Indian Journal of Experimental Biology Vol. 41, April 2003, pp. 304-310

Evaluation of Pongamia pinnata root extract on gastric ulcers and mucosal offensive and defensive factors in rats

T Prabha, M Dora Babu, S Priyambada, V K Agrawal & R K Goel*

Department of Pharmacology, Inst itute of Medical Sciences, Banaras Hindu University, Varanasi 221 OOS, India

Received 15 January 2003; revised 27 February 2003

Effect of methanolic extract of P. Pinnaw roots (PPRM) was studied against various experimental gastric ulcer models and offensive and defensive gastric mucosal factors in rats. An initial dose-response study using 12.S-S0mglkg P. Pinnaw root extract, when given orally in two divided dose for 4 days + Sth full dose on the day of experiment 60 min before the experiment, indicated 2Smg/kg as an optimal regimen and was used for further study. PPRM showed significant protection against aspirin and 4 hr PL, but not against ethanol-induced gastric ulceration. It showed tendency to decrease acetic acid­induced ulcer after IO days treatment. Ulcer protective effect of PPRM was due to augmentation of mucosal defensive factors like mucin secretion, life span of mucosal cells, mucosal cell glycoproteins, cell proliferation and prevention of lipid per oxidation rather than on the offensive acid-pepsin secretion.

P. pinnata (linn) Pierre (Syn P. glabra Vent) is a me­dium sized glabrous tree, found through out India and further distributed East wards mainly in the littoral regions of South Eastern Asia and Australia '. Differ­ent parts of this plant have been recommended in Ay­urvedic literature as a remedy for various ailments. The seeds and seed oil of this plant have been used for treating various inflammatory and infectious diseases such as leucoderma, leprosy, lumbago, muscular and rheumatism2

. Leaves are hot, digestive, laxative, anthelmintic and cure piles, wounds and other in­flammations3

. A hot infusion of leaves is used as a medicated bath for relieving rheumatic pains and for cleaning ulcers in gonorrhea and scroflilous enlarge­ment4

. Recently the ethanolic extract of leaves of this plant is shown to have prominent anti-inflammatory activity but devoid of any ulcerogenic potential as the commonly used NSAIDs generally showed increased propensity to gastric ulceration5

• Recently, we have reported the ulcer protective effects of extracts of both seeds and roots of P. pinnata (PP) and the ethanolic extract of the roots of this plant tended to decrease acid-pepsin and increase mucin secretion6

. Although several factors are reported to be involved in ulcero­genesis, the causative factor is basically the imbalance between offensive and defensive mucosal factors7

.

Several of the drugs used for the treatment of ulcers are those, which reduce or neutralize the offensive

*Correspondent author: Fax: ++91- OS42-2367S68; E-mail: rkgocl_bhu@yahoo.co.in

acid secretions, while some other drugs like carbe­noxolone, sucralfate and prostaglandin analogs in­crease the defensive factors with out affecting the of­fensive acid secretion8

. They are termed as cytopro­tective drugs and are proved useful in ulcer therapy . Several natural drugs have been reported to possess anti-ulcerogenic activity by virtue of their predomi­nant effect of mucosal defensive factors9

- '2. The pre­sent work is thus, in continuation of our earlier work and includes various experimental ulcer models and parameters of mucosal offensive (acid-pepsin output and reactive oxygen species) and defensive factors (gastric mucin secretion and gastric mucosal cell shedding, cell proliferation or glycoproteins) uStng methanolic extract of P. pinnata roots.

Materials and Methods Collection and extractions - Roots of Pongamia

pinnata were collected locally in the month of No­vember and were duly authenticated by Dr S K Ti­wari, Department of Kayachikitsa, BHU, Varanasi , India. The roots were macerated (1000 g) and ex­tracted with methanol (3 I, 5 days) by cold percola­tion. The % yield of dried extract was 3.2%.

Animals-Albino rats (CF strain) of either sex, weighing between 150-200 g were used for the pre­sent study. They were obtained from the Central Animal House of the Institute. They were kept in col­ony cages at room temperature (250 ± 30 C) and RH (45-56%) under 12 hr light/dark cycles for 5 days be­fore treatment. The animals were allowed freely to

PRABHA et of.: PONGAMIA ROOT EXTRACT & GASTRIC ULCERS 305

access rodent pellet diet (Hind lever) and water ad libitum.

Drug treatment - Methanolic extract of root of P. pinnata (PPRM) was given in graded doses of 12.5, 25 and 50 mg/kg in two divided doses, daily for 4 days and the last full dose was given on 5th day of ex­periment, 60 min before subjecting 18 hr fasted rats to 2 hr cold restraint stress. Preliminary results indicated an optimal ulcer protective effect with 25 mg/kg. Therefore, for further study this dose and duration was chosen for detailed study, while the control ani­mals received 1 % carboxy methylcellulose. All the animals received the drugs orally with the help of an orogastric tube at a concentration of 1.0 mlllOO g. Any deviation in the duration of treatment has been mentioned in the ResultslTables of the present study.

Anti-ulcer study Cold-restraint stress (CRS)-induced ulcers 13 - To

18 hr fasted rats, cold restraint stress was given by strapping the rats on a wooden plank and keeping them for 2 hr at 4°_6°C. The animals were then sacri­ficed by cervical dislocation and ulcers were scored on the dissected stomachs '4 and ulcer index was cal­culated as described earlier9

. Statistical analysis was done using Wilcoxon Sum Rank testIS.

Aspirin (ASP) induced ulcers-ASP at a concen­tration of 200 mg/kg (20 mg/ml) was administered to the animals on 5th day of experiment and ulcers were scored after 4 hr '6. The stomach was taken out and cut open along the greater curvature and ulcers were scored by a person unaware of the experimental pro­tocol in the glandular portion of the stomach as de­scribed above.

Ethanol (EtOH) induced ulcers-Gastric ulcers were induced in rats by administering EtOH (1 ml1200 g, I hr)'7 on 5th day of experiment and the animals were sacrificed by cervical dislocation 1 hr after ethanol administration. Stomach was incised along the greater curvature and examined for ulcers. The ulcer index was scored, based upon the product of length and width of the ulcers present in the glan­dular portion of the stomach (mm2

/ rat). Statistical analysis of data was done using unpaired Student's 1

test. Pylorus ligated (PL) induced ulcers - Rats were

kept for 18 hr fasting and care was taken to avoid coprophagy. On the 5th day of experiment, animals were anaesthetized after the drug treatment described above, using pentobarbitone (35 mg/kg, ip), the ab­domen was opened and pyloric end of stomach was

ligated without causing any damage to its blood sup­ply. The stomach was replaced carefully and the ab­domen wall was closed in two layers with interrupted sutures. The animals were deprived of water during the post-operative period 18. After 4 hr, stomachs were dissected out and contents were collected into tubes for estimation of biochemical parameters. The ulcers were scored and statistically analysed as described above for cold restraint stress ulcers.

Ulcer healing study Acetic acid induced ulcers-For induction of ace­

tic acid ulcer, the method was followed as reported earlier'l. Rats were anaesthetized with pentobarbitone (35 mg/kg, ip). The abdomen was opened and the stomach was visualized. A cylindrical glass tube (6 mm in diam) was tightly placed upon the anterior serosal surface of the glandular portion of stomach 1 cm away from the pyloric end. 50% acetic acid (0.06 mllanimal) was instilled into the tube and al­lowed to remain for 60 sec on the gastric wall. After removal of acid solution, the abdomen was closed in two layers and animals were caged and fed normally . PPRM (12.5 mg/kg) was given on day one, orally, twice daily, 4 hr after the application of acetic acid and continued either up to 5 or 10 days after induction of ulcer. The animals were then sacrificed after 18 hr of the last dose of drug either on 6th or 11th day of ex­periment to assess the ulcer size and healing. Ulcer index was calculated based upon the product of length and width (mm2/rat) of ulcers. Statistical significance was calculated using unpaired Student's t test.

Gastric secretion study-Studies on offensive fac­tors such as acid and pepsin and defensive factors such as mucin secretion and cell shedding were car­ried out in gastric secreticn. The gastric juice was col ­lected 4 hr after PL and centrifuged for 5 min at 2000 rpm. The supernatant was collected and the volume of gastric juice was expressed as mlllOOg body weight. Acid output was determined by titrating with 0.01 N NaOH, using phenolphthalein as indicator and ex­pressed as tlEq/4hr. Peptic output was determined using hemoglobin as substrate '9 and expressed as tlEq/4 hr. Dissolved muco-substances were estimated in 90% alcoholic precipitate of gastric juice. The pre­cipitate, thus obtained was either dissolved in 1 ml of 0.1 N NaOH or 1 ml of 0.1 N H2S04 . The former was used for estimation of protein2o

, total hexoses21,

hexosamine22 and fucose23, while the latter was used

for estimation of sialic acid24. The results have been

expressed in tlg/ml of gastric juice. Ratio of total

306 INDIAN J EXP SIOL, APRIL 2003

carbohydrate (TC; sum of total hexoses, hexosamine, fucose and sialic acid) to protein (P) has been taken as the index of mucin activit/5

. DNA content was esti­mated and expressed as M-g/ml gastric juice26

. Statisti­cal analysis of data was done using unpaired Student's t test.

Gastric mucosal study- Estimation of cellular mucin as glycoproteins was carried out in the gastric mucosa of 4 hr pyloric ligated rats. Mucosal scrapings of glandular portion of rat stomach were homogenized in normal saline (20 mg/ml) and treated with 90% ethanol in the same manner as described for mucin estimation in the gastric juice. The precipitate thus obtained, was subjected for estimation of carbohy­drates and protein using the methods described above for gastric juice contents. The results are expressed as /lg/l OOmg wet tissue and TC: P ratio has been taken as the index of glycoprotein activit/7

. Statistical analysis of data was done using unpaired Student's t test.

Cell proliferation-Estimation of DNA was car­ried out in gastric mucosal scrapings. Mucosal scrap­ings of glandular portion of rat stomach were ho­mogenized in 2.5 ml of ice-cold 0.6 N perchloric acid. DNA 27 and protein2o were then estimated. The

concentration of DNA has been expressed as M-g DNA/mg protein.

Estimation of free radical generation The fundic part of the gastric mucosal scrap was

homogenized (5%) in ice-cold 0.9% saline with a Pot­ter-Elvehjem glass homogeniser for 30 sec. The ho­mogenate was then centrifuged at 800 g for 10 min followed by centrifugation of the supernatant at 12,000g for 15 min and the obtained mitochondrial fraction was used for following estimations. 28

Lipid peroxidase - LPO product malondialdehyde (MDA) was estimated using 1,1 ,3,3-tetraethoxy­propane as the standard and has been expressed as nmole/mg of protein29

.

Superoxide dismutase (SOD) activity-SOD was estimated by following the procedure of Kakkar et al.30

. Inhibition of reduction of nitro blue tetra­zolium (NBT) to blue coloured formozan in presence of phenazine metha sulphate (PMS) and NADH was measured at 560 nm using n-butanol as blank. One unit of enzyme activity was defined as the amount of enzyme that inhibits rate of reaction by 50 % in 1 min under the defined assay conditions and the results have been expressed as units (U) of SOD activity/mg of protein.

Catalase (CAT) activity - Decomposition of H20 2

in presence of catalase was followed at 240 nm 31• One

unit of (U) CAT was defined as the amount of en­zyme required to decompose 1M-mole of H20 2 per min, at 25°C and pH 7.0. Enzyme activity has been ex­pressed as units (U) of CAT activity/mg of protein .

Results Methanolic extract of P. pinnata roots (PPRM;

12.5-50 mg/kg) when given in two divided doses , orally daily for 4 days and a full dose, 60 min before the experiment on day 5, showed significant ulcer protection (control ulcer index: 35.2±6.8; PPRM 12.5-50 mg/kg, % protection-48.3-67.9%; P<O.I­P<O.OI). A dose of 25 mg/kg for 5 days i.e. 12.5 mg/kg, bd x 4 days + 25.0 rng/kg stat on 51h day 60 min before, was then selected for further study. This dose showed significant ulcer protection against aspi­rin and 4 hr pylorus ligation (PL), but not against ethanol induced gastric ulceration (Table 1). Sucral­fate (SIT, 250 mg/kg bd, po, x 4 days + 500 mg/kg, stat, 60 min before on day 5,) showed significant ulcer protection in all the three above models (Table I). Further, PPRM tended to heal acetic acid induced gas­tric ulcers after 10 days treatment only, while SIT decreased the ulcer index both after 5 and 10 days treatment (Table 2).

On PL induced gastric secretion, PPRM tended to increase acid-pepsin secretion, while SIT signifi­cantly decreased the pepsin out put (Table 3). PPRM tended to increase mucin activity in terms of total car­bohydrate and protein ratio (TC: P), but SIT in­creased it significantly. There was no significant ef­fect on individual carbohydrates (total hexoses, hexosamine, fucose or sialic acid) or total carbohy­drates and protein with PPRM. However, SIT either tended to increase or increased the above parameters (Table 3). DNA concentration of gastric juice is an

Table I - Effect of methanolic extract of fresh roots of P. pin­/lata (PPRM) and sucralfate (SFT) on ethanol (100%, I m1l200g, po, I hr), aspirin (200 mg/kg, po, 4 hr) and 4 hr pylorus ligation (PL) induced gastric ulcers in rats

[Values are mean±SE from 8 animals in each group]

Oral treatment Ulcer index

(mg/kg x 5 days) Ethanol Aspirin PL

Control (I % CMC) 18.3±3.2 25 .1 ±4.0 1O.0±0.9 PPRM 25 20.5 ±3.6 15.1 ± 1.8' 5.3 ±0.8b

SFT 500 9.7 ± 1.6" 7.3±3.7b 2.1 ±OS

P values: ' <0.05, b <0.0 I, c <0.00 I compared to respective control groups

PRABHA et al.: PONGAMIA ROOT EXTRACT & GASTRIC ULCERS 307

important marker of gastric mucosal damage or cell shedding. DNA content was significantly decreased by both PPRM and SF[ (Table 3).

On mucosal glycoproteins, PPRM had little or no effect on individual carbohydrate or total carbohy­drates but tended to decrease protein content leading to significant increase in TC: P ratio while, SF[ tended to increase, or increased the individual carbo­hydrate. Again, SF[ tended to decrease protein con­tent leading to significant increase in TC: P ratio, in­dicating that both the treatments increased the glyco­protein content of gastric mucosa (Table 4). DNA/mg protein is a reliable index for cell proliferation. PPRM tended to increase the cell proliferation (32.4% in­crease), while SF[ had little effect (13.1 % increase; Table 4). Free radical and lipid peroxidation (LPO) play an important role in ulcerogenesis. PL tended to

Table 2 - Effect of PPRM and SFT on 50 % acetic acid-induced gastric ulcer healing

[Values arc mean ± SE from 8 animals in each group I

Oral treatment (mg/kg x 5 days)

Control (I % CMC) PPRM 25

SF[ 500

Acetic acid-induced ulcers (healing) 5 days 10 days

Ulcer % incidence Ulcer % incidence index perforations index perforations

13.1 ± 1.1 40.0 7.0±1.2 0

15.7±2.2 37.5 4.5 ± 1.3 0

8.9 ± 1.7" 12.5 2.1±0.9b 0

P values: "<0.05, b<O.O I compared to respective control groups

increase LPO, while superoxide dismutase (SOD) and catalase (CAT) levels were little affected. PPRM tended to reverse the above increase in LPO without any effect on SOD and CAT levels compared to con­trol PL group (Table 5).

Discussion The present study indicated the ability of roots of

P. pinnata to protect the formation of gastric ulcer significantly in various experimental models except ethanol-induced ulcer model in rats. Significant pro­tection was also observed with SF[, a known cytopro­tective drug used clinically in healing of gastric ulcers and preventing the recurrence of peptic ulcers32

. Ear­lier, we reported the ulcer protective effect of ethano­lic extract of P. pinnata roots in CRS- and 4 hr PL­induced ulcer models and the effect could be due to its effect on mucin secretion, which is one of the im­portant mucosal defensive factors7

. Recently Sriniva­san and associate while studying the anti­inflammatory activity of ethanolic extract of PP leaves in rats, demonstrated anti-inflammatory activ­ity of this plant and this effect was not associated with any ulcerogenic effect which most of NSAIDS like aspirin show commonl/. In an earlier observations from our laboratory, some of the plant products like kaempferol, a flavanoid and amentoflavanone too demonstrate both anti-inflammatory and anti­ulcerogenic activity. It thus , indicates that in plant kingdom both the activities can run side by side3

3.34

Table 3 - Effect of PPRM (25 mg/kg,po x 5 days) and SFT (500 mg/kg,po, x 5 days) on gastric juice volume, acid, pepsin and DNA content in 4 hr PL rats

[Values are mean ± SE of 8 animals in each group]

Gastric juice Control PPRM SFT

Acid-pepsin secretion

Volume (mIIlOO g) 1.99±0. 15 2.21 ± 0.15 1.87 ± 0. 13 Acid output (JiEq/4 hr) 163.4 ± 16.8 187.1 ± 15.8 149.3 ± 11.9 Pepsin output (Jimole/4 hr) 549.2 ± 52.8 603 .2 ± 71 .2 383.2 ± 47 .2"

Mucin activity (Jig/Ill/)

Total hexoses 350.0 ± 30.8 379.7 ± 24.1 401.2 ± 19.3 Hexosamine 178.7±9.1 173.1 ± 9.9 201.4 ± 13.2 Fucose 80.3 ±4.2 108.9 ± 14.6 99.3 ± 7.3 Sialic acid 15.9 ± 1.3 20.0 ± 2.1 29 .1 ± 5.4" Total carbohydrates (TC) 624.9 ± 36.0 681.7 ± 28.5 731.0 ± 33.4" Protein (P) 638.9 ± 40.5 573.8 ± 31.5 550.3 ± 23 .9 TC: P 1.02 ± 0.09 1.23 ± 0.10 1.33 ± 0.11 "

Cell shedding (Jig DNA/IIlI)

DNA (Jig/ml) 162.0 ± 8.2 114.2 ± 7.7c 108.1±8.9c

P values: a< 0.05, c<O.OOI compared to respective control

308 [ND[AN J EXP B[OL, APR[L 2003

Table 4 - Effect of PPRM (25 mg/kg,po, x 5 days) and SF[ (500 mg/kg,po, x 5 days) on gastric mucosal glycoproteins and cell proliferation in 4 hr PL rats

[Values are mean ± SE of 8 animals in each group]

Gastric mucosa Control PPRM SF[

Clycoproteills (/lg 1100 mg wet tissue)

Total hexoses 3109 ± 182 3075 ± 252 3448 ± 273 Hexosamine 1604 ± 76 1661 ± [40 1795 ± 157 Fucose 227 ± 25 247 ± 26 284 ± 23 Sialic acid 88 ± 8 81 ± II 133 ± 13" Total carbohydrates (TC) 5028 ± 248 5065 ± 373 5661 ± 301 Protein (P) 6240 ± 434 5189 ± 318 5213 ± 379 TC: P 0.81± 0.04 0.98 ± 0.06" l.l 0 ± 0.11" Cell proliferation Protein (/lgll 00 mg tissue) 7564 ± 831 6363 ± 361 6419 ± 483 DNA(/lg/IOO mg tissue) 786.7 ± 33.5 92.1 ± 49.0 798.3 ± 37.0 /lg DNNmg protein 109.1 ± 9.0 144.4± 15.4 123.4± 11.3

P value: "< 0.05 compared to respective control

Table 5 - Effect of PPRM on gastric mucosal lipid peroxidation (LPO), superoxide dismutase (SOD) and catalase (CAT) levels in 4 hr PL rats

rValues are mean ± SE of 8 animals in each group]

Oral treatment LPO (mg/kg x 5 days) (MDA, nmole/mg

of protein)

Control (I % CMC) 0.28 ± 0.02 PL (1% CMC) 0.41 ± 0.04" PL + PPRM 25 0.34 ± 0.02

P value: "< 0.05 compared to respective control

and these drugs could be used for their anti­inflammatory activities without the danger of gastric ulceration.

Damage produced by ethanol to gastric mucosa is due to number of contributing factors and includes effects on mucosal blood flow, platelet thrombi, dam­age to capillary endothelium and release of arachi­donic metabolites7

• PL-induced ulcers are thought to be due to increased presence of acid and pepsin in the stomach7

. Stress induced ulcers are caused by a num­ber of factors both physical and psychological in na­ture35. Increase in gastric motiliti6

, vagal over activ­ity37, mast cell degradation3~, decreased gastric muco­sal blood flow 39 and increased leukotrienes synthesis7

were also reported for gastric ulcers caused by etha­nol4o and stress41

• Hence, diverse mechanism might be involved in anti-ulcerogenic activity of PPRM. The extract had no effect on acid- pepsin secretion but tended to increase mucin activity. It significantly in­creased mucosal glycoproteins and life span of muco­sal cells as evidenced by decrease in DNA content of gastric juice and both could play an important role for its ulcer protective effects. Endogenous prostagland-

SOD CAT (units/mg of protein)

123.2 ± 10.3 34.7 ± 3.9 131.0± 15.2 33.6 ± 4.9 126.2 ± 8.9 40.0 ± 4.1

ins, mucosal blood flow or bicarbonate secretion play an important role in ulcer protection and it could be possible that this extract might have significant effect on any of these parameters.

Rapid proliferation of gastric mucosa plays an im­portant role in mucosal protection during normal state following mucosal damage. Following extensive damage of the surface epithelial cells, repair occurs with in a few hours through a process called restitu­tion and which could be followed by cell prolifera­tion. Increased life span of cell as evidenced by a de­crease in cell shedding and increased tendency to cell proliferation as evidenced by an increase in DNA/mg protein, thus indicated gastric mucosal renewal and restitution by P. pinnata.

Increase in lipid peroxidation (LPO) levels indi­cate, increase of reactive oxygen species (ROS), the major radical being superoxide anion (02), H20 2 and hydroxyl radical (OH). These induce cell degradation by increasing peroxidation of cell membrane lipids causing loss of structural and functional integrity of cell membranes. Increase in superoxide dismutase (SOD) level is in response to increased tissue O2

PRABHA et al.: PONGAMIA ROOT EXTRACT & GASTRIC ULCERS 309

speeding up their dismutation and converting It Im­mediately into I-h02 (Ref. 42). Accumulation of H20 2 occurs in the mitochondria and cytosol and if not scavenged by catalase (CAT)43, can lead to increased generation of OH Jadical. In 4 hr PL-induced gastric damage, LPO was significantly increased with little effect on SOD and CAT levels. However, treatment of rats with PPRM tended to reverse change in LPO activity, indicating decreased lipid peroxidation and damage to cells. Thus, the present study indicated the ulcer protective effects of PPRM. The protection af­forded by PPRM could be due to the augmentation of defensive mucosal factors and inhibition of lipid per­oxidation.

Acknowledgement The author (RKG) is thankful to UGC, New Delhi

for financial assistance.

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