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The effect of a traditional preparation of copper, tamrabhasma, on experimental ulcers and gastric secretion
Journal of Ethnopharmacology, 5 (1982) 79 - 89 0 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
THE EFFECT ~~~~~~~~~~~, SECRETION
A. K. SANYAL*, Department Varanasi-221
79
OF A TRADITIONAL PREPARATION OF COPPER, ON EXPERIMENTAL ULCERS AND GASTRIC
B. L. PANDEY and R. K. GOEL
of Pharmacology, 005 (India)
(Received June 18,198O;
Institute
of Medical
Sciences,
Banaras
Hindu
University,
accepted September 15,198O)
Summary Use of a traditional preparation of copper, tamrubhama, has been suggested in Ayurvedic texts for peptic ulcer. The anti-ulcerogenic effect of copper per se has not been reported in the literature. In the present study the anti-ulcerogenic effect of ~~~~u~~~s~~ was observed in 8-h immobilised, 4-h pylorus-ligated, and aspirin-induced gastric ulcers in rats. The anti-ulcerogenie effect of the drug was also studied in histamine-induced gastric and duodenal ulcers in male guinea pigs. The minimal oral effective anti-ulcerogenit dose of ta~ra~~as~a has been determined to be 1 mg/kg. The drug in this dose caused a decrease in the total acid and pepsin output and an increase in the carbohydrate/protein ratio, indicating increased mucus secretion in the gastric secretion of rats. Acute (1 g/kg, orally) and sub-acute (100 mg/ kg, orally, for 7 days) toxicity studies did not show any toxicity including any histological or biochemical evidence of liver or kidney toxicity. The results of the present experimental studies thus lend credence to the suggested use of ta~ru~~as~a in a~~lapitta, a condition resembling peptic ulcer.
Introduction The treatment of peptic ulcer is still unsatisfactory notwithstanding the introduction of drugs like cimetidine and carbenoxolone. Many new ideas are being tried out to find a better remedy. The geographic~ distribution of the disease in India (Tovey, 1979) had earlier prompted one of us to look for such anti-ulcerogenic agents in natural products. The result of such a study was the discovery of an anti-ulcerogenic effect of vegetable banana in different experimental models (Sanyal et al., 1961,1964, 1965), an observation recently confirmed by other workers (Elliot and Heward, 1976).
*To whom correspondence
and reprint requests should be addressed.
80
A superficial indication that dietary copper may influence the incidence of the disease is obtained from the fact that dietary copper is relatively lower where the disease is more prevalent in India, and vice uersa. Vegetable banana is reported to contain significant quantities of copper (Aykroyd, 1963). These observations prompted us to study the anti-ulcerogenic effect, if any, of an indigenous, traditionally prepared copper preparation, tamrubhasma, which has been cited in the Ayurvedic texts (Bhavaprakash, 1969) and which is an important constituent of a formulation used by practitioners of traditional medicine as a good remedy for amlupitta, a clinical entity resembling peptic ulcer. A survey of the literature indicates that the anti-ulcerogenic effect of copper has not been reported, though decreased ulcerogenicity has been reported with copper complexes of non-steroidal anti-inflammatory agents compared to the effect of the latter drugs alone (Boyle et al., 1976; Rainsford and Whitehouse, 1976). Materials and Methods The present study was conducted with a preparation of tumrubhusmu (TMB) obtained from the Ayurvedic Pharmacy of this University. An earlier published study (Raghunathan, 1976) had given the composition of TMB as: CuO 244.45% <66.13%; Fe,O, <6.03%; and sulphur <2.75%. The anti-ulcerogenic effects of TMB were studied on albino rats weighing 120 - 150 g of either sex and on male guinea pigs weighing 450 - 600 g. The animals were maintained on Hind Lever pellet diet. Animals were fasted for 18 h before experimentation but water was allowed ad libitum. Experimental gastric and duodenal ulcers were produced in rats and guinea pigs as follows. (A) Gastric ulcers in albino ruts (1) Pylorus-ligated gastric ulcers were produced by pylorus ligation for 4 h as described earlier (Sanyal et al., 1971). (2) Acetylsalicylic acid-induced gastric ulcers were produced by administering the drug as a suspension in water in a dose of 200 mg/kg, once daily for three days by mouth with the help of an oro-gastric polythene tube. The pylorus was then ligated on the fourth day and rats were sacrificed after 4 h by decapitation. The control group received an equal volume of water only. (3) To produce restraint ulcers, rats were immobilised on a wooden plank by adhesive plaster, adopting the method described by Fregly (1953), for 8 h. After the period of immobilisation, the rats were sacrificed by decapitation. (B) Histamine-induced ulcers in guinea pigs (1) Gastric ulcers were induced in guinea pigs with a single injection of histamine, following the method of Eagleton and Watt (1965). (2) Duodenal ulcers were produced by repeated injections of histamine adopting the method of Eagleton and Watt (1967).
81
In the rats, the number of ulcers per stomach was noted and the severity of the ulcers was scored after histological confirmation as follows: 0, no ulcer; +, changes limited to superficial layers of mucosa and no congestion; ++, half of the mucosal thickness showed necrotic changes; +++, more than two-thirds of the mucosal thickness destroyed with marked necrosis and congestion, muscularis remaining unaffected; ++++, complete destruction of the mucosa with necrosis and haemorrhage, muscularis still remaining unaffected. The pooled group ulcer score was then calculated by the method of Robert et al. (1968). In the guinea pigs, the presence or absence of ulcers was noted in each group and statistical significance was determined by the chi-square test. The effect of TMB on gastric secretion was studied in 4-h pylorus-ligated rats (see above). The gastric juice was collected and centrifuged and the volume of the supernatant was expressed as ml per 100 g body wt. Total acidity was determined by titrating with 0.01 N NaOH, using phenolphthalein as indicator, and is expressed as pequivjml. The total acid output is expressed as pequiv per 4 h. Peptic activity was determined by a modified method of Anson (1938) using haemoglobin as substrate, as described earlier (Debnath et al., 1974), and is expressed in terms of pmol of tyrosine per ml. Mucin activity was estimated in the mucosubstances precipitated by treating the gastric secretion with 90% ethanol in a 1:9 ratio. 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 protein (Lowry et al., 1951), total hexoses, hexosamine and fucose (Winzler, 1958), while the latter was used for the estimation of sialic acid (Warren, 1959). The results are expressed in pg/ml. The ratio of total carbohydrate (sum of total hexoses, hexosamine, fucose and sialic acid) to protein has been taken as the index of mucin activity. Toxicity studies Acute toxicity.
The effect of an oral dose of 1 g/kg TMB on the general and mortality up to 72 h in albino rats was studied. Sub-acute toxicity. 100 mg/kg TMB were administered orally as a single dose daily for 7 days. On the eighth day the rats were sacrificed. Besides noting the effect of the drug on general behaviour for 7 days, the effect of the drug on total red blood cells (RBC) and total and differential white blood cell (WBC) counts; haemoglobin, blood cholinesterase (Quastel, 1961) and on the histology of liver and kidney was studied. behaviour
Results The effect of TMB on immobilization-induced gastric ulcers in albino rats was studied with an arbitrarily selected oral dose of 50 mg/kg administered once daily for 3 days prior to experimentation. This dose significantly reduced the mean incidence and severity of ulcers. A dose-response study was
*p <0.05,
“*p
<0.025,
***,
cO.01.
1 0 0 0 0.2 0.2 0.166 0.4 0.8
7.375 2 1.2 0.4 0.6 1.8 2.5 10.2 5.2
9.375 -f 2.4 2.25 + o.gs** 1.4 t 1.666** 1.6 * 0.8** 1.0 1. 0.045*** 2.0 + 1.05** 3.166 r 1.47* 11 t 2.966 6.6 _t 1.25
8 100 8 50 5 40 5 60 5 60 5 60 6 50 5 100 5 100
Control 50 40 20 10 5 1 0.5 0.1
2.375 0.25 0.2 1.2 0.2 0 0.5 0.4 0.6
+++
Severity + ++
Average number of ulcers
n Incidence of ulcers (%I
on 8-h immobilization-induced
TMB dose (mg/W
Anti-ulcerogenic effect of Tamrubhasma
TABLE 1
0.75 0 0 0 0 0 0 0 0
++++ 18.125 + 4.46 2.5 t 1.05*** 1.6 r l.l*** 2.8 r 1.5,9*** 1.6 ? 0.748*** 2.4 t 1.03*** 4 Yh1.86** 12.2 f 2.8 8.8 + 1.74
Mean severity
gastric ulcer in albino rats
40 11 11 16.4 14.6 16.4 15.496 43.2 35.4
Group score
2
15
10
10
10
Pylorus-ligated control
TMB + PL
Aspirin control
TMB + ASP + PL 60
group.
2.5 t 0.82*
12.1 t 4.236
1 + 0.47
40
100
1.93 + 0.66
Average number of ulcers
60
(%)
Incidence of ulcers
aPL = pylorus-ligated; ASP = aspirin. *P <0.05, **P <0.025 relative to control
+ PL
n
Treatmenta
1.3
1
1.1
0.1
0.9 11
0.93
0.8
Ulcer severity + ++
0.2
0
0
0.26
+++
0
0
0
0
++++
3.9 f 1.24**
12.4
35.9
6.2
1.1 + 0.48 13.8 f 3.83
9.26
3.33 t 1.02
Group score
and on aspirin-induced
Mean severity
Effect of 1 mg/kg TMB administered orally once daily for 3 days on 4-h pylorus-Iigated (200 mg/kg daily for 3 days) gastric ulcers in albino rats
TABLE
84
then conducted on this experimental model with lower doses of TMB administered to different groups of rats. A dose of less than 1 mg/kg was not found to protect immobilization-induced ulcerogenesis significantly. The anti-ulcerogenic effect of doses higher than 1 mg/kg was not found to offer significant additional protection. The oral dose of 1 mg/kg once daily for 3 days was thus selected for further studies. The results of the dose-response study are summarised in Table 1. The mean number and severity of pylorus-ligated ulcers were reduced by a 1 mg/kg dose of TMB but were not significantly different from the control group. Similarly, the group ulcer score was also reduced (Table 2). A dose of 50 mg/kg of TMB, however, significantly reduced the average severity of ulcer. The incidence and severity of aspirin-induced ulceration was significantly reduced by 1 mg/kg TMB. The ulcer score was also markedly reduced (Table 2). The incidence of histamine-induced gastric ulcer in guinea pigs, though reduced, was not statistically significant with a dose of 1 mg/kg. The incidence of histamine-induced duodenal ulcer was, however, very significantly reduced, no TMB-treated animal showing any ulcer (Table 3). TABLE
3
Presence or absence of histamine-induced gastric or duodenal ulcers in guinea pigs Group
Gastric
n
Ulcer Present
X2 Absent
ulcer
Histamine control TMB + Histamine Duodenal Histamine control
10
6
4
10
3
7
10
8
2
ulcer
10 TMB + Histamine
10
0
10
TMB (lmg/kg oral) significantly reduced the volume of gastric fluid, total acid output and total pepsin output. The concentration of peptic activity per ml of gastric secretion was, however, significantly increased by TMB (Table 4). The drug significantly decreased the total hexoses and increased the fucose and sialic acid without significantly affecting the hexosamine concentration. Protein concentration was also significantly decreased by TMB. The carbohydrate/protein ratio was significantly increased in the gastric juice of TMB-treated rats compared to the untreated controls (Table 5).
2.08** kO.08 1.26**** to.1
10
10
10
Aspirin
TMB
Aspirin TMB
224.5**“* t 29.86
111.6 ti16.08 192.2* t 20.0 95.2 2 11.79
364.4 +17.3
146.13 f 6.66
272.4 t 21.8 to control
229.04**** ~15.26
/_lequiv per 4 h
Total acid /Jequiv/ml
*p < 0.05, **p < 0.025, ***p < 0.01, ****p < 0.001, relative +p < 0.01, ++ p < 0.001, relative to aspirin-treated group.
2.86 * 0.4
2.54 to.16
15
Control
+
Volume ml/l00 g
No. of samples
Treatment
group.
Effect of TMB (1 mg/kg orally for three days) on volume, total acid and peptic and aspirin (200 mg/kg orally for three days) treated albino rats -
TABLE 4
activity
454.56** 149.72
397.15** + 58.76
484.4+ 149.56 -.
744.24 t 53.44
362.25*** + 30.76
168.0++ t22.16
615.6 45.4
per 4 h
(control)
pm01 tyrosine
242.66 116.37
per ml
of gastric juice in untreated
pm01 tyrosine
Peptic -
activity
439.5*** i51.23
10
10
10
Aspirin
TMB
Aspirin TMB
433.8 553.3
180.23 F 39.95
216.5 + 28.01
234.5 + 25.22 141.0 + 20.9
Hexosamine
@g/ml)
54.48++ + 10.76
42.68 +4.7 23.6*“* 22.6 70.0** ilO.
Fucose
62.3* ~8.5
group.
735.84 * 62.89
739.s** i 64.58
17.03* ~2.18
40.45+++ 16.31
1066.43 + 102.69 537.99**** t 50.65
36.56 ~8.67
Sialic acid
Total Carbohydrate Wglmt)
of gastric juice in untreated
*p < 0.05, **p < 0.025, ***p < 0.01, ****p < 0.001 relative to control +p < 0.05, ++p < 0.01, +++ p < 0.001 relative to aspirin-treated group.
+
741.04 t87.23 405.5*** + 54.65
15
Control
Total Hexoses
Carbohydrate
No. of samples
Treatment
Effect of TMB (1 mg/kg orally for three days) on mucin activity (200 mg/kg orally for three days) treated albino rats
TABLE 5
241.06 + 35.79
354.4 1-49.62 147.5x*** f 19.22
284.57 ?:26.0
Total Protein (fig/ml)
(control)
3.59+ r0.35
1.6**** io.21 5.4 f 0.33
4.03 20.34
Carbohydrate/ protein ratio
and aspirin
87
Aspirin pretreatment significantly decreased the volume of gastric fluid and total acid output. However, it increased the peptic activity concentration of the gastric secretion in the treated animals. As for mucin, aspirin treatment caused a significant decrease in the concentration of all the carbohydrates (total hexoses, hexosamine, fucose and sialic acid) in the gastric juice, Protein concentration showed an increase, though this was not statistically significant. The net effect of aspirin on the carbohydrate/protein ratio was a significant decrease. In aspirin-treated rats, TMB significantly decreased the total acid and peptic output. TMB, however, significantly increased the c~bohy~ate/protein ratio in the gastric juice of aspirin-treated rats {Table 5). Toxicity stuc!ies The single oral dose of 1 g/kg TMB did not produce any significant change in the general behaviour of rats and also did not cause any death within 72 h of administration. An oral dose of 100 mg/kg daily for 7 days produced a slight increase in the weight of the animals, a significant increase in the haemoglobin concentration and total WBC count with a non-significant increase in total RBC count. Weight of adrenal gland and blood cholinesterase level nonsignificantly increased from 23.15 + 1.19 mg per 100 g and 44.53 rt 6.6 mmol acetylcholine (Ach) per ml per h, respectively, in untreated controls to 30.045 * 5.84 mg per 100 g, and 59.4 + 4.98 mmol Aeh per ml per h in TMB-treated animals. Histological studies of the liver and kidney did not show any detectable change in TMB-treated animals. Discussion The present study was undertaken primarily to study the anti-ulcerogenie effect, if any, of a traditional copper preparation tumrubhasma (TMB), which is cited in Ayurvedic texts as a remedy for, amongst other diseases, amlapitta which closely resembles peptic ulcer. TMB has been found to possess an anti-ulcerogenic property in different experimental preparations of gastric and duodenal ulcers in rats and guinea pigs. The minimal antiulcerogenic dose has been determined as 1 mgfkg orally. Higher doses do not seem to increase the anti-ulcerogenic property of TMB significantly. The experiment& evidence of the anti-ulcerogenic effect of TMB in pylorusligated, aspirin-induced and immobilization-induced gastric ulcers in albino rats and histamine-induced gastric and duodenal ulcers in guinea pigs, lends credence to the suggested use of TMB in amlapitta. On gastric secretion, TMB in a 1 mg/kg oral dose caused a decrease in the acid and pepsin output and a qualitative and quantitative change in the dissolved mucin content of the gastric juice was also observed after treatment with TMB. The concentrations of fucose and sialic acid were found to be significantly raised in the gastric juice of the TMB-treated group compared to untreated controls showing a qualitative change. The increased fucose and sialic acid in the gastric juice of TMB-treated rats may contribute to-
88
wards the anti-ulcerogenic effect of TMB (Curt and Pringle, 1969; Baume et al., 1967). Further, the significant increase in the carbohydrate/protein ratio in the gastric juice of TMB-treated rats showed that the drug caused a quantitative increase in the dissolved mucin content of the gastric juice. TMB also showed a similar effect in aspirin-treated animals. Aspirin decreased the concentration of all the individual carbohydrates and of the carbohydrate/protein ratio, though the decrease in the hexosamine concentration was not significant. A similar decrease in the carbohydrate/protein ratio and of individual carbohydrates (except that no significant change was observed with total hexoses instead of hexosamine) had been earlier reported in the non-dialysable and lyophilised fraction of the mucus in aspirin-treated (Menguy and Masters, 1965) and indomethacinand phenylbutazone-treated (Menguy and Desbaillets, 1968) dogs. These results tend to confirm that aspirin-like drugs cause ulceration by affecting the mucosal barrier and that the carbohydrate protein ratio of the gastric juice is a good index of the mucus barrier. TMB tended to reverse this process as evidenced by increased fucose and sialic acid content and an increase in the carbohydrate/protein ratio. Thus, TMB increased the efficiency of the mucosal barrier, which appears to have contributed to its anti-ulcerogenic effect. Peptic ulcer is now widely believed to be due to an imbalance between offensive acid-pepsin and defensive mucus factors. TMB has been found to increase the mucus and to decrease the acid-pepsin output in the rat. These effects of TMB on parameters that influence the initiation and perpetuation of ulceration may be considered as highly desirable properties of an antiulcerogenic drug. TMB thus has the potential of an anti-ulcerogenic agent. The more so in view of the fact that no acute and sub-acute toxic manifestations of TMB could be detected. Blood cholinesterase levels, which are indicative of liver toxicity (Oser, 1965), did not change significantly, nor was any histological evidence found implicating liver or kidney damage by TMB. The data of the present toxicity studies tend to agree with the observation that oral copper administration is unlikely to cause any toxic effect because it does not give rise to a cumulative toxicity (Schroeder, 1973). Treatment of peptic ulcer is, however, a long-drawn-out affair and as such, chronic toxicity studies in at least two species should be undertaken as part of preclinical studies. It may at the same time not be out of place to mention that TMB incorporated in Ayurvedic formulations is available as “over the counter” preparations in India and from which no serious toxicity has been reported. References Anson,
M. L., Estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology, 23 (1938) 79 - 89. Aykroyd, W. R., In C. Gopalan and S. C. Balasubramanian (eds.), The Nutritive Value of Indian Foods and the Planning of Satisfactory Diets, 6th edn., Indian Council of Medical Research Publication, New Delhi, 1963, pp. 87 - 117. Baume, P. E., Baxter, C. H. and Nicholls, A., Concentration and partial characterization of human salivary gastrone. American Journal ofDigestive Diseases, 12 (1967) 965 _ 972.
89 Bhavaprakash Nighantu of Shri Bhava Misra (1500 - 1600 A.D.) Datuumde Varga, B. S. Misra and R. L. Vaisya (eds.), 5th edn., Chaukhambha Sanskrit Sansthan, Varanasi, 1969, pp. 605 - 606. Boyle, E., Freeman, P. C., Goudie, A. C., Mangan, F. R. and Thomson, M., The role of copper in preventing gastrointestinal damage by acidic anti-inflammatory drugs. Journal of Pharmacy and Pharmacology, 28 (1976) 865 - 868. Curt, J. R. N. and Pringle, R., Viscosity of gastric mucus in duodenal ulceration. Gut, 10 (1969) 931 - 934. Debnath, P. K., Gode, K. D., Govinda Das, D. and Sanyal, A. K., Effect of propranolol on gastric secretion in albino rats. British Journal of Pharmacology, 51 (1974) 213 216. Eagleton, G. B. and Watt, J., Acute gastric ulceration in the guinea pig induced by a single intraperitoneal injection of aqueous histamine. Journal of Pathology and Bacteriology, 90 (1965) 679 - 682. Eagleton, G. B. and Watt, J., Acute duodenal ulceration in the guinea pig induced by repeated intramuscular injection of aqueous histamine. Journal of Pathology and Bacteriology, 93 (1967) 694 - 696. Elliot, R. C. and Heward, G. J. F., The effect of banana supplemented diet on gastric ulcer in mice. Pharmacological Research Communications, 8 (1976) 167 - 171. Fregly, M. J., Minimal exposures needed to acclimatize rats to cold. American Journal of Physiology, 173 (1953) 393 - 402. Lowry, 0. H. Roseborough, N. I., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193 (1951) 265 - 275. Menguy, R. and Desbaillets, L., The gastric mucus barrier: Influence of protein bound carbohydrate in mucus on the rate of proteolysis of gastric mucus. Annals of Surgery, 168 (1968) 475 - 482. Menguy, R. and Masters, Y. F., Effects of aspirin on gastric mucous secretion. Surgery, Gynecology and Obstetrics, 120 (1965) 92 - 98. Gser, B. L., In Hawk’s Physiological Chemistry, 14th edn.,McGraw-Hill, New York, 1965, p. 1131. Quastel, J. H., In Methods in Medical Research, Vol. 9, Year Book Publishers, Inc., Chicago, 1961, p. 73. Raghunathan, K., In Pharmacopeal standards for Ayuruedic Formulations, Central Council for Research in Indian Medicine and Homeopathy, New Delhi, 1976, p. 363. Rainsford, K. D. and Whitehouse, M. W., Concerning the merits of Cu-aspirin as potential anti-inflammatory agents. Journal of Pharmacy and Pharmacology, 28 (1976) 83 - 86. Robert, A., Nezamis, J. E. and Phillips, J. P., Effects of prostaglandin El on gastric secretion and ulcer formation in the rat. Gastroenterology, 55 (1968) 481 - 487. Sanyal, R. K., Das,.P. K., Sinha, S. and Sinha, Y. K., Banana and gastric secretion, JournaE of Pharmacy and Pharmacology, 13 (1961) 318 - 319. Sanyal,
A. K., Gupta, K. K. and Chowdhury, N. K., Studies on peptic ulceration. Part I: Role of banana in phenylbutazone induced ulcers. Archives Internationales de Pharmucodynamie et de Therapie, 149 (1964) 393 - 400. Sanyal, A. K., Banerjee, C. R. and Das, P. K., Studies on peptic ulceration. Part II: Role of banana in restraint and prednisolone induced ulcer in albino rats. Archives Internationales de Pharmacodynamie et de Therapie, 155 (1965) 244 - 248. Sanyal, A. K., Debnath, P. K., Bhattacharya, S. K. and Gode, K. D., The effect of cyproheptadine on gastric activity. An experimental study, In C. J. Pfeiffer (ed.), Peptic Ulcer, Munksgaard, Copenhagen, 1971, pp. 312 - 318. Schroeder, H. A., Cited by G. Oster and M. P. Salgo, Copper in mammalian reproduction. Advances in Pharmacology and Chemotherapy, 14 (1973) 327 - 409. Tovey, F., Peptic ulcer in India and Bangladesh. Gut, 20 (1979) 329 - 347. Warren, L., The thiobarbituric acid assay of sialic acids. Journal of Biological Chemistry, 234 (1959) 1971 _ 1975. Winzler, R. J., Determination of serum glycoproteins. Methods of Biochemical Analysis, 2 (1958) 279 - 311.
THE EFFECT ~~~~~~~~~~~, SECRETION
A. K. SANYAL*, Department Varanasi-221
79
OF A TRADITIONAL PREPARATION OF COPPER, ON EXPERIMENTAL ULCERS AND GASTRIC
B. L. PANDEY and R. K. GOEL
of Pharmacology, 005 (India)
(Received June 18,198O;
Institute
of Medical
Sciences,
Banaras
Hindu
University,
accepted September 15,198O)
Summary Use of a traditional preparation of copper, tamrubhama, has been suggested in Ayurvedic texts for peptic ulcer. The anti-ulcerogenic effect of copper per se has not been reported in the literature. In the present study the anti-ulcerogenic effect of ~~~~u~~~s~~ was observed in 8-h immobilised, 4-h pylorus-ligated, and aspirin-induced gastric ulcers in rats. The anti-ulcerogenie effect of the drug was also studied in histamine-induced gastric and duodenal ulcers in male guinea pigs. The minimal oral effective anti-ulcerogenit dose of ta~ra~~as~a has been determined to be 1 mg/kg. The drug in this dose caused a decrease in the total acid and pepsin output and an increase in the carbohydrate/protein ratio, indicating increased mucus secretion in the gastric secretion of rats. Acute (1 g/kg, orally) and sub-acute (100 mg/ kg, orally, for 7 days) toxicity studies did not show any toxicity including any histological or biochemical evidence of liver or kidney toxicity. The results of the present experimental studies thus lend credence to the suggested use of ta~ru~~as~a in a~~lapitta, a condition resembling peptic ulcer.
Introduction The treatment of peptic ulcer is still unsatisfactory notwithstanding the introduction of drugs like cimetidine and carbenoxolone. Many new ideas are being tried out to find a better remedy. The geographic~ distribution of the disease in India (Tovey, 1979) had earlier prompted one of us to look for such anti-ulcerogenic agents in natural products. The result of such a study was the discovery of an anti-ulcerogenic effect of vegetable banana in different experimental models (Sanyal et al., 1961,1964, 1965), an observation recently confirmed by other workers (Elliot and Heward, 1976).
*To whom correspondence
and reprint requests should be addressed.
80
A superficial indication that dietary copper may influence the incidence of the disease is obtained from the fact that dietary copper is relatively lower where the disease is more prevalent in India, and vice uersa. Vegetable banana is reported to contain significant quantities of copper (Aykroyd, 1963). These observations prompted us to study the anti-ulcerogenic effect, if any, of an indigenous, traditionally prepared copper preparation, tamrubhasma, which has been cited in the Ayurvedic texts (Bhavaprakash, 1969) and which is an important constituent of a formulation used by practitioners of traditional medicine as a good remedy for amlupitta, a clinical entity resembling peptic ulcer. A survey of the literature indicates that the anti-ulcerogenic effect of copper has not been reported, though decreased ulcerogenicity has been reported with copper complexes of non-steroidal anti-inflammatory agents compared to the effect of the latter drugs alone (Boyle et al., 1976; Rainsford and Whitehouse, 1976). Materials and Methods The present study was conducted with a preparation of tumrubhusmu (TMB) obtained from the Ayurvedic Pharmacy of this University. An earlier published study (Raghunathan, 1976) had given the composition of TMB as: CuO 244.45% <66.13%; Fe,O, <6.03%; and sulphur <2.75%. The anti-ulcerogenic effects of TMB were studied on albino rats weighing 120 - 150 g of either sex and on male guinea pigs weighing 450 - 600 g. The animals were maintained on Hind Lever pellet diet. Animals were fasted for 18 h before experimentation but water was allowed ad libitum. Experimental gastric and duodenal ulcers were produced in rats and guinea pigs as follows. (A) Gastric ulcers in albino ruts (1) Pylorus-ligated gastric ulcers were produced by pylorus ligation for 4 h as described earlier (Sanyal et al., 1971). (2) Acetylsalicylic acid-induced gastric ulcers were produced by administering the drug as a suspension in water in a dose of 200 mg/kg, once daily for three days by mouth with the help of an oro-gastric polythene tube. The pylorus was then ligated on the fourth day and rats were sacrificed after 4 h by decapitation. The control group received an equal volume of water only. (3) To produce restraint ulcers, rats were immobilised on a wooden plank by adhesive plaster, adopting the method described by Fregly (1953), for 8 h. After the period of immobilisation, the rats were sacrificed by decapitation. (B) Histamine-induced ulcers in guinea pigs (1) Gastric ulcers were induced in guinea pigs with a single injection of histamine, following the method of Eagleton and Watt (1965). (2) Duodenal ulcers were produced by repeated injections of histamine adopting the method of Eagleton and Watt (1967).
81
In the rats, the number of ulcers per stomach was noted and the severity of the ulcers was scored after histological confirmation as follows: 0, no ulcer; +, changes limited to superficial layers of mucosa and no congestion; ++, half of the mucosal thickness showed necrotic changes; +++, more than two-thirds of the mucosal thickness destroyed with marked necrosis and congestion, muscularis remaining unaffected; ++++, complete destruction of the mucosa with necrosis and haemorrhage, muscularis still remaining unaffected. The pooled group ulcer score was then calculated by the method of Robert et al. (1968). In the guinea pigs, the presence or absence of ulcers was noted in each group and statistical significance was determined by the chi-square test. The effect of TMB on gastric secretion was studied in 4-h pylorus-ligated rats (see above). The gastric juice was collected and centrifuged and the volume of the supernatant was expressed as ml per 100 g body wt. Total acidity was determined by titrating with 0.01 N NaOH, using phenolphthalein as indicator, and is expressed as pequivjml. The total acid output is expressed as pequiv per 4 h. Peptic activity was determined by a modified method of Anson (1938) using haemoglobin as substrate, as described earlier (Debnath et al., 1974), and is expressed in terms of pmol of tyrosine per ml. Mucin activity was estimated in the mucosubstances precipitated by treating the gastric secretion with 90% ethanol in a 1:9 ratio. 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 protein (Lowry et al., 1951), total hexoses, hexosamine and fucose (Winzler, 1958), while the latter was used for the estimation of sialic acid (Warren, 1959). The results are expressed in pg/ml. The ratio of total carbohydrate (sum of total hexoses, hexosamine, fucose and sialic acid) to protein has been taken as the index of mucin activity. Toxicity studies Acute toxicity.
The effect of an oral dose of 1 g/kg TMB on the general and mortality up to 72 h in albino rats was studied. Sub-acute toxicity. 100 mg/kg TMB were administered orally as a single dose daily for 7 days. On the eighth day the rats were sacrificed. Besides noting the effect of the drug on general behaviour for 7 days, the effect of the drug on total red blood cells (RBC) and total and differential white blood cell (WBC) counts; haemoglobin, blood cholinesterase (Quastel, 1961) and on the histology of liver and kidney was studied. behaviour
Results The effect of TMB on immobilization-induced gastric ulcers in albino rats was studied with an arbitrarily selected oral dose of 50 mg/kg administered once daily for 3 days prior to experimentation. This dose significantly reduced the mean incidence and severity of ulcers. A dose-response study was
*p <0.05,
“*p
<0.025,
***,
cO.01.
1 0 0 0 0.2 0.2 0.166 0.4 0.8
7.375 2 1.2 0.4 0.6 1.8 2.5 10.2 5.2
9.375 -f 2.4 2.25 + o.gs** 1.4 t 1.666** 1.6 * 0.8** 1.0 1. 0.045*** 2.0 + 1.05** 3.166 r 1.47* 11 t 2.966 6.6 _t 1.25
8 100 8 50 5 40 5 60 5 60 5 60 6 50 5 100 5 100
Control 50 40 20 10 5 1 0.5 0.1
2.375 0.25 0.2 1.2 0.2 0 0.5 0.4 0.6
+++
Severity + ++
Average number of ulcers
n Incidence of ulcers (%I
on 8-h immobilization-induced
TMB dose (mg/W
Anti-ulcerogenic effect of Tamrubhasma
TABLE 1
0.75 0 0 0 0 0 0 0 0
++++ 18.125 + 4.46 2.5 t 1.05*** 1.6 r l.l*** 2.8 r 1.5,9*** 1.6 ? 0.748*** 2.4 t 1.03*** 4 Yh1.86** 12.2 f 2.8 8.8 + 1.74
Mean severity
gastric ulcer in albino rats
40 11 11 16.4 14.6 16.4 15.496 43.2 35.4
Group score
2
15
10
10
10
Pylorus-ligated control
TMB + PL
Aspirin control
TMB + ASP + PL 60
group.
2.5 t 0.82*
12.1 t 4.236
1 + 0.47
40
100
1.93 + 0.66
Average number of ulcers
60
(%)
Incidence of ulcers
aPL = pylorus-ligated; ASP = aspirin. *P <0.05, **P <0.025 relative to control
+ PL
n
Treatmenta
1.3
1
1.1
0.1
0.9 11
0.93
0.8
Ulcer severity + ++
0.2
0
0
0.26
+++
0
0
0
0
++++
3.9 f 1.24**
12.4
35.9
6.2
1.1 + 0.48 13.8 f 3.83
9.26
3.33 t 1.02
Group score
and on aspirin-induced
Mean severity
Effect of 1 mg/kg TMB administered orally once daily for 3 days on 4-h pylorus-Iigated (200 mg/kg daily for 3 days) gastric ulcers in albino rats
TABLE
84
then conducted on this experimental model with lower doses of TMB administered to different groups of rats. A dose of less than 1 mg/kg was not found to protect immobilization-induced ulcerogenesis significantly. The anti-ulcerogenic effect of doses higher than 1 mg/kg was not found to offer significant additional protection. The oral dose of 1 mg/kg once daily for 3 days was thus selected for further studies. The results of the dose-response study are summarised in Table 1. The mean number and severity of pylorus-ligated ulcers were reduced by a 1 mg/kg dose of TMB but were not significantly different from the control group. Similarly, the group ulcer score was also reduced (Table 2). A dose of 50 mg/kg of TMB, however, significantly reduced the average severity of ulcer. The incidence and severity of aspirin-induced ulceration was significantly reduced by 1 mg/kg TMB. The ulcer score was also markedly reduced (Table 2). The incidence of histamine-induced gastric ulcer in guinea pigs, though reduced, was not statistically significant with a dose of 1 mg/kg. The incidence of histamine-induced duodenal ulcer was, however, very significantly reduced, no TMB-treated animal showing any ulcer (Table 3). TABLE
3
Presence or absence of histamine-induced gastric or duodenal ulcers in guinea pigs Group
Gastric
n
Ulcer Present
X2 Absent
ulcer
Histamine control TMB + Histamine Duodenal Histamine control
10
6
4
10
3
7
10
8
2
ulcer
10 TMB + Histamine
10
0
10
TMB (lmg/kg oral) significantly reduced the volume of gastric fluid, total acid output and total pepsin output. The concentration of peptic activity per ml of gastric secretion was, however, significantly increased by TMB (Table 4). The drug significantly decreased the total hexoses and increased the fucose and sialic acid without significantly affecting the hexosamine concentration. Protein concentration was also significantly decreased by TMB. The carbohydrate/protein ratio was significantly increased in the gastric juice of TMB-treated rats compared to the untreated controls (Table 5).
2.08** kO.08 1.26**** to.1
10
10
10
Aspirin
TMB
Aspirin TMB
224.5**“* t 29.86
111.6 ti16.08 192.2* t 20.0 95.2 2 11.79
364.4 +17.3
146.13 f 6.66
272.4 t 21.8 to control
229.04**** ~15.26
/_lequiv per 4 h
Total acid /Jequiv/ml
*p < 0.05, **p < 0.025, ***p < 0.01, ****p < 0.001, relative +p < 0.01, ++ p < 0.001, relative to aspirin-treated group.
2.86 * 0.4
2.54 to.16
15
Control
+
Volume ml/l00 g
No. of samples
Treatment
group.
Effect of TMB (1 mg/kg orally for three days) on volume, total acid and peptic and aspirin (200 mg/kg orally for three days) treated albino rats -
TABLE 4
activity
454.56** 149.72
397.15** + 58.76
484.4+ 149.56 -.
744.24 t 53.44
362.25*** + 30.76
168.0++ t22.16
615.6 45.4
per 4 h
(control)
pm01 tyrosine
242.66 116.37
per ml
of gastric juice in untreated
pm01 tyrosine
Peptic -
activity
439.5*** i51.23
10
10
10
Aspirin
TMB
Aspirin TMB
433.8 553.3
180.23 F 39.95
216.5 + 28.01
234.5 + 25.22 141.0 + 20.9
Hexosamine
@g/ml)
54.48++ + 10.76
42.68 +4.7 23.6*“* 22.6 70.0** ilO.
Fucose
62.3* ~8.5
group.
735.84 * 62.89
739.s** i 64.58
17.03* ~2.18
40.45+++ 16.31
1066.43 + 102.69 537.99**** t 50.65
36.56 ~8.67
Sialic acid
Total Carbohydrate Wglmt)
of gastric juice in untreated
*p < 0.05, **p < 0.025, ***p < 0.01, ****p < 0.001 relative to control +p < 0.05, ++p < 0.01, +++ p < 0.001 relative to aspirin-treated group.
+
741.04 t87.23 405.5*** + 54.65
15
Control
Total Hexoses
Carbohydrate
No. of samples
Treatment
Effect of TMB (1 mg/kg orally for three days) on mucin activity (200 mg/kg orally for three days) treated albino rats
TABLE 5
241.06 + 35.79
354.4 1-49.62 147.5x*** f 19.22
284.57 ?:26.0
Total Protein (fig/ml)
(control)
3.59+ r0.35
1.6**** io.21 5.4 f 0.33
4.03 20.34
Carbohydrate/ protein ratio
and aspirin
87
Aspirin pretreatment significantly decreased the volume of gastric fluid and total acid output. However, it increased the peptic activity concentration of the gastric secretion in the treated animals. As for mucin, aspirin treatment caused a significant decrease in the concentration of all the carbohydrates (total hexoses, hexosamine, fucose and sialic acid) in the gastric juice, Protein concentration showed an increase, though this was not statistically significant. The net effect of aspirin on the carbohydrate/protein ratio was a significant decrease. In aspirin-treated rats, TMB significantly decreased the total acid and peptic output. TMB, however, significantly increased the c~bohy~ate/protein ratio in the gastric juice of aspirin-treated rats {Table 5). Toxicity stuc!ies The single oral dose of 1 g/kg TMB did not produce any significant change in the general behaviour of rats and also did not cause any death within 72 h of administration. An oral dose of 100 mg/kg daily for 7 days produced a slight increase in the weight of the animals, a significant increase in the haemoglobin concentration and total WBC count with a non-significant increase in total RBC count. Weight of adrenal gland and blood cholinesterase level nonsignificantly increased from 23.15 + 1.19 mg per 100 g and 44.53 rt 6.6 mmol acetylcholine (Ach) per ml per h, respectively, in untreated controls to 30.045 * 5.84 mg per 100 g, and 59.4 + 4.98 mmol Aeh per ml per h in TMB-treated animals. Histological studies of the liver and kidney did not show any detectable change in TMB-treated animals. Discussion The present study was undertaken primarily to study the anti-ulcerogenie effect, if any, of a traditional copper preparation tumrubhasma (TMB), which is cited in Ayurvedic texts as a remedy for, amongst other diseases, amlapitta which closely resembles peptic ulcer. TMB has been found to possess an anti-ulcerogenic property in different experimental preparations of gastric and duodenal ulcers in rats and guinea pigs. The minimal antiulcerogenic dose has been determined as 1 mgfkg orally. Higher doses do not seem to increase the anti-ulcerogenic property of TMB significantly. The experiment& evidence of the anti-ulcerogenic effect of TMB in pylorusligated, aspirin-induced and immobilization-induced gastric ulcers in albino rats and histamine-induced gastric and duodenal ulcers in guinea pigs, lends credence to the suggested use of TMB in amlapitta. On gastric secretion, TMB in a 1 mg/kg oral dose caused a decrease in the acid and pepsin output and a qualitative and quantitative change in the dissolved mucin content of the gastric juice was also observed after treatment with TMB. The concentrations of fucose and sialic acid were found to be significantly raised in the gastric juice of the TMB-treated group compared to untreated controls showing a qualitative change. The increased fucose and sialic acid in the gastric juice of TMB-treated rats may contribute to-
88
wards the anti-ulcerogenic effect of TMB (Curt and Pringle, 1969; Baume et al., 1967). Further, the significant increase in the carbohydrate/protein ratio in the gastric juice of TMB-treated rats showed that the drug caused a quantitative increase in the dissolved mucin content of the gastric juice. TMB also showed a similar effect in aspirin-treated animals. Aspirin decreased the concentration of all the individual carbohydrates and of the carbohydrate/protein ratio, though the decrease in the hexosamine concentration was not significant. A similar decrease in the carbohydrate/protein ratio and of individual carbohydrates (except that no significant change was observed with total hexoses instead of hexosamine) had been earlier reported in the non-dialysable and lyophilised fraction of the mucus in aspirin-treated (Menguy and Masters, 1965) and indomethacinand phenylbutazone-treated (Menguy and Desbaillets, 1968) dogs. These results tend to confirm that aspirin-like drugs cause ulceration by affecting the mucosal barrier and that the carbohydrate protein ratio of the gastric juice is a good index of the mucus barrier. TMB tended to reverse this process as evidenced by increased fucose and sialic acid content and an increase in the carbohydrate/protein ratio. Thus, TMB increased the efficiency of the mucosal barrier, which appears to have contributed to its anti-ulcerogenic effect. Peptic ulcer is now widely believed to be due to an imbalance between offensive acid-pepsin and defensive mucus factors. TMB has been found to increase the mucus and to decrease the acid-pepsin output in the rat. These effects of TMB on parameters that influence the initiation and perpetuation of ulceration may be considered as highly desirable properties of an antiulcerogenic drug. TMB thus has the potential of an anti-ulcerogenic agent. The more so in view of the fact that no acute and sub-acute toxic manifestations of TMB could be detected. Blood cholinesterase levels, which are indicative of liver toxicity (Oser, 1965), did not change significantly, nor was any histological evidence found implicating liver or kidney damage by TMB. The data of the present toxicity studies tend to agree with the observation that oral copper administration is unlikely to cause any toxic effect because it does not give rise to a cumulative toxicity (Schroeder, 1973). Treatment of peptic ulcer is, however, a long-drawn-out affair and as such, chronic toxicity studies in at least two species should be undertaken as part of preclinical studies. It may at the same time not be out of place to mention that TMB incorporated in Ayurvedic formulations is available as “over the counter” preparations in India and from which no serious toxicity has been reported. References Anson,
M. L., Estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology, 23 (1938) 79 - 89. Aykroyd, W. R., In C. Gopalan and S. C. Balasubramanian (eds.), The Nutritive Value of Indian Foods and the Planning of Satisfactory Diets, 6th edn., Indian Council of Medical Research Publication, New Delhi, 1963, pp. 87 - 117. Baume, P. E., Baxter, C. H. and Nicholls, A., Concentration and partial characterization of human salivary gastrone. American Journal ofDigestive Diseases, 12 (1967) 965 _ 972.
89 Bhavaprakash Nighantu of Shri Bhava Misra (1500 - 1600 A.D.) Datuumde Varga, B. S. Misra and R. L. Vaisya (eds.), 5th edn., Chaukhambha Sanskrit Sansthan, Varanasi, 1969, pp. 605 - 606. Boyle, E., Freeman, P. C., Goudie, A. C., Mangan, F. R. and Thomson, M., The role of copper in preventing gastrointestinal damage by acidic anti-inflammatory drugs. Journal of Pharmacy and Pharmacology, 28 (1976) 865 - 868. Curt, J. R. N. and Pringle, R., Viscosity of gastric mucus in duodenal ulceration. Gut, 10 (1969) 931 - 934. Debnath, P. K., Gode, K. D., Govinda Das, D. and Sanyal, A. K., Effect of propranolol on gastric secretion in albino rats. British Journal of Pharmacology, 51 (1974) 213 216. Eagleton, G. B. and Watt, J., Acute gastric ulceration in the guinea pig induced by a single intraperitoneal injection of aqueous histamine. Journal of Pathology and Bacteriology, 90 (1965) 679 - 682. Eagleton, G. B. and Watt, J., Acute duodenal ulceration in the guinea pig induced by repeated intramuscular injection of aqueous histamine. Journal of Pathology and Bacteriology, 93 (1967) 694 - 696. Elliot, R. C. and Heward, G. J. F., The effect of banana supplemented diet on gastric ulcer in mice. Pharmacological Research Communications, 8 (1976) 167 - 171. Fregly, M. J., Minimal exposures needed to acclimatize rats to cold. American Journal of Physiology, 173 (1953) 393 - 402. Lowry, 0. H. Roseborough, N. I., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193 (1951) 265 - 275. Menguy, R. and Desbaillets, L., The gastric mucus barrier: Influence of protein bound carbohydrate in mucus on the rate of proteolysis of gastric mucus. Annals of Surgery, 168 (1968) 475 - 482. Menguy, R. and Masters, Y. F., Effects of aspirin on gastric mucous secretion. Surgery, Gynecology and Obstetrics, 120 (1965) 92 - 98. Gser, B. L., In Hawk’s Physiological Chemistry, 14th edn.,McGraw-Hill, New York, 1965, p. 1131. Quastel, J. H., In Methods in Medical Research, Vol. 9, Year Book Publishers, Inc., Chicago, 1961, p. 73. Raghunathan, K., In Pharmacopeal standards for Ayuruedic Formulations, Central Council for Research in Indian Medicine and Homeopathy, New Delhi, 1976, p. 363. Rainsford, K. D. and Whitehouse, M. W., Concerning the merits of Cu-aspirin as potential anti-inflammatory agents. Journal of Pharmacy and Pharmacology, 28 (1976) 83 - 86. Robert, A., Nezamis, J. E. and Phillips, J. P., Effects of prostaglandin El on gastric secretion and ulcer formation in the rat. Gastroenterology, 55 (1968) 481 - 487. Sanyal, R. K., Das,.P. K., Sinha, S. and Sinha, Y. K., Banana and gastric secretion, JournaE of Pharmacy and Pharmacology, 13 (1961) 318 - 319. Sanyal,
A. K., Gupta, K. K. and Chowdhury, N. K., Studies on peptic ulceration. Part I: Role of banana in phenylbutazone induced ulcers. Archives Internationales de Pharmucodynamie et de Therapie, 149 (1964) 393 - 400. Sanyal, A. K., Banerjee, C. R. and Das, P. K., Studies on peptic ulceration. Part II: Role of banana in restraint and prednisolone induced ulcer in albino rats. Archives Internationales de Pharmacodynamie et de Therapie, 155 (1965) 244 - 248. Sanyal, A. K., Debnath, P. K., Bhattacharya, S. K. and Gode, K. D., The effect of cyproheptadine on gastric activity. An experimental study, In C. J. Pfeiffer (ed.), Peptic Ulcer, Munksgaard, Copenhagen, 1971, pp. 312 - 318. Schroeder, H. A., Cited by G. Oster and M. P. Salgo, Copper in mammalian reproduction. Advances in Pharmacology and Chemotherapy, 14 (1973) 327 - 409. Tovey, F., Peptic ulcer in India and Bangladesh. Gut, 20 (1979) 329 - 347. Warren, L., The thiobarbituric acid assay of sialic acids. Journal of Biological Chemistry, 234 (1959) 1971 _ 1975. Winzler, R. J., Determination of serum glycoproteins. Methods of Biochemical Analysis, 2 (1958) 279 - 311.