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Antiulcerogenic potential of Strychnos potatorum Linn seeds on aspirin plus pyloric ligation-induced ulcers in experimental rats.

Abstract

Strychnos potatorum (Fam: Loganiaceae) Linn seeds are useful in the treatment of gastropathy in Indian traditional system of medicine. The present study describes the antiulcerogenic potential of S. potatorum Linn seeds on aspirin plus pyloric ligation (Aspirin + PL)-induced gastric ulcer model to substantiate its folklore claim. The seed powder (SPP) and aqueous extract of the seeds (SPE) at two doses 100 and 200mg/kg, p.o. prevented ulcer formation by decreasing acid secretory activity and increasing the mucin activity in rats. The antiulcerogenic potential was further confirmed by the histopathological studies of stomach mucosa. The results indicate that SPP and SPE exhibit antiulcerogenic activity by both antisecretory and mucoprotective actions. The mucoprotective action of SPP and SPE may be due to the presence of polysaccharides in seeds. The antiulcerogenic potential of SPP and SPE was compared with the standard antiulcer drug, ranitidine.

[c] 2007 Published by Elsevier GmbH.

Keywords: Antiulcerogenic; Strychnos potatorum; Aspirin plus pyloric ligation; ranitidine

Introduction

Strychnos potatorum Linn (Fam: Loganiaceae), commonly known as Katakam in Ayurvedic system of medicine, is a moderate-sized tree found in southern and central parts of India, Srilanka and Burma (Kirtikar and Basu, 1933). The seeds are used in hepatopathy, nephropathy, gastropathy, bronchitis, chronic diarrhoea, dysentery, diabetes and eye diseases (Asima and Satyesh, 2001). The ripe seeds are used for clearing muddy water. They are reported to be very effective as coagulant aids. Alum aided by the seeds has been found to be very effective in removing the suspended impurities from coal-washery wastes. The clarification is due to the combined action of colloids and alkaloids in the seeds (Wealth of India, 1976).

Presence of diaboline (major alkaloid) and its acetate (Singh and Kapoor, 1975), [beta] sitosterol, stigmasterol, oleanolic acid and its 3[beta] acetate, saponins containing oleanic acid (Singh and Dhar, 1977), triterpenes (Harkishan Singh et al., 1975), mannogalactans (polysaccharides) (Corsaro et al., 1995), etc. have been reported in the seeds. The objective of the present study was to evaluate the antiulcerogenic potential of SPP and SPE in Aspirin + pyloric ligated (PL) ulcer model in rats.

Materials and methods

Animals used

Wistar albino rats (140[+ or -]20 g) of either sex procured from Tamilnadu University of Veterinary and Animal Sciences (TANUVAS) were used for the study. The animals were grouped and arranged three rats per cage and kept in polypropylene cages, maintained at a temperature of 22 [+ or -] 2 [degrees]C, atleast 1 week before commencing the experiment. They were fed with standard pelleted feed (TANUVAS) and water. The study received the approval from the Institutional Animal Ethical Committee (IAEC) of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Preparation of the extract

The S. potatorum seeds for the study were collected from crude drug market, Chennai, and the authenticity of the seed specimen was confirmed by Dr. S. Jayaraman, Botanist, Plant Anatomy Research Centre, Chennai, Tamilnadu. A voucher specimen of the seed was deposited at the Department of Pharmacology & Environmental Toxicology, Dr. ALM PGIBMS, University of Madras, Chennai, India.

The air-dried seeds were coarsely powdered and subjected to hot water extraction for 2h at 100 [degrees]C. It was then filtered and the filtrate was evaporated to dryness. A gray-colored semisolid mass was obtained, which was dried under vacuum and kept in a dessicator. The percentage yield of the extract (SPE) was 22.5% w/w from the starting raw material. The seed powder (SPP) as such was also used for the treatment. For the experimental study, both SPP and SPE were triturated with distilled water and administered immediately by oral intubation.

[FIGURE 1 OMITTED]

Phytochemical analysis

SPP and SPE were analyzed for the presence of phytochemicals by qualitative chemical analysis (Kokate et al., 1997). The HPTLC patterns for SPP and SPE fractions were developed. Polysaccharide fraction isolated (Corsaro et al., 1995) from SPE was hydrolyzed with 1 M sulphuric acid at 100 [degrees]C for 3h and the solution was hydrolyzed with Ba(OH)[.sub.2]. The precipitate was filtered and the filtrate was concentrated, examined for simple sugars by developing HPTLC pattern on solvent system n-butanol:methanol:water (5:2.5:2) and sprayed with diphenylamine reagent.

Antiulcerogenic activity

The animals were divided into seven groups, each containing six animals. Group I served as PL control, which received vehicle only. Group II served as Aspirin (200mg/kg, p.o.) + PL control. Group III received Ranitidine (50mg/kg, p.o.) as standard drug + Aspirin + PL. Groups IV and VI received SPP and SPE alone at the dose of 200 mg/kg, p.o + PL. Groups V and VII received SPP and SPE at the dose of 200mg/kg, p.o + Aspirin + PL.

Groups III-VII received the assigned drug treatment for the respective 10 days daily. From days 8 to 10, animals of Groups II, III, V, VII received aspirin orally as an aqueous suspension at the dose of 200mg/kg, 2h after the administration of the drugs (Goel et al., 1986). Animals in all groups were fasted for 18 h after the assigned treatment, anesthetized and the pyloric was ligated (Shay et al., 1945). The rats were sacrificed after 4h by excess anesthesia (ether). The stomach was removed, opened along greater curvature and the gastric lesions were observed. The gastric ulcers were counted and the ulcer index was determined (Minanao et al., 1987). The gastric juice was collected, centrifuged and the volume of the supernatant was expressed as mL/100 gm.b.wt. Free acidity and total acidity were determined by titrating with 0.01 N NaOH using Topfer's reagent and phenolphthalein as indicators (Parmar et al., 1984). The free and total acids were expressed as mEq/L. The total acid output was determined and expressed as mEq/L.

[FIGURE 2 OMITTED]

Estimation of biochemical parameters

Mucin activity was estimated in the mucosubstances precipitated twice by treating the gastric secretion with 90% ethanol in a 9:1 ratio. The precipitate thus obtained was divided into two parts, one part was dissolved in 1 mL of 0.1 N NaOH, and the other part was dissolved in I mL of 0.1 N [H.sub.2]S[O.sub.4]. The former was used for the estimation of protein (Lowry et al., 1951), total hexoses (Dische and Schettles, 1948), hexosamine (Dische and Baren Freund, 1950) and fucose (Winzler, 1958), while the latter was used for the estimation of sialic acid (Warren, 1959). The results were expressed as [micro]g/mL. Finally, the total carbohydrate to protein (TC:P) ratio, i.e., mucin activity was determined.

Histopathological examination

For histopathological examination, the stomach was washed thoroughly with saline, dehydrated in gradual ethanol (50-100%), cleared in xylene and embedded in paraffin. Sections (4-5 [micro]m) were prepared and then stained with hematoxylin and eosin (H-E) dye for photomicroscopic observation (magnification 100 x ).

Statistical analysis

The data represent Mean [+ or -] SEM. Results were analyzed statistically using one-way ANOVA followed by Tukey's multiple comparison. The minimum level of significance was set at p < 0.05.

Results and discussion

On qualitative phytochemical analysis, SPP and SPE revealed the presence of steroids, triterpenes, saponins, polyphenolics and sugars. The HPTLC pattern of simple sugars hydrolyzed from the polysaccharide fraction showed five well-resolved peaks (Fig. 1) and were also compared with the standard sugars mannose, galactose and maltose, which confirmed the presence of mannogalactans (i.e., mannose and galactose) (Fig. 2). The acute and chronic toxicity studies of SPP and SPE was reported (Sanmuga priya and Venkataraman, 2006) and it was found to be non-toxic upto the dose level of 2000 mg/kg/p.o.

Aspirin causes mucosal damage by interfering with prostaglandin synthesis, increasing acid secretion and back diffusion of [H.sup.+] ions (Rao et al., 2000). In pyloric ligation, the digestive effect of accumulated gastric juice and interference of gastric blood circulation are responsible for the induction of ulceration (Patel et al., 2000). Aspirin was administered to PL rats; thus, aspirin further aggravated the acidity and the resistance of the gastric mucosa was decreased thereby causing extensive damage to the glandular regions of the stomach. The effect of the drugs SPP and SPE on Aspirin + PL rats are presented in Tables 1 and 2. Table 1 shows the effect of drugs on secretory parameters like total and free acidity, total acid output, gastric volume and ulcer index. From the table, it is clear that the drugs exhibited significant antisecretory activity by reducing the secretory parameters when compared with the control group.

Table 2 indicates the effect of drugs on mucin activity of Aspirin + PL rats. In Group II (Aspirin + PL) rats, there was significant increase (p < 0.001) in protein concentration, but decrease in individual as well as total carbohydrate levels, when compared with control. The drug treatment significantly decreased the protein level and increased the total carbohydrate (TC) level. The index of mucin activity TC:P was found to be decreased in the Aspirin + PL rats. The drugs SPP and SPE at the tested dose levels significantly (p<0.001) increased the TC:P ratio. The standard drug Ranitidine showed less significance (p<0.01) when compared with the test drugs.

[FIGURE 3 OMITTED]

Increased mucus secretion by the gastric mucosal cells can prevent gastric ulceration by several mechanisms, including lessening of stomach wall friction during peristalsis and gastric contractions, improving the buffering of acid in gastric juice and by acting as an effective barrier to back diffusion of H + ions (Venables, 1986). Both the drugs SPP and SPE were found to augment the mucin secretion as evidenced from the increase in concentration of the individual as well as total carbohydrate levels. The drugs also increased the TC:P ratio, which reflects the functional integrity of the mucosal barrier and has been accepted as a reliable index of mucin secretion. The increase in protein content of the gastric juice resulting in a decrease in the TC:P ratio in the Aspirin + PL animals indicates damage of the gastric mucosa, as a result of which the plasma proteins may leak into the gastric juice. As the drug treatment significantly reduced the protein concentration and increased the total carbohydrate content, it may be suggested that the drugs SPP and SPE may act by strengthening the mucosal barrier of the gastric mucosa. The Group VII (SPE, 200mg/kg p.o.) showed greater activity that is comparable to the standard drug, Ranitidine. The presence of mannogalactans (Corsaro et al., 1995) in SPP and SPE may be responsible for the mucoprotective action in the specified ulcer model. The antiulcer activity of various polysaccharides in experimental ulcers has also been reported (Sun et al., 1992; Matsumoto et al., 2002). The antiulcerogenic potential of SPP and SPE was further evidenced by the histopathological studies (Fig. 3).

In conclusion, both SPP and SPE at the dose of 200 mg/kg, p.o. exhibited antiulcerogenic activity, which may be attributed to the presence of mucilaginous polysaccharides (mannogalactans).

References

Asima, C., Satyesh, C.P., 2001. The Treatise of Indian Medicinal Plants, vol. 4. Publications and Information Directorate, CSIR, Delhi, pp. 85-87.

Corsaro, M.M., Giuddiciani, I., Lanzetta, R., Marciano, C.E., Monaco, P., Parilli, M., 1995. Polysaccharides from seeds of Strychnos species. Phytochemistry 39 (6), 1377-1380.

Dische, Z., Baren Freund, I., 1950. A spectrophotometric method for the micro determination of hexosamine. J. Biol. Chem. 184, 517.

Dische, Z., Schettles, L.B., 1948. A specific color reaction for methyl pentoses and spectrophotometric micro method for determination. J. Biol. Chem. 175, 595.

Goel, R.K., Gupta, S., Shankar, R., Sanyal, A.K., 1986. Antiulcerogenic effect of banana powder (Musa sapientum var paradisica) and its effect on mucosal resistance. J. Ethnopharmacol. 18, 33-44.

Harkishan Singh, Vijay K Kapoor, 1975. Investigation of Strychnos spp. III study of triterpenes and sterols of Strychnos potatorum seeds. Planta Med. 28, 392-396.

Kirtikar, K.R., Basu, B.D., 1933. Indian Medicinal Plants, vol. 3. L.M. Basu Publications, Allahabad, p. 1647.

Kokate, C.K., Purohit, A.P., Gokhale, S.B., 1997. Pharmacognosy, fifth ed. Nirali Prakashan Publications, India, pp. 109-137.

Lowry, O.H., Rosenbrough, N.J., Farr, A.I., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

Matsumoto, T., Sun, X.B., Hanawa, T., Kodaira, H., Ishii, K., Yamada, H., 2002. Effect of the antiulcer polysaccharide fraction from Bupleuram falcatum L. on healing of gastric ulcer induced by acetic acid in rats. Phytother. Res. 16, 91-93.

Minanao, J.F., Serrano, J.S., Pascaul, J., Sancibrian, M., 1987. Effects of GABA on gastric acid secretion and ulcer formation in rats. Life Sci. 41, 1651-1658.

Parmar, N.S., Hennings, G., Gulati, O.P., 1984. The Gastric antisecretory activity of 3-methoxy 5, 7, 3, 4-tetra hydroxyl flavan (ME)--a specific histidine decarboxylase inhibitor in rats. Agents Actions 15, 143.

Patel, A.V., Santani, D.D., Goyal, R.K., 2000. Antiulcer activity and the mechanism of action of magaldrate in gastric ulceration models of rat. Ind. J. Physiol. Pharmacol. 44 (3), 350-354.

Rao, Ch.V., Sairam, K., Goel, R.K., 2000. Experimental evaluation of Bacopa monniera rat gastric ulceration and secretion. Ind. J. Physiol. Pharmacol. 44, 35-41.

Sanmuga priya, E., Venkataraman, S., 2006. Toxicological investigations on Strychnos potatorum Linn seeds in experimental animal models. J. Health Sci. 52 (4), 339-343.

Shay, H., Komarov, S.A., Fels, S.S., Meranze, D., Gruenstein, M., Siplet, H., 1945. A simple method for the uniform production of gastric ulceration. Gastroenterology 5, 43.

Singh, H., Kapoor, V.K., Phillipson, J.D., Bisset, N.G., 1975. Diaboline from Strychnos potatorum. Phytochemistry 14, 587-588.

Singh, A.K., Dhar, D.N., 1977. Studies on the chemical constituents of the seeds of Strychnos potatorum L. Part II. Planta Med. 32, 362-367.

Sun, X.B., Matsumoto, T., Yamada, H., 1992. Purification of an antiulcer polysaccharide from the leaves of Panax ginseng. Planta Med. 58 (5), 445-448.

Venables, C.W., 1986. Mucus, pepsin and peptic ulcer. Gut 27, 233-238.

Warren, L., 1959. The thiobarbituric acid assay of sialic acid. J. Biol. Chem. 234, 1971-1975.

Wealth of India, 1976. Raw Materials. Sp-W, vol. 10. Publications and Information Directorate, CSIR, New Delhi, pp. 66-67.

Winzler, R.J., 1958. Determination of serum glycoproteins. Methods Biochem. Anal. 2, 279.

E. Sanmugapriya (a), S. Venkataraman (b,*)

(a) Department of Pharmacology & Environmental Toxicology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai-600 113, India

(b) C.L. Baid Mehta Foundation for Pharmaceutical Education and Research, Jyoti nagar, Old Mahabalipuram Road, Thorapakkam, Chennai-600 096, Tamilnadu, India

*Corresponding author. Tel.: + 44 24960151; fax: +44 24960425.

E-mail address: svraman@md4.vsnl.net.in (S. Venkataraman).
Table 1. Effects of SPP and SPE on acidity parameters in aspirin +
pyloric ligation-induced ulcers

Groups Total acidity (mEq/1/4 h) Free acidity (mEq/1/4 h)

Group I 138.52[+ or -]3.47 113.16[+ or -]5.68
Group II 260.83[+ or -]3.81a* 193.16[+ or -]6.35a*
Group III 159.83[+ or -]7.45b* 145.33[+ or -]8.56 b (@)
Group IV 121.33[+ or -]6.23b* 102.56[+ or -]7.25b*
Group V 180.66[+ or -]5.35b (#) 148.62[+ or -]6.45b (@)
Group VI 115.32[+ or -]4.35b* 98.56[+ or -]2.65b*
Group VII 154.66[+ or -]3.56b* 135.49[+ or -]4.62b (#)

Groups Total acid output (mEq/1/4 h) Gastric volume (mL/100 g)

Group I 378.01[+ or -]12.50 2.66[+ or -]0.06
Group II 958.75[+ or -]10.40a* 4.44[+ or -]0.07a*
Group III 526.00[+ or -]9.68b (#) 3.28[+ or -]0.11a*b*
Group IV 358.23[+ or -]8.32b* 2.45[+ or -]0.06b*
Group V 596.46[+ or -]9.47b (@) 3.37[+ or -]0.09a*b*
Group VI 342.56[+ or -]5.64b* 2.36[+ or -]0.02b*
Group VII 518.41[+ or -]7.40b (#) 3.05[+ or -]0.05a (#)b*

Groups Ulcer index

Group I 1.10[+ or -]0.02
Group II 4.32[+ or -]0.07a*
Group III 2.99[+ or -]0.09a*b*
Group IV 1.01[+ or -]0.09b*
Group V 2.74[+ or -]0.14a*b*
Group VI 0.97[+ or -]0.11b*
Group VII 2.56[+ or -]0.03a (#)b*

Values are Mean[+ or -]s.e.m., n = 6 animals in each group. Group
I -- vehicle control (PL), Group II -- aspirin (200 mg/kg body wt./p.o.)
+ PL control, Group III -- ranitidine (50 mg/kg body wt./p.o.) + aspirin
+ PL, Groups IV and VI -- SPP and SPE alone at the dose 200 mg/kg body
wt./p.o. + PL, Groups V and VII -- SPP and SPE treated (200 mg/kg body
wt./p.o.) + aspirin + PL.
Comparisons were made between: (a) Group I vs. II, III, TV, V, VI and
VII; (b) Group II vs. III, IV, V, VI and VIT; (c) Group III vs. V and
VII. Symbols represent statistical significance: *p<0.001, (#) p<0.01,
(@) p<0.05.

Table 2. Effects of SPP and SPE on mucin activity in aspirin + pyloric
ligation induced ulcers

Groups Protein ([micro]g/mL) Hexose ([micro]g/mL)

Group I 266.84[+ or -]7.05 856.02[+ or -]5.06
Group II 474.77[+ or -]5.81a* 468.59[+ or -]9.14a*
Group III 354.07[+ or -]4.12a (#)b (#) 560.64[+ or -]8.21a*b (@)
Group IV 250.15[+ or -]10.2b* 874.64[+ or -]5.59b*
Group V 335.13[+ or -]6.61a (#)b*c (@) 669.06[+ or -]10.80a (#)b*
 c (@)
Group VI 249.91[+ or -]12.79b* 885.76[+ or -]5.33b*
Group VII 281.75[+ or -]6.34a (@)b*c (#) 752.74[+ or -]3.77a (@) b*c*

Groups Hexosamine ([micro]g/mL) Sialic acid ([micro]g/mL)

Group I 241.16[+ or -]5.09 66.75[+ or -]1.72
Group II 135.20[+ or -]4.31a* 26.39[+ or -]1.33a*
Group III 165.53[+ or -]3.31a*b (#) 35.25[+ or -]1.17a*b (@)
Group IV 250.49[+ or -]6.13b* 69.84[+ or -]2.22b*
Group V 186.26[+ or -]3.08a (#)b*c (@) 56.07[+ or -]1.25a (@) b*c*
Group VI 256.28[+ or -]5.49b* 70.33[+ or -]1.79b*
Group VII 226.38[+ or -]5.49a (@)b*c* 69.28[+ or -]2.19b*c*

 Total carbohydrates
Groups Fucose ([micro]g/mL) ([micro]g/mL)

Group I 97.10[+ or -]0.80 1261.05[+ or -]4.85
Group II 89.80[+ or -][5.36.sup.n.s] 720.00[+ or -]7.09a*
Group III 98.08[+ or -][0.56.sup.n.s] 959.50[+ or -]11.05a* b (#)
Group IV 97.00[+ or -][0.77.sup.n.s] 1286.76[+ or -]11.03b*
Group V 99.42[+ or -][0.44.sup.n.s] 1108.83[+ or -]9.85a (#)b*c (@)
Group VI 103.21[+ or -][1.01.sup.n.s] 1298.32[+ or -]10.56b*
Group VII 98.12[+ or -][0.79.sup.n.s] 1196.32[+ or -]7.63a (@)b*c (#)

Groups TC:P

Group I 4.73[+ or -]0.12
Group II 1.51[+ or -]0.02a*
Group III 2.42[+ or -]0.03a*b (#)
Group IV 4.90[+ or -]0.21b*
Group V 4.21[+ or -]0.56a (@)b (#)c*
Group VI 5.20[+ or -]0.21b*
Group VII 4.56[+ or -]0.16b*c*

Values are Mean[+ or -]s.e.m., n = 6 animals in each group. Group
I -- vehicle control (PL), Group II -- aspirin (200 mg/kg body
wt./p.o.) + PL control, Group III -- ranitidine (50 mg/kg body
wt./p.o.) + aspirin + PL, Groups IV and VI -- SPP and SPE alone at the
dose 200 mg/kg body wt./p.o. + PL, Groups V and VII -- SPP and SPE
treated (200 mg/kg body wt./p.o.) + aspirin + PL. Comparisons were made
between: (a) Group I vs. II, III, IV, V, VI and VII; (b) Group II vs.
III, IV, V, VI and VII; (c) Group III vs. V and VII.
Symbols represent statistical significance: *p<0.001, (#) p<0.01,
(@) p<0.05.
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Author:Sanmugapriya, E.; Venkataraman, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:9INDI
Date:May 1, 2007
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