Indian Journal of Experimental Biology Vol. 39, February 2001, pp. 137-142

Effect of Centella asiatica Linn on physical and chemical factors induced gastric ulceration and secretion in rats

K Sairam, Ch V Rao & R K Goel*

Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India

Received 7 February 2000; revised 14 November 2000

Centella asiatica is commonly mentioned as a Rasayana in Ayurveda, an ancient system of Indian medicine for various ailments including abdominal di sorders. Rasayanas have been advocated for use in rejuvenation therapy. The present study was conducted to evaluate the possible anti-ulcerogenic activity of fresh juice of C. asiatica (CAJ) against ethanol-, aspirin-, cold-restraint stress- and pyloric ligation induced gastric ulcers in rats. The drug given orally in doses of 200 and 600 mg/kg twice daily for live days. showed significant protection against all the above experimental ulcer models and the results were comparable with those elicited by sucralfate (SF, 250 mg/kg, po, BD X 5 days). CAJ showed little or no effect on offensive acid-pepsin secretion. However, at 600 mg/kg CAl significantly increased gastric juice mucin secretion and increased the mucosal cell glycoprotiens signifying increase in cellular mucus. It also decreased cell shedding indicating fortification of mucosal barrier. Thus, the ulcer protective effect of CAl may be due to strengthening of the mucosal defensive factors.

Gastric ulceration is caused by many factors like stress, drugs, alcohol etc 1• A rational therapy for peptic ulcer still remains elusive and search for safer potential drugs is being carried out. Use of natural drugs in gastric ulcers is well documented2

.5

. Most of these drugs augment the mucosal defensive factors, which are thought to be important for protection of gastric mucosa2.3 . Peptic ulcers are reported to be due to an imbalance between offensive acid-pepsin secretion and defensive mucosal factors like mucin secretion and cell shedding6

. Centella asiatica Linn Urban (CA, Hindi-Mandukaparni), a creeping perennial is mentioned in Ayurveda for treatment of abdominal disorders and epilepsy, and as a carminative and a cardiotonic7

• The usefulness of CAin various diseases has been reported8

.14

. Also CA had inhibited gastric ulceration induced by cold restraint stress in rats and the activity was attributed to the GABA-ergic system 15

•

The present experimental work has been undertaken to study the effect of juice of fresh whole plants of Centella asiatica (CAJ) on different models of gastric ulcer in rats and its possible effects on offensive and defensive mucosal factors.

Materials and Methods Animals-Albino rats ( C-F strain) of either sex

weighing between 150-180 g were procured from the central animal house of the Institute and housed in

*Correspondent author

well ventilated colony cages in the departmental animal house at 25°±2°C and 45-56% RH, 10 :14 hr L:D cycles for one week for acclimatization. The animals were fed with standard rodent pellet diet (Hind Lever) and water ad Libitum.

Collection ofplant-Centella asiatica of cultivated variety was obtained during the month of March from the Ayurvedic gardens of the Institute and was identified with the reference herbarium maintained in the Department of Dravyaguna. Whole plants ( 1 kg) were size reduced, crushed and 750 ml of juice thus obtained was filtered. The dry weight in terms of solid content in the juice was 4 %. The fresh juice was stored in a refrigerator at -20°C in a glass container and was used within a week of its extraction. The juice was warmed each time at 37°C before administration to the animals.

Experimental study- CAJ in doses of 200 and 600 mg/kg (in terms of dry weight) and sucralfate (SF) in the dose of 250 mg/kg were administered, po twice daily at 1000 and 1600 hrs for five days. On the 6th day of experiment, the 18 hr fasted rats were subjected to the following experimental gastric ulcer studies.

Anti- ulcer study (a) Ethanol (EtOH)-induced ulcers: The gastric

ulcers were induced in rats by administering EtOH (1 mV200 g, 1 hr )16 and the animals were sacrificed by cervical dislocation and stomach was incised along the greater curvature and examined for ulcers. The

138 INDIAN J EXP BIOL. FEBRUARY 200 1

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 by using unpaired Student 's t test.

(b) Aspirin (ASP)- induced ulcers: ASP in dose of 200 mg/kg (20 mg/ml) was administered to the animals and ulcers were scored after 4 hr17 .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 g landular portion of the stomach as described below.

Ulcer index has been calculated by adding the total number of ulcers per stomach and the total severity of ulcers per stomach. The total severity of the ulcers was determined by recording the severity of each ulcer in pluses(+). Severity of the ulcers were scored afte r hi sto logical confirmation as follows 3

: 0, no ulcer; +, pin point ul cer and histological changes limited to superficial layers of mucosa and no congestion; ++, ulcer size less than 1 mm and half of the mucosal thickness showed necrotic changes; +++, ulcer size J -2 mm with more than two-thirds of the mucosal thickness destroyed with marked necrosis and congestion, muscularis remaining unaffected; ++++, ulcer either more than 2 mm in size or perforated wi th complete destruction of the mucosa \Vith necrosis and haemorrhage, muscularis still remaining unaffected (Figs. l -4).The pooled group ulcer score was then calcul ated 18

. Statistical analysis was done by using Wilcoxon Sum Rank tes t19

•

(c) Cold-restraint stress (CRS)- induced ulcers : On day six , to 18 hr fasted r:lts cold restraint stress was given by strapping the rats on a wooden plank and keeping them at 4°-6°C for 2 hr. The animals were then sacrificed by cervical di slocation and ulcers were scored on the di ssected stomachs20 as described above.

(cl) Pylorus-ligated (PL)- induced ulcers: Drugs were ad min istered for 5 clays as described above. On clay 6 after the last close, the rats were kept for J 8 hr fas ting and care was taken to avoid coprophagy . Animals were anaesthe ti zed using pentobarbitone (35 mg/kg, ip), the abdomen was opened and pylorus ligat ion was clone 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-operati ve periocl21

. After 4 hr, stomachs were dissected out and contents were co llected into tubes for estimation of biochemical parameters. The ulcers were scored as described under ASP-induced ulcers.

Gastric secretioll study - Studies on offensive factors such as acid and pepsin and defensi ve factors such as mucin secretion and cell shedding . were carried out in gastric secretion. The gastric juice was collected 4 hr after PL and centri fuged for 5 min at 2000 rpm. The supernatant was collected and the volume of gastric juice was expressed as ml/IOOg body weight. Acid concentration and output were determined by titrating with 0.0 1 N NaOH, usi ng phenolphthalein as indicator and is expressed as pEq/ml and pEq/4hr respectively . Peptic activity was determined using hemoglobin as substrate22 and has been expressed as pmol/ml and p mol/4 hr for concentration and output respectively. Di ssolved mucosubstances were estimated in 90 % alcoholic precipitate of the gastric juice. The precipitate, thus obtained was either di ssolved in I ml of 0.1 N NaOH or I ml of O.I N H2S04. The former was used for the estimation of protein23

, total hexo es24, hexosamine25

and fucose26, while the latter was used for the

estimation of sialic acid27 • The results are expressed

in pg/ml of gastric juice. The ratio of total carbohydrate (TC) (sum of to tal hexoses, hexosamine, fucose and sialic acid) to protein (P) has been taken as the index of mucin activit/8

. DNA content was estimated and expressed as )lg/ml gastric juicell OOg

. h f 09 we1g t o rat~ .

Gastric mucosal s tudy-Estimation of cellular mucin as glycoproteins was carried out in the gastric mucosa in 4 hr pyloric ligated rats . Mucosal scraping of glandular portion of rat stomach were homogenized in normal saline (20 mg/ml) and treated wi th 90% ethanol in the same man ner as descri bed for mucin esti mation in the gastric juice. The precipitate thus obtained, was subjected for the est imation of carbohydrates and protein us ing the methods described above for gastric juice contents' . The results are expressed as pg/ lOOmg wet ti ssue and TC:P ratio has been taken as the index of glycoprotein activity. Statistical ana lys is of data was done by using unpaired Student's t test.

Results CAJ in doses of 200 and 600 mg/kg, g iven twice

dai ly for 5 days showed a significant protection agai nst the experimental ulcers induced by EtOH , ASP, CRS, and PL and was comparable with SF a known ulcer protective drug (Table 1 ).

CAJ (200 and 600 mg/kg) showed little or no change in gastric juice volume, ac id and pepsin

SAIRAM eta/.: ANTI-ULCEROGENIC EFFECT OF CENTELLA ASIATICA JUICE 139

concentration or output, while SF showed significant reduction mainly in pepsin concentration and output (Table 2). A significant decrease in DNA content was observed both with CAJ and SF (Table 2).

On mucin secretion, both CAJ (600 mg/kg) and SF had pronounced effect on various fractions of mucoprotiens. They tended to decrease protein (P), had little or no effect on fucose and sialic acid, while they increased the total hexoses and hexoseamine significantly leading to increase in total carbohydrates

(TC) and TC:P ( Table 3). Similarly, higher dose of CAJ and SF tended to increase or increased total hexoses, hexosamine, sialic acid and total carbohydrates leading to significant increase in TC : P ratio of mucosal glycoproteins (Table 3).

Discussion The juice of CA (fresh whole plants) was found to

posses significant antiulcer activity against EtOH-, ASP-, CRS-, and PL-induced gastric ulcers in rats.

Figs 1-4- Photomicrograph of rat mucosa (1)- stained with haemotoxy lin-eosin showing "+ ' ulcer. Note the hi stological changes limited to the superficial layers with no congestion. (2)-showing '++' ulcer. Note the necrotic changes in about half of the mucosal thickness. (3)- showing '+++' ulcer. Note the necrotic changes in about two-thirds of the mucosal thickness. (4)- showing '+++' ulcer. Note the perforations with complete destruction with necrosi s and haemorrhage, and muscularis remains unaffected. [Figs 1-4 X 100; US­ulcer site; SML- superficial mucosal layer; MM-mucularis mucosae]

140 INDIAN J EXP BIOL, FEBRUARY 2001

Ulcers caused by chemical inducers like ethanol and aspirin could be due to their direct effect or release of noxious substances including free radicals30

.

Disruption of prostanoid synthesis is another contributing factor for aspirin- induced ulcers. Various physical and psychological stresses cause gastric ulcers in human and experimental animals31

•32

.

The effect of CAJ on various models suggests its diverse role in ulcer protection, which may include its reported effect on wound healing9

, as an antioxidant10

and GABA-ergic activities 15•

In the healthy stomach, there is a balance between aggressive factors and the protection afforded by pre­epithelial, epithelial and sub- epithelial mechanisms of mucosa. Secretion of mucus and bicarbonate by surface epithelial constitute a mucus-bicarbonate barrier, which is regarded as first line of defense against potential ulcerogens. Although pathogenesis of ulcer is multifactorial, ulcer is considered to be due to derangement of balance between aggressive and defensive factors. Hence further study was undertaken to study the status of aggressive factors namely acid

Table 1-Effect of juice of CA (CAJ) on ethanol (EtOH, 100%, 1 ml/200 g, po, 1 hr)-, aspirin (ASP, 200 mg/kg, po, 4 hr)-, 2 h cold

restraint stress (CRS)- and 4 hr pylorus ligated (PL)- induced gastric ulcers in rats

[Values are mean± SE of 6 animals in each group]

Treatment

(mg/kg, bd x 5 days)

Control CAJ

SF

Control CAJ

SF

Control CAJ

SF

Control CAJ

SF

/ 200 600 250

Ulcer Index

EtOH induced ulcers (mm2/rat)

15 .6±2.1 1.0 ± l.Ob

O±Ob 5.8 ±2.0"

ASP-induced ulcers

21.5±4.3 200 5.0±2.5" 600 1.3 ± 1.0" 250 3.5±3.0a

CRS-induced ulcers

29.8±2.4 200 8.0± 1.4b 600 6.4±2.0b 250 11.2±1.2b

PL-induced ulcers

15.0±2.6 200 3.2 ± 1.5 a 600 1.6 ± I.Ob 250 5.6±0.7a

P values: "< 0.01, b < 0.001

Protection (%)

93.6 100.0 62.8

76.7 94.0 83 .7

73.2 78.5 62.4

78.7 89.4 62.7

and pepsin and important defensive factors such as mucin secretion, cellular mucus and cell shedding. Cell shedding has been taken as an indicator of life span of mucosal cells29

.

In the gastric juice, evaluation of offensive factors showed that CAJ did not have any significant effect on acid and pepsin secretion, while SF decreased the concentration and output of pepsin. This activity may be due to its adherence to the gastric mucosa and interaction of SF polyanions with the substrate proteins thus, preventing pepsin from binding to the substrate, which is the first step in peptic hydrolysis. SF has also been reported to exert its anti-peptic effect by directly adsorbing to the enzyme33

.

The entire surface of the gastric mucosa is covered by a continuous layer of mucus gel. The mucin has several functions apart from being the first line of defense against noxious gastric contents. The defense is mostly due to active secretion of bicarbonates and retardation of diffusion of hydrogen ions and penetration of large molecules like pepsin, which are inactivated by the alkaline environmene4

. The thickness of mucus gel is determined by the dynamic balance between mucus secretion and surface erosion by proteolysis and mechanical destruction.

PL-induced gastric ulcers are caused by enhanced acid-pepsin secretion leading to auto digestion of the gastric mucosa and break down of mucosal barrier6

. A copious amount of gastric mucus is secreted during superficial mucosal damage and provides a favorable microenvironment in repair by restitution. Hence estimation of mucin secretion is valuable for the study of mucosal defensive mechanisms against ulcerogens. TC: P ratio is taken as reliable index for mucin secretion 17 and was significantly increased by CAJ. Increase in total carbohydrates reflects increase in dissolved carbohydrates and this was mainly due to significant increase in total hexoses and hexosamine. SF also significantly increased the TC:P indicating increased mucin secretion, which was mostly due significant increase in mucopolysachyharides such as total hexoses and hexosamine.

Surface mucus cells and mucus neck cells secrete mucous by exocytosis35.The main components of gastric mucous are the acidic glycoprotein, sialic acid and neutral mucopolysaccarides like total hexoses, hexosamine and fucose. The glycoproteins are of importance for their specific properties such as gel formation and viscosity. TC:P of the mucosa, which is taken as a reliable marker for cellular mucus29 was significantly increased by higher dose of CAJ,

SA I RAM eta/.: ANTI-ULCEROGENIC EFFECT OF CENTELLA ASIATICA JUICE 141

indicating increase in mucin content of the mucosa. This was primarily due to increase in mucopolysaccharides, specifically of sialic acid, which was significant. SF also increased sialic acid, total hexoses and total carbohydrates in the gastric mucosa, there by increasing TC:P indicating increase in glycoprotein content. Sialic acid, an acidic glycoprotein has been reported to have a mucosal defensive effects36

. Hence the augmentation of the mucosal barrier by CAJ was both due to increase in cellular mucus and secretion of dissolved mucus in the gastric secretion . This protective effect in tum caused less shedding of mucosal cells as evident from decrease in DNA content of the gastric juice, which is taken as a reliable indicator for cell shedding29

. SF similarly decreased the DNA content of the gastric juice. The increase in mucin can be attributed to several factors although its exact mechanism is still obscure. Cholinergic37

, GABA-ergic38 and seratonergic39 systems have been reported to stimulate mucus secretion. Prostagladins are important for maintaining the integrity of the gastric mucosa40 by several mechanisms including inhibition of acid output, stimulation of mucus and

reduction in cellular exfoliation6. In contrast, the

protective effect of SF was also due to anti-peptic activity apart from augmentation of various mucosal defensive factors. This was consistent with earlier findings of SF being categorized as mucosal protective agent, as it enhanced the mucosal defensive factors without any significant change in acid secretion 16

• SF is reported to produce these effects by a more complex mode of action. SF stimulates PG synthesis and release and thereby promotes bicarbonate secretion, although the increase in mucus secretion is independent of PGs6

.

CAJ can also be categorized as a mucoprotective agent like SF as it also shows a similar profile of action. Further studies on epithelial protective factors like cell tum over and restitution, subepithelial factors like mucosal blood flow and tissue acid- base balance and other factors like serotonin, GABA, PGs, nitric oxide, cAMP would throw more light on the precise mechanism of anti-ulcerogenic activity of CAJ.

The present investigation establishes the ulcer protective effect of juice of fresh plants of Centella asiatica and the protective effect may be due to augmentation of defensive mucosal factors.

Table 2- Effect of CAJon gastric juice volume, acid, pepsin. and DNA contents in 4 hr PL rats

[Values are mean ± SE of 6 animals in each group]

Treatment Volume Acid Pepsin DNA (mg/kg. bd x 5 days) (ml/IOOg) Concentration Output Concentration Output (pg/ml/100 g)

().!Eq/ml) ().!Eq/4hr) ()lmol/ml) ()lmol/4 hr)

Control 1.32 ±0.09 62.5 ±4.0 82.5 ± 11.7 484.2±22.4 639.1 ±49.7 292.3 ±24.4 CAJ 200 1.42 ±0.15 53.3 ±3.3 75.7 ± 10.6 446.3±26.6 633.7±83.2 202.2 ± 29.6'" 600 1.76 ± 0.23 52.5±3.1 92.4± 12.6 426.3± 14.3 750.3 ±52.8 159.2± 18.7 b

SF250 1.53 ±0.15 55.3± 3.1 84.6 ± 8.7 279.5±31.6 < 427.6±66.0" 131.8± 13.2c

P values: "< 0.05, b<O.OI, c< O.OOI

Table 3-Effect of CAJon gastric juice mucoproteins and glycoproteins in 4 hr PL rats

[Values are mean± SE of 6 animals in each group]

Treatment Protein Total hexoses Hexosamine Fucose Sialic acid TC TC : P (mg/kg, bd x 5 days) (P) (a) (b) (c) (d) (a+b+c+d)

Mucoprotei n (pg/ml)

Control 332.7± 27.9 203.8 ± 18.3 113.04 ± 12.9 68.1±5.7 26.6 ± 3.2 411.5 ± 49.6 1.25 ± 0.17 CAJ 200 274.1± 41.2 241 .0± 24. 1 155.8 ± 20.4 63.4 ± 6.8 24.9 ± 6.2 485. 1 ± 69.4 1.79 ±.0.23

600 245.6± 39.2 282.9± 25.0" 205.8 ± 24.9 b 65.8±9. 1 31.5 ± 2.5 558.0 ± 51.8 2.29 ± 0.32 u

SF 250 237.5± 35.9 297.0± 22.6b 184.2 ± 20.9 u 71.0 ± 11.2 28.6 ± 3.1 580.8 ± 63.9 2.48 ± 0.26 b

Glycoprotein (pg/IOOmg wet tissue)

Control 4252± 373 2714 ± 262 1391 ± 132 268 ± 11 99 ± 8 4472 ± 395 1.06 ± 0. 09 CAJ 200 4349± 306 3150 ± 241 1713 ± 145 240 ± 16 126 ±II 5229 ± 413 1.23 ± 0.11

600 3876 ± 245 3260 ± 275 1801 ± 151 280 ± 13 140± 13 " 5481 ± 3 17 1.45 ± 0.1 3"

SF 250 4031± 284 3554 ± 253" 1454 ± 106 307 ± 39 170 ± 29" 5485 ± 284 1.40 ± 0.12"

P values: "<0.05. b< O.OI

142 INDIAN J EXP BIOL, FEBRUARY 2001

Acknowledgement RKG is thankful to Indian Council of Medical

Research, New Delhi for grant-in-aid.

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