Indian Journal of Experimental Biology Vol. 39. Apri l 200 I . pp. 350-354

Effect of Convolvulus pluricaulis Chois on gastric ulceration and secretion in rats

K Sairam. eh v Raa & R K Gae( Department of Pharmacology. Institute of Medical Sciences. Banaras Hindu University. Varanasi 221005 .

Receil'ed 5 May 2000; rel'ised 23 October 2000

COI/I 'O/I'U/US p/uric(/u/is is an indigenous plant commonly mentioned in Ayurveda. an ancient system of Indian med i­cine. as a rasayana which is mainly advocated for use in rejuvenati on therapy. The present study was conduc ted to evaluate the potential anti -ulcerogenic effect of juice of fresh whole plants of C p/uric(lll/is (CPJ) against various experimental gas­tri c ulcer mouels induced by ethanol. aspirin. 2 hI' co ld restrai nt stress and 4 hr pyloric ligation in rats. The drug was given orally twice daily for live days in the doses of 375 and 750mg Ikg body weight. CPJ showed anti- ulcerog\!nic effect at both doses in all the experimental gastric ulcer models and was comparable to the reference drug sucralfate (250mg/kg). Gastric juice secretion and mucosa l studies were undertaken to find ou t the possible mechanism of act ion of aJ1l iuleer effect by studying i ts effec ts both on offensive and defensive mucosal factors. The an tiu lcerogenic effect o f CPJ was fOllnd to be due to augmentation of mucosal defen ive factors like mucin secretion. lifespan of mllcosal cell s and glycoprol iens rather than on the offensive factors like acid-pepsin.

CO/l volvulus pluricaulis Chois (CP) is an indigenou hairy perennial native plant of some parts of northern India and Biharl. It has been commonly mentioned as a rasayana in several Ayurvedic texts includ ing Caraka Salllhira and Cakradllfla . A ra ayana is one which promotes longevi ty and prevents diseases by providing strength and immunity 2. In Caraka Salllhita, it is mentioned as one of the ingredients of Bramha rasayana, whi ch is advocated for use in rejuvenation therap/: The juice of CP mixed with other ingredients is used to allev iate insanit/. CP is reported to have and hypnoti c activity and is clinically effective in anxiety neurosis5

.fl

.

Precise etiology of ulcer is unclear and a sati factory regi men still remains el usive. Many factors are thought to be involved in the pathogenesis of ulcer7

. Ulcers are thought to be due to an imbalance in (a) offensive factors like acid and pepsin and (b) defensive factors like mucin secretion, cell proliferation, prostagladins etcs. Most of the available drugs are thought to act on the offensive factors, such as antacids wh ich neutrali ze acid secretion and H~

receptor blockers'> viz. ranitidine, fa motid ine etc., anticholinergics like pirenzepine, telezipine etc., and proton pump inhibitors lO like omeprazo le etc., which interfere with acid secretion, whereas sucralfate the cytoprotective drug is also reported to have anti ­peptic activit/ I . A number of herbal drugs including

* Correspondent author: rax: 0542-3675(,X. E-mai l :rkgoel@banaras.ef.llct. in

banana powder l2 and Tambarabasma. a traditional preparation of copper l3 have been reported to have antiulcer act ivity by virtue of their beneficial effects on mucosal defensive factors like mucin secretion. Stress is one of the important factors for gastric ulceration 14 . CP being a rasayana akin to the modern adaptogens, which are mostly used for stress related disorders, it was thought prudent to ex perimentally evaluate the potential anti- ulcer ac tivity of jui ce of COll volvulus pluricaulis (CPJ). Further, studi es were done to probe its possible role in offensive and defensive factors in volved in ulcerogenesis.

Materials and Methods

Animals-Albino rats of CF strain of either sex weighing between 150 - 180 g were procured from the Central Animal House of the Ins titute and were housed in well ventilated co lony cages in the departmental animal house at 25° ± 2° C and 45-55 % RH , 10: 14 hI' L: 0 cycle for one week for acclimatization. The animals were fed with standard rodent pellet diet (Hind Lever) and water ad libitum.

Collectio/l oj pla/lt-C. plllricalllis of cultivated variety was obtained in the month of April from the Ayurvedie gardens of the Institute and was idcntifi ed \ ith the reference herbarium maintained in the Dep:lrtmcilt of Dravyagun:l. Whole green plants ( I kg.) wa~ . izL' reduced, cru shed l.lnd 200 1111 or juiCE thll~ oblaincd was filtered. The dry weight in term. 01 solid con ten t ill thc juice was 2.5 %. The fresh juice

SAIRAM et al : ANTI-ULCEROGENIC EFFECT OF JUICE OF CONVOLVULUS PLURICAUU 351

was stored in a refrigerator at _20° C in a glass containers of 20 ml capacity and was used with in a week of its extraction. The juice was warmed each time at 37° C before administration to the animals.

Experimental study--CPJ in doses of 0.75 and 1.5 ml/ 100 g ( equivalent to 325 and 750 mg/kg,in terms of dry weight) and sucralfate (SF) in the dose of 250 mg/kg were administered orally, twice daily at 1000 and 1600 hrs respectively for 5 days. On the 6th day of experiment, the 18 h fasted rats were subjected to the following experimental gastric ulcer studies:

a) Ethanol (ETH)- induced ulcers: The gastric ulcers were induced in rats by administering orally 100 % ETH ( Im1l200 g )11 and the animals were sacrificed by cervical dislocation after 1 hr of ETH administration and 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 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 on the day of the experiment and ulcers were scored after 4 hr as described earlierl5.The number of ulcers per stomach was noted and the severity of the ulcers was scored after histological confirmation as follows : 0, no ulcer; +, pin point ulcer 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 1-2 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 with complete destruction of the mucosa with necrosis and haemorrhage, muscularis still remaining unaffected. The pooled group ulcer score was then calculated1s. Statistical analysis was done by using Wilcoxon Sum Rank test 19.

c) Cold-restraint stress (CRS)- induced ulcers: On day six, cold restraint stress was gi ven to 18 hr fasted rats by strapping the rats on a wooden plank and keeping them at 4°_6°C for 2 hr. The animals were then sacri ficed by cervical dislocation and ulcers were scored on the dissected stomachs 16 as described above.

d) Pylorus-ligated (PL)- rats: Drugs were admini stered fo r a period of 5 days . On day six after the las t dose, the rats were kept fo r 18 hr fas ting and

care was taken to avoid coprophagy. Animals were anaesthetized using pentobarbitone (35 mg/kg, ip), the abdomen was opened and pylorus ligation was done 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 17 . After 4 hr, stomachs were dissected out and contents were collected into tubes for estimation of biochemical parameters. 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 mentioned in aspirin induced ulcers.

Gastric secretion-The gastric juice was collected 4 hr after PL and centrifuged for 5 min at 2000 rpm and the volume of the supernatant was expressed as mlllOOg body weight. Total acid output was determined by titrating with 0.01 N NaOH, using phenolphthalein as indicator and is expressed as llEq/4hr. Peptic activity was determined using haemoglobin as substrate20 and has been expressed as llmollml and llmo1l4 hr for concentration and output respectively. Dissolved mucosubstances were estimated in the 90 % alcoholic precipitate of the 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 the estimation of protein2 1

, total hexoses22, hexosamine23 and fucose24

,

while the latter was used for the estimation of sialic acid25

• The results are expressed in llg/ml. The ratio of total carbohydrate (TC) (sum of total hexoses, hexosamine, fucose and sialic acid) to protein (P) has been taken as the index of mucin activitl6

. DNA content were estimated and expressed as Jlg/ml gastric juice/IOOg weight of rat27

•

Gastric mucosal studies-Samples of gastric mucosa were homogenized in normal saline and treated with 90% ethanol and were subjected for the estimation of carbohydrates and proteins using the methods described above for gastric juice contents. Statistical analys is of data was done by using unpaired Student's t test.

Results CPJ in the doses of 375 - 750 mg/ kg, twice daily,

given for 5 days orally, showed signi ficant protection against various experimental gastric ulcers induced by ethanol, aspirin, 2hr cold restraint stress and 4h pyloric ligation in rats and was comparable to the

352 INDIAN J EXP BIOL, APRIL 2001

reference drug sucralfate ( 250 mg/ kg, twice daily, orally for 5 days) (Table I) .

In gastric secretion studies, CPJ (375 - 750 mgl kg) did not show any significant effect on volume, concentration and output of acid and pepsin secretion. However, SF showed significant reduction in concentration and output of pepsin (Table.2) with little or no effect on volume and acid secretion. A significant reduction in DNA content of the gastric juice was observed with both the doses of CPJ and SF (Table 2).

Table I-Effect of juice of C. pillricGlliis (CPJ ) on ethanol (ETH. 100%. I ml1200 g. po. I hr)-, aspirin (ASP. 200 mg/kg. po, 4 hr)-, 2 hr cold restrai nt stress (CRS)-. and 4 hr pylorus ligated (PL)-

induced gastric ulcers in rats. [Values are mean ± SE from 8 animals in each group]

Treatment Ulcer index

(mg/kg, bd x 5 days)

ETH (mm1/rat)

Control 25.3±5.9

CPJ 375 9.0±3.?"

750 4.3± 1.9h

SF 250 6.2±1.4b

ASP

Control 17.8±3.3

CPJ 375 11 .0±2.6

750 6.4±2.2·

SF 250 4.8±2.5b

CRS

Control 18.9±2.8

CAJ 375 11.0±1.9·

750 9.3±1.8"

SF 250 8.2± I.7b

PL

Control 14.6±3.2

CPJ 375 6.0±2.0·

750 4.1±1.7·

SF 250 4.3±1.8·

P values: • < 0.05, b < 0.0 I

Protection

(% )

64.4

83.0

75.5

38.2

64 .0

73.0

41.8

50.8

56.6

58.9

71.9

70.5

Effect of different doses of CPJ and SF on mucin secret ion and mucosal glycoprotiens is shown in Table 3. On mucin secretion, CPJ at the lower dose produced significant changes in hexosamine and TC: P, whereas the higher dose produced significant increase not only in hexosamine and TC:P but also in levels of other individual carbohydrates like total hexoses and sialic acid and total carbohydrates compared to the control group. SF at 250 mg / kg produced a significant change in sialic acid, total carbohydrates and TC:P ratio, while other parameters were not significantly altered.

On mucosal glycoproteins, a significant increase was observed in TC:P ratio with higher dose of CPJ and SF. The significant increase in TC:P was due to their effects on individual carbohydrates and protein content which either tended to increase or decrease respectively, except for a significant increase in s ialic acid content with SF.

Discussion The juice of fresh plants of C. pfllricQulis has been

found to possess significant antiulcer activity in various experimental gastric ulcer models and was comparable to the reference drug sucralfate. The protective effect of CPJ on different models may be due to activities covering a wide range, as many factors are though t to be involved in pathogenesis of gastric ulcers in different models. The damage produced by ethanol to gastric mucosa is due a number of contributing factors, which include effects on mucosal blood flow, platelet thrombi, damage to capillary endothelium and release of arachidonate metabolites, specifically LTCJD4 and PAF 8. NSAlDs produce gastric ulcers by inhibiting fo rmation of PGs, which are essential for integrity of mucosa as they have various mucosal defensive actions29

. Further PGs increase the conversion of hydroxy fatty acids in the lipo-oxygenase pathway and xanthone-oxidase activity in the mucosa28

. PL induced ulcers are

Table 2- Effect of CPJ on gastric juice volume, acid, pepsin, and DNA contents in 4 hr PL rats. [Values are mean ± SE from 8 animals in each group]

Treatment Volume Acid Pepsin DNA

(mglkg. bd x 5 (miIlOOg) Concentration Output Concentration Output (>lg/mlllOO g) days) (~Eq/ml) (~Eq/4hr) (~mollml ) (~moIl4 hr)

Control 1.18±0. 13 111 .6±8.0 137.6± 14.0 652.5±58.2 787.4±72.9 273.5±23.2

CPJ 375 1.39±0.11 98.3±5.4 142.5±17.6 639.5±49.4 893.3±71 .5 2oo.2±20.8"

750 1.16±0.14 89.2±8.7 104.7± 16.5 635.4±52.2 747.2±78.9 I 69.8±27.6"

SF 250 1.12±0.14 96.2±8.4 I09.4±IO.2 476.1±65.0· 535.2±81 .3· I 66.5±22.2b

P values: • < 0.05, b < 0.0 I

SAIRAM et al : ANTI-ULCEROGENIC EFFECf OF JUICE OF CONVOLVULUS PLURICAULI 353

Table 3--Effect of CPJ on gastric juice mucoproteins and mucosal glycoproteins in 4 hr PL rats [Values are mean ± SE from 8 animals in each group]

Treatment (mg/kg, Protein Total hexoses Hexosamine Fucose Sialic acid TC TC:P bd x 5 days) (P) (A) (B) (C) (D) (A+B+C+D)

Mucoprotei n (~g/m l )

Control 434.1± 30.6 291 .3 ± 26.3 174.3 ± 17.0 57.1 ± 3.6 37.7 ± 2.8 560.4 ± 36.8 1.29 ± 0.11

CPJ 375 426.8± 23.0 375.7± 35.7 230.0 ± 18.6" 66.2 ± 5.2 43.9 ± 3.5 715.8 ± 69.4 1.68 ± 0.3 1"

750 38 1.7 ± 17.4 403.3± 40.7" 263.8 ± 23.0 b 65.8 ± 4.7 54.2 ± 6.2" 787.9 ± 63.0b 2.06± 0.25 "

SF 250 370. 1± 27.1 352.2± 31.3 229.4 ± 27.0 67 .2 ± 5.9 59.4±8.1" 708.2 ± 50.8" 1.9 1 ±0.21 "

Glycoprotein (~g/lOOmg wet tissue)

Control 5234± 459 2059 ± 245 1266± 83

CPJ 375 4675± 397 2380 ±260 1397 ± 102

750 4367 ± 312 2649 ± 20 1 1431±103

SF250 4589± 320 2624 ± 282 1380 ± 124

P values: "< 0.05, b < 0.0 I

thought to be caused due to increased presence of acid and pepsin in the stomach8

. Stress induced ulcers are caused by a number of factors both physical and psychological 30. Increase in gastric motili ty31, vagal overactivity32. mast cell degranulation33, decreased gastric mucosal blood flow34 and decreased PG synthesis30 are also reported to be involved in genesis of stress induced ulcers. Involvement of free radicals are also reported for gastric ulcers caused by ethanol35

and stress36. Hence diverse mechanisms may be involved in anti ulcerogenic activity of CPJ.

As genesis of gastric ulcer is thought to be due to an imbalance in offensive and defensive factors, a study on the above parameters in the gastric secretion revealed that CPJ did not show antacid or anti peptic acti vity , but a significant change in the quality and quantity in dissolved mucin content and decrease in cell shedding was observed. SF showed significant anti peptic activity as seen from the reduction in peptic concentration and output as reported earlierll. The gastric secretion study showed that the concentration of total hexoses, hexoseamine and sialic acid were significantly increased signifying the increase in rate of mucin secretion and is further evidenced by an increase in total carbohydrates: protein ratio (TC:P) which is taken as a reliable marker for mucin secretion thus, indicating the enhancement of mucosal resistance factors by CPJI 5. The protective effect could also be due to the strengthening of the mucosal barrier as observed from the decrease in DNA content, indicating decreased cell shedding27 and decreased protein content in the gastric juice suggesting decreased leakage from the damaged mucosa by

198 ± 19 208 ± 22 373 1 ± 302 0.71 ± O. 07

197 ± 17 214 ± 21 4188 ± 254 0.90 ± 0.08

245 ± 20 227± 17 4552±284 1.04±0.10"

210 ± 19 299 ± 26" 4713± 392 1.03 ± 0.09"

ulcerogens37 .The effect of CPJ was comparable to the ulcer protective drug SF.

On the gastric mucosa CPJ significantly increased glycoprotein content as indicated by an increase in the TC:P rati027 . The changes induced by CPJ in the mucosal carbohydrates further corroborate the evidence of increase in muci n secretion and the protection afforded by CPJ to damage by different ulcerogens may be due to its fortification of defensive mucin secretion.

The present study, for the first time reports the anti ulcerogenic effect of CPJ. The effect may be due to the augmentation of defensive mucosal factors rather than on the offensive factors .

Acknowledgement RKG is thankful to ICMR for grant-in-aid.

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