offensive (gastric acid secretion) and defensive (gastric mucosal integrity)

factors[21]

(Hoogerwerf and Pasricha, 2006). Consequently, reduction of gastric acid

production as well as re-inforcement of gastric mucosal production has been the major

approaches for therapy of peptic ulcer disease. As a result, more and more drugs, both herbal

and synthetic are coming up offering newer and better options for treatment of peptic ulcer.

The type of drugs varies from being proton-pump inhibitor to H2 antagonist or a

cytoprotective agent. At the same time, each of these drugs confers simpler to several side

effects like arrhythmias, impotence, gynaecomastia, enterochromaffin-like cell (ECL),

hyperplasia and haemopoeitic changes [22]

(Akthar et al., 1992).

The present study showed that the methanolic extract of Sterculia scaphigera and its

methanolic fraction possess gastroprotective activity as evidenced by its significant inhibition

in the formation of ulcers induced by physical (pyloric-ligation ) and chemical factors

(ethanol). Pylorus ligation-induced ulcers are due to autodigestion of the gastric mucosa and

break down of the gastric mucosal barrier[23]

(Sairam et al., 2002).

The incidence of ethanol-induced ulcers developing predominantly in the glandular part of

stomach was reported to stimulate the formation of leukotriene C4 (LTC4), mast cell

secretory products[24]

(Oates and Hakkinen,1988); and reactive oxygen species[25]

(Mizui et al.,

1987) resulting in the damage of rat gastric mucosa[26]

(Peskar et al., 1986).

Ethanol-induced depletion of gastric wall mucus has been prevented by Sterculia

scaphigera.

In rat, ulcerprotective activity of Sterculia scaphigera is quite similar to Ranitidine [H2-

antagonist], a reference ulcerprotective agent. Both of them improved the parameters of

pyloric-ligation and ethanol induced ulcer including total acidity, volume of acid secretion,

pH of gastric content and ulcer index.

In rat, ulcerprotective activity of Sterculia scaphigera is quite similar to Ranitidine [H2-

antagonist], a reference ulcerprotective agent. Both of them improved the parameters of


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pyloric-ligation and ethanol induced ulcer including total acidity, volume of acid secretion,

pH of gastric content and ulcer index.

The antiulcer activity of various polysaccharides in experimental ulcers has also been

reported[27,28]

(Sun et al., 1992; Matsumoto et al., 2002).

Sterculia scaphigera significantly (P < 0.01) reduced the ulcer index and afforded significant

protection against ethanol-induced ulcer. The antioxidant properties of Sterculia scaphigera

may have scavenged the free radicals produced by the metabolism of ethanol and thereby

heal the ulcers.

Ethanol-induced gastric lesion formation may be due to stress in gastric blood flow that

contributes to the development of the hemorrhage and necrotic aspects of tissue injury[29]

(Guth et al., 1984). This chemical agent also increases Na+ and K+ flux into the lumen and

increases pepsin secretion along with histamine release.

Ethanol induces oxidative stress in rat gastrointestinal mucosa, increasing lipid peroxidation

and DNA fragmentation and leading to gastric lesions. It also depresses tissue levels of DNA,

RNA and proteins, altering blood flow, producing necrosis and hemorrhage- like gastric

tissue[30]

(Kwiecien et al., 2002).

The antiulcerogenic potential of Sterculia scaphigera was further evidenced by the

histopathological studies ( Fig.1 and Fig.2) The Qualitative phytochemical investigations of

Methanolic extract & Methanolic fraction of Sterculia scaphigera have shown the presence

of alkaloids and flavonoids.Methanolic extract & fraction of Sterculia scaphigera offers

protective effect against pyloric ligation & ethanol induced ulcer in experimental rats. The

mechanism of action is yet to be investigated but may be due to the antioxidant effects found

to be present in the extract.

Hence, it can be suggested that the antiulcer activity of the extract may be attributed to its

antisecretory and antioxidant activities.


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SCREENING OF METHANOLIC EXTRACT OF STERCULIA SCAPHIGERA WALL SEEDS FOR ULCERPROTECTIVE & ANTIOXIDANT ACTIVITY

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