Bitter Melon Studies

Potentiation of Tolbutamide Action by Jasad Bhasma and Karela (Momordica charantia)

filipinoweb — Thu, 10 Aug 2006 04:23:19 +0000

5 September 1962 Ind. Jour. Med. Res. Vol.50 pp.715-719

by R. D. Kukarni, B. B. Gaitonde, (with tech. Assistance of Shri N. K. Dadkar)
Some measure of success has been achieved in the oral treatment of Diabetes mellitus with some introduction of the sulphonylurea…

Potentiation of Tolbutamide Action by Jasad Bhasma and Karela (Momordica charantia)

Author: R. D. Kukarni, B. B. Gaitonde, (with tech. Assistance of Shri N. K. Dadkar)

Type of Publication: Pre-clinical

Date of Publication: September 5, 1962

Publication: Ind. Jour. Med. Res. Vol.50 pp.715-719, 5 September 1962

Organization: Department of Pharmacology, Grant Medical College, Bombay

Some measure of success has been achieved in the oral treatment of Diabetes mellitus with some introduction of the sulphonylurea derivatives in its therapy. However, usefulness of these agents is limited to the treatment of the elderly obese diabetes without ketosis. The safest of the series, Tolbutamide, is the eldest potent also. Several indigenous drugs have been tested for their antidiabetic action both in the laboratory (Aiman, 1955; 1956; Shrotri and Aiman, 1960; Mukerji, 1957) and in the clinic (Sathe et al., 1960; Vad, 1960). The effectiveness of such indigenous drugs, where it has been demonstrated, is far less as compared with the synthetic compounds. Two indigenous drugs- Jasad Bhasma (J.B.) and Karela (K)- have been shown to be effective in large number of diabetes by Sathe et al. (loc. cit.) and Vad (loc. cit.), respectively. The present investigation was planned to test the effect of these drugs on blood sugar of rabbits and also to see whether these drugs could potentiate the action of Tolbutamide.

MATERIAL AND METHODS

The work was carried out on rabbits weighing between 1 kg. and 2 kg. The rabbits were fasted over a period of 48 hours as this period is necessary to obtain a true fasting state (Kulkarni et al., 1959). Blood samples were collected from marginal ear vein for glucose estimation which was done by Somogyi’s method.

The experiments were performed to study the effects of single administration of the drug on the blood sugar profile of rabbits (acute experiments) and to study the effect of the chronic administration of the drugs on the fasting blood sugar level. In the acute experiments the blood samples were collected initially and then 2 hours, 4 hours, 6 hours, and 8 hours after the drug administration. In chronic experiments blood samples were collected before the initiation of therapy and then at weekly intervals before the administration of the drug on that day. In chronic experiments the drug was administered twice a day. In all cases the drugs were given orally through a stomach tube.

Jasad Bhasma was prepared for us by the Himalaya drug Co., Bombay. The same sample was used throughout the studies. It satisfied the test for the Bhasmas, viz, that floated on water as a thin transparent film. The market samples tried earlier, contained a variable amount of zinc oxide as impurity. In acute experiments the doses used were 100 mg., and 500 mg. per kg. In chronic experiments 500 mg./kg. given in two divided doses daily.

The dried juice of Karela was supplied by the Himalaya Company, Bombay. The doses used were similar to those of Jasad Bhasma.

Tolbutamide powder supplied by the Hoechst Fedco Pharma, Ltd., Bombay, was used. It was administered in the doses of 250 mg. and 500 mg./kg., earlier alone or together with 200 mg. each of Karela and Jasad Bhasma.

The dosage range used here is found to be optimum from the various pilot experiments which are not included in this report.

RESULTS

Jasad Bhasma in the dose of 500 m./kg. does not cause any fall in the fasting blood sugar level at the end of 8 hours after administration (t=0.26,P>0.5). Karela also, in the dose of 500 mg./kg. does not cause significant fall in the fasting blood glucose level (t=0.26, P>0.5). Combination of two drugs like wise does not cause a significant fall in the blood sugar of rabbits (t=0.9, P>0.5).

On chronic administration, Jasad Bhasma causes a fall in fasting blood sugar of rabbits. The fall is evident at about 2 weeks and goes on progressively increasing till at the end of 6 weeks there is an average of 22 per cent fall in the fasting blood sugar. This fall, though moderate, is statistically highly significant (t=9; P<0.01). Karela has no such effect on chronic administration. When Jasad Bhasma and Karela administered together there is a progressive fall of blood sugar over six weeks but the fall at the end of 6 weeks is little over 28 per cent which is slightly more than that obtained with Jasad Bhasma alone. This small difference is also statistically highly significant (t=3.2; P<0.01).

Tolbutamide in the dose of 500 mg./kg. causes a fall in the blood sugar with a maximum of 20 per cent reaching at 4 hours. The blood sugar then gradually returns to the normal level at 8 hours. When Tolbutamide is given together with Jasad Bhasma and Karela, a similar degree of fall of blood sugar occurs in 4 hours but thereafter the blood sugar remains at the same level and even at the end of 8 hours is appreciably lower than the initial level. The difference in the blood sugar levels of the rabbits goven Tolbutamide and those given Tolbutamide + J. B. + K, 8 hours after the administration, is statistically significant (t=5.6; P<0.01).

When Tolbutamide is given in the dose of 250 mg./kg., there is a mean fall of 11 per cent in the fasting blood sugar at the end of 4 hours. When the same dose of Tolbutamide is given together with Jasad Bhasma and karela, there is a fall of 28 per cent in the fasting blood sugar level at the end of 4 hours. This difference in the percentage fall of blood sugar is significant (t=4.2; P<0.01).

DISCUSSION

Jasad Bhasma used in this study did not cause any fall in the blood sugar level of the fasting rabbits. On prolonged administration, the same preparation caused a progressive fall in the fasting blood sugar of the rabbits over a period of 6 weeks. Though the extent of the fall was not very great, it was appreciable. This finding is consistent with the clinical observation of sathe et al.(loc. cit.) that the effect of Jasad Bhasma is progressively greater on prolonged administration. The commercial preparations of Jasad Bhasma do not give satisfy the requirements of a Bhasma. A few studies with zinc oxide revealed that it did not possess the activity of Jasad Bhasma.

Dried Karela juice used in these studies did not have any hypoglycaemic activity on acute or chronic administration. However, when Karela juice was admistered together with Jasad Bahsma over a prolonged period, hypoglyceamic effect was greater than when Jasad Bhasma was administered alone. However,the difference is very small and only suggest the synergistic action of the two drugs. At the stage it cannot, however, be definitely said that such an action does exist.

When Tolbutamide is administered in the dose of 250 mg./kg., it produces a fall of about 10 per cent in the fasting blood sugar. However, when this same dose is given with Jasad bahsma and Karela there is a fall of about 28 per cent. Since sugar in acute experiments, this definitely suggests a potentiation of Tolbutamide action by the two indigenous drugs. When Tolbutamide is given in the dose of 500 mg./kg., it produces a fall of 24 per cent in the fasting blood sugar over 4 hours and the blood sugar returns to normal in about 8 hours. When this dose is given together with Jasad Bhasma and Karela the extent of fall does not increase but the duration of the hypoglycaemic action is prolonged.

This leaves no doubt regarding the enhancement of Tolbutamide action by Jasad bahsma and Karela. However, the mechanism of this enhancement is not clear. It appears from the results obtained so far that the two indigenous drugs might be interfering with the excretion of Tolbutamide. It has also been observed that while a single dose of 100 mg. of Tolbutamide is without effect on the blood sugar, the combination of this dose with the two indigenous drugs causes a small but definite fall in the blood sugar. This makes it possible that there may be a true potentiation of the action of Tolbutamide. The detailed wok on the mechanism of this enhancement of action is in progress.

This observations of ours various therapeutic possibilities. Tolbutamide which is the safest of the oral antidiabetic drugs has the drawback of being least potent. It is also rapidly excerted from the body. If the activity of this compounds is enhanced and its duration of action prolonged, it might be the ideal drug for the treatment of the type of diabetes for which the oral antidiabetic drugs are now useful.

SUMMARY

The work was undertaken to investigate the place of some indigenous antidiabetic drugs in therapy vis-à-vis Tolbutamide. Rabbits starved over a period of 48 hours were used for fasting blood sugar estimation. Jasad Bhasma, dried Karela juice and Tolbutamide were given either individually or in combination orally. The effect of single administration on the fasting blood sugar of rabbits was studied over an eight-hour period. The drugs were administered chronically and the effect on blood sugar estimated over 6 weeks period. Jasad Bhasma and Karela had no effect on single adminstration. But Jasad Bhasma potentiated the hypoglycaemic action of single dose of Tolbutamide. Jasad Bhasma caused some fall in fasting blood sugar on prolonged administration and enhanced the effect of Tolbutamide. Karela had no such effect.

REFERENCE

Aiman, R. (1955). Enquiry into the antidiabetic property of indigenous drugs. Technical Report of I. C. M. R.

Idem (1956). Ibid.

Kulkarni, R. D., Shrotre, D. S., and Aiman, R. (1959). Period of fasting and testing of hypoglycaemic agents in rabbits. Ind. Jour. Phys. & Pharma., 3, 67.

Mukerji, B. (1957). Indigenous antidiabetic drug: A review. Jour. Sci. & indust. Res., 16A.

Sathe, R. V., Talwalkar, N. G., and Ajagaonkar, S. S. (1960). Investigations in the use of Jasad bhasma: An Ayurvedic preparation of zinc in the treatment of Diabetes mellitus. Ind. Jour. Med. Res., 48, 720.

Shrotri, D. S., and Aiman, R. (1960). The relationship of the post absorptive state to the hypoglycaemic action: Studies on Ficus bengalensis and Ficus glomerata. Ibid., 48, 162.

Vad, B. G. (1960). Place of Momordica charantia in the treatment of Diabetes mellitus. Maharashtra Med. Jour., 6, 734.

Effects of Indigenous Anti-Diabetic Drugs Against the Acute Hyperglycemic Response of Anterior Pituitary Extract in Glucose Fed Albino Rats

filipinoweb — Thu, 10 Aug 2006 04:21:11 +0000

July 1963 Ind. Journal Med. Res. 51. 4, pp.716-724

by S. S Gupta
In previous communications, Gupta et al. (1962) reported on the inhibitory effect of daily administration of a few Ayurdevic anti-diabetic…

Effects of Indigenous Anti-Diabetic Drugs Against the Acute Hyperglycemic Response of Anterior Pituitary Extract in Glucose Fed Albino Rats

Author: S. S Gupta

Type of Publication: Pre-Clinical

Date of Publication: July 1963

Publication: Ind. Journal Med. Res. 51. 4, pp.716-724, July 1963

Organization: Department of Pharmacology, Gandhi Medical College, Bhopal

In previous communications, Gupta et al. (1962) reported on the inhibitory effect of daily administration of a few Ayurdevic anti-diabetic remedies- Gymnema sylvestre and Tribang shila against the hyperglycemic response of anterior pituitary extract, while Gupta and Seth (1962) demonstrate their ameliorating effect in idio-hypophyseal diabetic condition in rats. Since the diabetogenic effect of anterior pituitary extract is effected through inhibition of glucose utilization by the tissues (Young, 1951), it would be of interest to investigate the effect of some commonly used indigenous anti-diabetic drugs against the acute hyperglycemic response of the anterior pituitary extract in fasting animals fed with glucose through stomach tube.

MATERIALS AND METHODS

The present investigations were conducted on adult male albino rats of Nowegian strain weighing between 175 g. and 200 g. The animals were kept in separate cages and fed ad libidum on stock diet as reported previously (Gupta et al., loc. cit.). Their fasting blood sugar determined every 5^th day for a control period of 15 days, was found to vary within the fiducial limits of 4.43 mg. per cent only.

After collecting the fasting blood, all animals were injected subcutaneously 100 mg./kg. dose of the anterior pituitary extract. The animals were then divided into five groups of six rats each. One hur the injection, half the animals in each of the groups I to V-insulin, were given respectively, the alcoholic extract of Gymnema sylvestre and Coccinia indica 9100 mg./kg. each), aqueous infusion of Pterocarpus marsupium (20 ml./kg.), extract of Momordica charantia Linn. (5 ml./kg.) and tolbutamide (50 mg../kg.) orally through a stomach tube. The remaining half of the animals in each goup were administered equivalent amount of the solvent distilled water to serve as controls. All the animals, irrespective of the groups were fed 50 per cent glucose (2 ml./100 g.) through tube after half an hour of administering drugs. The administration of drugs and glucose was given repeated after collecting 6^th hour blood samples. The blood sugar was then determined in the individual rats at 3^rd, 6^th, 12^th, and 24^th hours of the anterior pituitary injections. Cross over test was performed after a week when the blood sugar had returned to normal. Blood sugar was estimated as per Folin’s modification of Hagedorn Jenson’s method.

Drugs used were as follows: Alcoholic extracts of Gymnema sylvestre and Coccinia indica were prepared by extracting the dried leaves and roots, respectively, in ethanol for 5 hours in a Soxhlet apparatus. The dried residue was then re-dissolved in 1 part of alcohol, 2 parts of Twen 80 and 2 parts of water to prepare 200 mg./kg. solutions for oral administration.

Aqueous infusion of Pterocarpus marsupium was prepared by soaking 100 g. saw dust of the wood of P. marsupium in 200 ml. of water for 18 hours. The supernatant fluid was decanted off and volume adjusted so that 2 ml. of infusion represented 1 g. of the crude wood.

Aqueous extract of Momordica charantia Linn, was prepared by expressing out the juice from 100 g. of fresh fruits in an extractor. The juice was then diluted with distilled water to make 100 ml. of the extract.

Tolbutamide was given after powdering the tablet and then suspending in gum acacia for oral administration. The drug preparations were stored in sealed containers in refrigerator at 4⁰ C. for a week for the cross over tests.

Anterior pituitary extract use in the present investigation was prepared by M/s Bengal Immunity Co., Calcutta, from 1 g. of fresh pituitary gland (ox) homogenized with glycerine 1 ml., parachlorometacresol 0.05 g. and distilled water sufficient quantity to make 10 mg./ml. extract for subcutaneous injections.

RESULTS

Effect of the indigenous anti-diabetic drugs- Gymnema sylvestre and Coccinia indica on the hyperglycaemic response of the anterior pituitary extract in glucose fed rats is summarized in Table I. Rise in blood sugar level at 3,6,12, and 24 hours after administering the anterior pituitary extracts in control and treated rats is compared in Graph 1.

In apreliminary experiment, in which only anterior pituitary extract was given to the fasting animals, the blood sugar level was found to be lowered (though not significanty P>0.05) in a few rats by 3^rd hour but it gradually increased by 6^th hour and remained appreciably high by 12^th hour. The increase in blood sugar was more marked in rats fed glucose solution twice during the day and the hyperglycaemic was observed to persist by 24 hours. The average percentage increase in blood sugar in the drug treated groups of rats was found to be lowe as compared to that controls as shown in Table II and Graph 2.

DISCUSSION

In the present investigations, inhibitory effect of the common indigenous oral anti-diabetic drugs-Gymnema sylvestre, Coccinia indica, Pterocarpus marsupium Momordica charantia Linn. have been observed against the acute hyperglycaemic response of anterior pituitary extract and compared with that of tolbutamide. The blood sugar level in each of the control sets, increased gradually after the 3^rd hour and reached maximum only after 12 hours of the administration of the anterior pituitary extract. The rise in blood sugar level during 3^rd hour of administation of anterior pituitary extract seems to be due to the direct effect of glucose absorbed from the gastro-intestinal tract, in view of the fact that within this period, anterior pituitary extract per se did not cause any significant change in the blood sugar level of fasting animals in our preliminary experiments. Therefore, the 3^rd hour blood sugar values (one and half hour after glucose administration) seem to be indicative of the glucose tolerance in the treated and untreated groups. Analysis of the data presented in the Table I would show that the indigenous anti-diabetic drugs, under study, have a favourable influence on glucose tolerance in view of the fact that the blood sugar level did not rise much in treated animals as compared to the controls (Graph I). This inhibitory effect was more marked in animals treated with the alcoholic extract of G. sylvestre, aqueous infusion of P. marsupium and M. cahrantia extract. This is in conformity with previous observations on the glucose tolerance after these drugs in normal fasting animals, as reported elsewhere (Gupta, 1963; Gupta and Seth, 1962a). On the other hand, insignificant or doubtful lowering of fasting blood sugar has been reported after some of these drugs (Chopra et al., 1928; Gupta and Seth, 1962). It is therefore, likely that the drugs might sentisize the pancreatic islets tissue for the secretion of insulin occurring in response to glucose hyperglyceamic (Best and taylor, 1955) or to the increased peripheral demand for insulin induced by the anterior pituitary hormones (Mirsky et al., 1959) which have been reported to cause initial hypoglycaemia in hypophy-sectomised rats (park et al., 1952). The inhibitory effect of Pterocarpus marsupium, however, amy also be related to the retardation of glucose absorption from the gastrointestinal tract by the drug as reported by Joglekar et al., (1959). The blood sugar level in tolbutamide –treated animals, on the other hand, was found to be higher than the controls. This decreased tolerance after the drugs seems to be in conformity with that reported by others (Holt and Holt, 1956; Leaderer and De Myer, 1957; Mohnike, 1957; Mukherjee et al., 1958).

The blood sugar level was observed to be significant (37.5 mg. per cent at P>0.05) raised in control animals by the sixth hour of the administration of the anterior pituitary extract. This hyperglycaemia seems to be related to the increased gluconeogenesis (Houssay, 1936; Long, 1973) as well as to the inhibition of the peripheral utilization of glucose effected by the anterior pituitary hormones (Young, lic. cit.) An appreciable inhibition of the hyperglycaemic response of the anterior pituitary extract was observed in animals treated with the oral anti-diabetic drugs under investigations, as would be apparent on comparing blood suagr levels of control and treated animals in Graphs 1 and 2. This inhibitory effect was most marked and significant (P<0.001) in animals of Group I, treated with the alcoholic extract of G. sylvestre which has also observed to inhibit epinephrine hyperglycaemia (Gupta, 1961) known to be mediated through gluconeogenesis (Vogt, 1944). Reduction in the blood sugar level in animals treated with the aqueous extract of M. charantia was also quite significant (P<0.05). The inhibition of the hyperglyceamic response was however, not found to be significant (P>0.05) after other drugs. The delayed reduction in the blood sugar of these anterior pituitary treated hyperglycaemic animals after tolbutamide was similar to that observed in diabetic patients (Krantz and Carr, 1961).

The hyperglycaemic effect of the anterior pituitary extract was well marked by the 12^th hour and seems to be related to the inhibition of the oxidation of the 2^nd dose of glucoseadministered after sixth hour. Increase in the blood sugar level was, however, less marked in drug treated animals as compared to their counterpart controls. Marked percentage inhibition of the hyperglyceamic response of the anterior pituitary extracts occurred after both tolbutamide and G. sylvestre (Table II), though the inhibitory effect of the latter only was found to be very highly significant (P=0.001). Reduction in blood sugar after other drugs under study, was also quite appreciable but their inhibitory effect was not found to be significant (P>0.05).

The blood sugar level was found to be elevated even after 24 hour, though appreciably levels were observed drug treated animals as compared to the controls. Inhibitory effect of tolbutamide and G. sylvestre was again found to be maximum and highly significant (P=0.01 and 0.001, respectively). Reduction in blood sugar level, next in order was found to be in the animals treated with M.charantia Linn (groupIV), P. marsupium (group III) and Coccinia indica (group II). The difference in the blood sugar of treated rats as compared to their counterpart controls in these groups was, however, significant (P<0.02) only in animals given the aqueous infusion of P. marsupium.

Thus, the marked and highly significant inhibitory effects of gymnema sylvestre and Tolbutamide against the hyperglycaemic response of the anterior pituitary extract observed after 24 hours, seem to substantiate the previous reports on these drugs (Gupta et al., loc. cit.; Mirsky et al., loc. cit.). Though this persistent hyperglycaemic response of the anterior pituitary has been demonstrated to be due to the inhibition of the glucose uptake by the tissues (park, 1952; Krahl, 1956(, but in absence of any positive evidence it would be difficult to postulate a direct influence of these drugs on glucose utilization in the hexokinase reaction, which is known to be inhibited by the anterior pituitary hormones (Price et al., 1946). An indirect effect of the drugs, through stimulation of the pancreatic insulin secretion, therefore, seems to be responsible for this inhibition, as has also been suggested by others (Mhaskar and Caius, 1930; Loubatieries, 1957). The persistent effect of G. sylvestre can, however, be related to the inhibition of the adrenocortico activity (Gupta and variyar, 1961) which is known to potentiate the inhibition of the hexokinase reaction caused by the anterior pituitary extract, while that of the tolbutamide to the inhibition of the insulinase activity as postulated by Mirsky et al.(1957).

The inhibitory effect of M. charantia though not found to be significant after 12 and 24 hours, in the present investigations has, however, been reported to potentiate and prolong the tolbutamide action on blood sugar in rabbits (kulkarni and gaitonde, 1962). This may be indicative of the potentiation of insulin secretion by the extract of M. charantia similar to that effected by tolbutamide. The anti-diabetic effect of P. marsupium and C. indica which also caused appreciable inhibition of the hyperglycaemic response of the anterior pituitary extract and have been reported to cause hypoglycaemia fasting animals (Ojha et al., 1949; De and Mukerjee, 1953) is also likely to be due to some indirect stimulation of pancreatic insulin secretion or to retardation of glucose absorption, in view of the fact that the hyperglycaemic effect of the anterior pituitary hormones is inhibited in fasted, insulin treated animals (Best et al., 1942; Luken et al., 1943).

The inhibition of the hyperglycaemic response of the anterior pituitary extract by some of these drugs, is likely to prevent the vicious cycle of hyperglycaemia responsible for the deterioration of the pancreatic lesion in diabetic (lukens, cited by Mukherjee et al., 1958). Thus, the beneficial effects of these indigenous drugs in some cases of milder diabetic states may be related to the inhibition of the noxicious influence of anterior pituitary hormones.

SUMMARY

Effect of oral administration of some of the indigenous oral anti-diabetic drugs- Gymnema sylvestre, Coccinia indica, Pterocarpus marsupium. Momordica charantia Linn. and tolbutamide has been investigated at 3,6,12 and 24 hours against the oral hyperglycaemic response of anterior pituitary extract injected subcutaneously in albino rats.
The alcoholic extract of G. sylvestre, aqueous infusion of P. marsupium and the aqueous extract of M. charantia Linn. inhibited the hyperglycaemia of the 3^rd hour (one and half hour after glucose administration) probably by influencing the glucose tolerance inthese animals.
Inhibition of the hyperglycaemic response of the anterior pituitary extract at 6,12 and 24 hours was most marked after both tolbutamide and G. sylvestre. Inhibitory effects of G. sylvestre were highly significant (P<0.001) at 6,12 and 24 hours, while that of tolbutamide at 24 hours. Aqueous extract of M.charantia and infusion of P. marsupium also produced significant inhibition of pituitary extract induced hypeglycaemia at 6^th and 24 hours, respectively.
Possible mechanism involved in the inhibition of the anterior pituitary hormone induced hyperglycaemia after the drugs has been discussed.

The author is thankful to the Dean, Gandhi Medical College, Bhopal, for providing facilities for this work and to Mr. M. C. variyar, for the statistical analysis of the data presented in the paper. He also wishes to thank M/s Bengal Immunity Co., Calcutta, for preparing anterior pituitary extract and to M/s Hoechst Pharmaceuticals Ltd., Bombay, for supplying Rastinon (tolbutamide) for the present investigations.

REFERENCES

Best, C. H., Campbell, J., Haist, R. E., and Ham, A. W. (1942). The effect of insulin and anterior pituitary extract on the insulin content of the pancreas and histology of the islets. Jour. Physiol., 101, 17.

Best, C. H., and Taylor, N. B. (1955). Carbohydrate Metabolism in Physiological basis of Medical Practices, 6^th ed., 668, Williams & Wilkins Company, Baltimore.

Chopra, R> N., Bose, J. P., and Chatterjee, N. R. (1928-29). Gymnema sylvestre in Diabetes mellitus. Ind. Jour. Med. Res., 16, 115.

De, U. N., and Mukerjee, B. (1953). Effect of Coccinia indica Wight and Arn. On alloxan diabetes in rabbits. Ind. Jour. Med. Sci.,7, 665.

Gupta, S.S. (1961). Inhibitory effect of Gynema sylvestre induced hyperglycaemia rats. Ibid., 15, 883-887.

Idem (1963). Effect of Gynema sylvestre and Pterocarpus marsupium on glucose tolerance inrats Ibid., 17, 501-506.

Gupta, S. S., Seth, C. B., and Variyar, M. C. (1962). Experimental studies on pituitary diabetes Part I. Inhibitory effect of a few Ayurdevic anti-diabetic remedies on anterior pituitary extract induced hyperglycaemia in albino rats. Ind. Jour. Med. Res., 50, 73-81.

Gupta, S. S., and Seth, C. B. 91962) Experimental studies on pituitary diabetes Part II. Comparison of blood sugar level in normal and anterior pituitary extract induced hyperglycaemic rats treated with a few Ayurdevic remedies. Ibid., 50, 708-714.

Idem (1962a) Effect of Momordica charantia Linn. (Karela) on glucose tolerance in albino rats. Ind Jour. Med. Ass.,39, 501-584.

Gupta, S. S., and Variyar, M. C. (1961) Inhibitory effect of gynema sylvestre (Gurmar) on adrenohypophyseal activity in rats. Ind. Jour. Med. Sci., 15, 658-659.

Holt, C. V., and Holt, L., von (1956). Pancreas endocrine et anti-diabetique par voie orals. Am. Endocr., Paris, 18, 171-173.

Houssay, B. A. (1936). Hypophysis and metabolism. New Eng. Jour. Med., 214, 961-971.

Joglekar, G. V., Chaudhary, N. Y., and Aiman, Ranita (1959). Effect of indigenous plant extracts on glucose absorption in mice-Abst. Ind. Jour. Physiol. Pharmacol., 3, 76.

Krantz, J. C. (Jr) and carr, C. J. (1961). Pharmacological Principles of Medical Practice, 5^th ed. 1318. Williams & Wilkins Company, Baltimore.

Kulkarni, R. D., and Gaitonde, B. B. (1962). Ind. Jour. Med. Res. (Under publication).

Krahl, M. E. (1956). In regulation of carbohydrates metabolism in isolated tissues (Renold, A. E., Ashmore, J., and Hasting, B. A.) Vitamins and Hormones, 14, 146.

Lederer, J., and De Myer, R. (1957). Influence de l’ingestion prolongu de B. Z. 66, a faible dose chez le rat. Ann. Endoc., Paris, 18, 252-257.

Long, C. N. H. (1937). Harvey Lectures. 32, 194-223.

Loubatieres, A. (1957). The mechanism of action of the hypoglycemic sulphonamides: A concept based on investigations in animals and in man. Diabetes, 6, 408-417, Ann. N.Y. Acad. Sci., 71, 192-206.

Luken, F. D. W., Dohan, F. C., and Wolcott, M. W. 91943). Pituitary diabetes in cat: recovery following phlorizin treatment. Endocrinology, 32, 475.

Mhaskar, K. S., and Caius, J. F. (1930). A study of Indian medicinal plants Gymnema sylvestre. Ind. Med. Res. Memoirs, No. 16.

Mirsky, A. Gitelson, S., and Peributti, G. (1959). The effect of tolbutamide on the diabetogenic action of somatatropin. Endocrinology, 64, 766.

Mirsky, J. A., Perisutti, G., and Gitelson, S. (1957). The role of insulinase in the hypoglycemic response to sulfonylureas. Ann. N. Y. Acad. Sci., 71, 103-111.

Mohnire, G. (1957). Daueranwendung von blutzuckesenkenden Harns-tofforivaten bei stoffwechselgesunden und alloxan-diabetischen Hunden. Dtsch. Med. Wschr., 82, 1576-1578.

Mukherjee, S. K., De, U. N., and Mukerji, B. (1958). Further studies with Nadisan: Effect on blood cholesterol blood glutathione and sugar tolerance in albino rats. Ind. Jour. Med. Res., 46, 185-192.

Idem (1958a). Studies in experimental diabetes: Effect of desiccated thyroid and corticotrophin and other combinations with insulin on alloxan diabetes in albino rats. Ind. Jour. Med. Res., 46, 403.

Ojha, K. N., Paramjit, R. P., and venkata-Chalan, K. (1949). Ind. Jour. Pharm., 11, 281.

Park, C. R. (1952). Phosphorus Metabolism (W. Mc Eloroy and B. Glass Ed.), 2, 634-653. John Hopkins Press, Baltimore.

Park, C. R., Brown, D. H., Comblath, M., Daughday. W. H., and Krahl, M. E. (1952). Cited by Renold et al., in Regulation of carbohydrate Metabolism, Vitamins and Hormones, 14, 147, 1956.

Price, W. H., Slein, N. W., Colowick, S. P., and Cori, G.T. (1946). Quoted by Shull, K. H. and Mayer, J. (1958). Experimental hyperglycaemic state not primarily due to lack of insulin, Vitamins and Hormones, 14, 197.

Vogt, M. (1944). Observations on some conditions affecting rate of hormonal output by adrenal cortex. Jour. Physiol. (London), 103, 317-332.

Young, F. G. (1951). Growth hormone and experimental diabetes, Jour. Clinical . Endocrinol., 11, 531-533.

Pharmacology of a Hypoglycaemic Principles Isolated from the Fruits of Momordica charantia Linn

filipinoweb — Thu, 10 Aug 2006 04:07:28 +0000

May 1966 The Indian Journal of Pharmacy Vol.28, No.5, pp.129-133

by M. M. Lolitkar, M. R. Rajarama Rao
Charantin, non-nitrogenous, neutral princile, giving colour tests for phytosterolins is isolated in a pure state from the fruits of Momordica…

Pharmacology of a Hypoglycaemic Principles Isolated from the Fruits of Momordica charantia Linn

Author: M. M. Lolitkar, M. R. Rajarama Rao

Type of Publication: Pre-Clinical

Date of Publication: May 1966

Publication: The Indian Journal of Pharmacy Vol.28, No.5, pp.129-133, May 1966

Organization: Department of Chemical Technology, University of Bombay

Charantin, non-nitrogenous, neutral princile, giving colour tests for phytosterolins is isolated in a pure state from the fruits of Momordica charantia. Charantin lowers blood sugar in fasting rabbits, the fall being gradual from the 1^st to 4^th hour, and recovering slowly to initial level. Charantin (50 mg./kg.) administered orally lowers blood sugar by 42 per cent at the 4^th hour, the mean fall during 5 hours being 28 per cent. The cumulative hypoglycaemic potency curve is not linear, but tends to flatten out as the dose is increased. Charantin is more potent than tolobutamide in hypoglycaemic activity but the pattern of blood sugar changes is similar to both. The hypoglycaemic activity of charantin is depancreatixed cats is less, but abolished, indicating a pancreatic as well as extra-pancreatic action. It exerts non-specific antispasmodic and mild cholinergic-blocking activity.

The unripe fruit of Momordica charantia (Hindi: karela, Eng.: bitter gourd) is commonly used as vegetable. It is popular as a folk-lore remedy for diabetes mellitus. One to two ounces of the juice of fresh unripe fruits is taken daily to control diabetes. Vad^1,2 reported clinical trial of the fresh juice in 160 cases of diabetes. The juice was found to control diabetes but was not capable of curing it. Rivera³ isolated an alkaloid, momordicine and a glycoside from an alcoholic extract of the fruits. Gaessler⁴ reported hypoglycaemic activity in a crude crystalline substance isolated from the fruits. Sharma et al.,⁵ Kulkarni et al. and Pabrai et al.⁷ gave conflicting reports on the hypoglycaemic activity of the juice of fresh unripe fruits. Lolitkar and Rao⁸ reported hypoglycaemic activity of a non-nitrogenous neutral principle isolated from an alcoholic extract of the dried fruits.

MATERIALS AND METHODS

Isolation of charantin: Fresh unripe fruits of Momordica charantia bought from the market were cut into small bits and dried in a hot air oven below 60⁰. The dried material was broken into a coarse powder and percolated successively with petroleum ether (b.p. 60-80⁰) and 80 per cent ethanol. The petroleum ether extract was rejected and the ethanolic extract was concentrated in vacuo, suspended in 95 per cent ethanol and rendered alkaline with KOH to around pH 10. After 48 hours, the suspension was diluted with water and extracted with ether. The ether extract was washed with water, 5 per cent HCI, with water again, and dried over anhydrous sodium sulphate. The ether was distilled off and the residue recrystallized several times from 95 per cent ethanol. The substance thus obtained will be referred to here as charantin. Yield was 0.035 per cent of the dried fruits.

Charantin is a non-nitrogenous neutral substance, melting at 269-272⁰ with decomposition, giving a play of colours changing from violet to blue to green and yellow with Libermann-Burchard test, decolourising dilute potassium permanganate and bromine water, soluble in ether, benzene and chloroform, sparingly soluble in ethanol and methanol and insoluble in petroleum ether, acetone and water. It answers colour tests for phytosterolins⁹ – a violet ring and greenish orange fluorescence in the bottom layer, on adding saturated solution of thymol to a solution of the compounds in conc. Sulphuric acid. On hydrolysis with HCI it yields glucose and sterol. On analysis it was found to contain C, 74.0; H, 10.4; and O, 15.6 per cent. It forms acetyl and methyl derivatives. Mol. Wt. Of acetyl derivatives is 608, by Rast method, and its elemental analysis is C, 70.7; H, 9.2; and O, 20.1 per cent. Elemental analysis of methyl derivatives is, C, 75.2; H,12.7; and O, 12.1 per cent. Mol wt. Of charantin was not done since it was insoluble in camphor. Determination of acetyl and methoxy groups was not done since the derivatives were not soluble in the solvents usually employed here. Probable mol. Formula of acetyl derivatives is C₃₆ H₅₆O₈. I. R. spectra of charantin and cholesterol have a close resembles, the former having more hydroxyl groups and double bonds. Thin layer chromatography of charantin on silica gel with methanol: benzene mixture (2:8), and spraying with conc. Sulphuric acid gave a single spot with RF value of 0.5.

Hypoglycaemic activity of charantin: Rabbits of either sex (Haffkine strain) weighing 1.5-3 kg. were fasted by with drawing food the previous day. Blood sugar was estimated by the method of Hagedron and Jensen.¹⁰ Cahrantin was suspended in 0.3 per cent. Twelve 80, and administered orally through a stomach tube, or intravenously. Alloxan diabetes was produced in rabbits by intravenous injection of alloxan, 200 mg./kg., and 4 out of 20 which survived after 5 days were used. Anaesthetized (pentobarbitone sodium 30 mg./kg. intraperitoneally) cats fasted for a day were also used. Pancreas of cats was removed through a median incision in the abdomen. Removal of samples of blood and administration of charantin was through the femoral vein. Blood pressure of anaesthetized (as above) cats was recorded on a kymographs using a mercury manometer connected to a cannula in the carotid or femoral artery. Respiration was recorded through a tambour connected to a cannula in the trachea.

Contraction of the nictitating membrane of cats was recorded on a kymograph with a frontal lever, the preganglionic cervical sympathetic being stimulated by a 10 volt current from a square-wave stimulator. Isolated hearts of frogs were perfused with frog Ringer, and charantin added to the cannula. Isolated duodenum of rabbit and ileum of guinea pig were suspended in an oxygenated organ bath of 50 ml. capacity at 35⁰ and contractions were recorded through frontal levers. Salivary secretion was induced in rabbits by subcutaneously injection of 15 mg./kh. Pilocapine nitrate. Charantin or atropine sulphate were administered intravenously. Tremors were induced in mice and rabbits by intraperitoneal injection of 10 mg./kg. Tremorine. Toxicity was studied in mice weighing 18-22 g. (Haffkine strain) cahrantin being administered intraperitoneally.

EXPERIMENTAL AND RESULTS

Hypoglycaemic activity. Control rabbits were given 5 ml. of 0.3 per cent Tween 80. Tween 80 had no effect on fasting blood sugar of rabbits. The mean fall in blood sugar was calculated from

IBS – FBS

the formula _________________ X 100 where

IBS

IBS= initial blood sugar, FBS- the mean of five reading taken during five hours following administration of the drug. Peak fall in blood sugar was calculated from the same formula but with FBS as one of the five readings where fall of blood sugar was maximal.Charantin produced a gradual and steady fall of blood sugar for 4 hours after which tended to recover to initial level. The duration of activity was more than five hours. Maximal activity was reached in the 4^th hour. There was no difference in the pattern of blood sugar change between intravenous and oral routes, the peak effect appearing at the 4^th hour in both. However, 15 mg./kg. charantin by intravenous route produced the same effect as 25 mg./kg. orally. Equivalent doses of tolbutamide were less effective than charantin. The pattern of blood sugar changes was similar to both. (Table1). Fig.1 presents cumulative hypoglycaemic potency curve for charantin, where each point indicates the cumulative hypoglycaemic potency for the duration of the testing period, i.e. five hours. There is a clear indication that higher doses of charantin may not produced a proportionate fall in blood sugar. The cumulative hypoglycaemic potency is calculated from the formula, ∑ P₁….P₅where P₁ = per cent fall in blood sugar at the first hou, P₂ at the second hour and so on. This formula is considered by Radding et al.¹¹ as more informative than a log. dose-response curve, giving some idea on the rate of absorption and metabolic dispersion of the compound.

The course of blood sugar changes in alloxan diabetic rabbits in response to charantin was rather erratic. In two of the rabbits there was a marked rise in the first hour after the drug, while the other two showed marked and steady fall. Besides, only mildly diabetic rabbits had survived 5 days after alloxan.

Charantin produced marked fall (25 per cent for 5 mg./kg.i.v.) of blood sugar in anaesthetized cats. The effect of charantin was considerably less (nearly half) in the depancreatized cat. There was no fall in blood sugar of depancreatized cat not treated with charantin.

Cardiovascular system. In anaesthetized cats 800 g./kg. of charantin lowered the blood sugar pressure by 5-10 per cent, and blocked the ise of blood pressure caused by occlusion for one minute of carotid arteries, but did not affect the changes produced by acetyl choline and adrenaline.

There was no change in the contraction of the nictitating membrane of cat after injection of 400-800 g./kg. of charantin and hence, no ganlionic blockade.

Small doses of charantin (5-10 g.) increased the amplitude of contraction of the isolated perfused heart of frog, and abolished the inhibitory action of acetylcholine (Fig.II).

The vehicle, 0.3 per cent Tween 80, did not have any effect on the blood pressure of cats or the heart of frog.

No significant changes in respiration were observed in anaesthetized cat after the administration of charantin.

Anti-sialogogue activity. Subcutaneous injection of pilocarpine nitrate (15 mg./kg.) produced in rabbits copious flow of saliva for over 2 hours. The flow ranged from 1.5-4 ml. per 10 minutes, and the flow was fairly constant during the experimental perion. Intravenous injection of 7.89 mg./kg. of atropine sulphate reduced this flow of saliva by 50 per cent.

Charantin did not have any effect on the amplitude of contractions of isolated duodenum of rabbits, but 500 g./ml. of charantin partially reduced the amplitude of spasm produced by acetyl choline (Fig.III).

Charantin inhibited the spasm produced by acetyl choline histamine and barium chloride on the isolated ileum of guinea-pig. ED₅₀ against acetyl choline was 0.343 g./ml. (atropine sulphate 0.006 g./ml.), against histamine 1.39 g./ml(mepyramine maleate 0.448 g./ml.), and against barium chloride 2.29 g./ml. (papavarine hydrochloride 7.65 g./ml.). Since charantin is insoluble in water, its potency in these experiments is not calculated.

Charantin did not appear to produce any behavioral changes in mice doses up to 400 mg./kg. intraperitoneally. This dose was also not lethal.

Tremorine (10 mg./kg.) injected intraperitoneally produced salivation and tremors in mice within 10 minutes, the tremors lasting over two hours. In the case of rabbits, 10 mg./kg. tremorine produced profuse salivations, and occasional waves of tremors lasting over two hours. Charantin (10-25 mg./kg.) by oral or intravenous route delayed the onset of tremors by 20-40 minutes, but did not affect salivation. Atropine sulphate (2 mg./kg.) given intraperitoneally promptly abolished both the tremors and salivation in these animals.

DISCUSSON

The colour tests and other data of charantin indicate that it is phytosterolin i.e., a sterol glycoside. Since its mode of action is not known, it may be inappropriate to calculate its potency using tolbutamide as a standard. The results however, indicate a similarity in the pattern of blood sugar changes in response to these two agents. On this basis it may be inferred that charantin is more potent than tolbutamide. The cumulative hypoglycaemic potency curve tends to fall from 25 mg./kg. to 50 mg./kg. Since 400 mg./kg. of charantin is not lethal to mice it is possible that higher doses of charantin may not be accompanied by a proportionate fall in blood sugar. This may be due to limited absorption or action or both. The reduced effect of charantin in the depancreatized cat indicates a pancreatic as well as extrapancreatic action. While charantin does not block the fall in blood sugar pressure after acetyl choline it blocks the inhibitory action of the latter on amphibian heart. It also delays the onset tremors due to Tremorine, inhibits the siologogue action of pilocarpine, and exerts antispasmodic activity. It is therefore to be inferred that the cholinergic blocking action of charantin is of a mild nature. The blocking action of charantin on the rise of blood pressure due to occlusion of the carotid arteries is not fully explained by the data collected. It is not, however, due to adrenergic or ganglionic blockage.

It is doubtful if charantin represents all the hypoglycaemic activity present in the fruit of Momordica charantia. For example, it takes more than 1,500 g. of fresh fruits to get 50 mg. of charantin. The beneficial clinical results reported from using daily 50-60 ml. of the juice of fresh fruits could not be entirely due to the few mg. of charantin present in them. Perhaps the whole fruit, instead of the juice, would produce better results clinically, provided it did not exert any toxic effect on prolonged use.

ACKNOWLEDGEMENTS

We are indebted to Dr. T. S. Gore and Dr. M. R. Padhye, for chemical and spectral analysis of charantin, and to Dr. R. K. Richards of Abbott Laboratories, Chicago, for the sample of Tremorine.

We are indebted to the Government of Maharashtra for financial assistance received during the course of this work.

REFERENCES

Vad, B. G., Maharashtra Medical J., 1960, 6, 733.

Vad, B. G., Symposium on Indigenous drugs held at Topiwala Medical College, Bombay, 1961.

Rivera, G., Am. J. Pharm., 1941, 113, 281.

Gaessler, W. G., Am. J. 1942, 72, 114.

Sharma, V. N., Sogani, R. K. and Arora, R. B., Indian J. med Research, 1960, 48, 471.

Kulkarni, R. D. and Gaitonde, B. B., Indian J. Med. Research, 1962, 50, 715.

Pabrai, P. R. and Sehra, K. B., Indian J. Pharm., 1962, 24, 209.

Lolitkar, M. M. and Rajarama Rao, M. R., J. Univ. Bomaby, 1961, 29, 223.

Ambike, S. H. and Rajarama Rao, M. R., unpublished data.

Hagedorn and Jensen, Biochem. Z., 1923, 135, 46.

Radding, R. S., Kern, L. R. and Owens, J. C., metabolism, 1962, 11, 411.

Isolation of a Guanylate Cyclase Inhibitor from the Balsam (Momordica charantia Abreviata)

filipinoweb — Thu, 10 Aug 2006 04:03:13 +0000

1977 Biochemical and Biophysical Research Communications Vol. 77, No.4

by David L. Vesley, William R. Graves, Timothy M. Lo, Mary Ann Fletcher, Gerald S. Levey
The balsam pear (Momordica charantia abreviata) is a plant that grows wild throughout subtropical regions of the world inclufing the…

Isolation of a Guanylate Cyclase Inhibitor from the Balsam (Momordica charantia Abreviata)

Author: David L. Vesley, William R. Graves, Timothy M. Lo, Mary Ann Fletcher, Gerald S. Levey

Type of Publication: Pre-Clinical

Date of Publication: 1977

Publication: Biochemical and Biophysical Research Communications Vol. 77, No.4, 1977

Organization: University of Miami School of Medicine , Miami , Florida

SUMMARY

The balsam pear (Momordica charantia abreviata) is a plant which grows wild thoughout subtropical regions including the Gulf Coast and Florida. The ripe fruit is highly toxic and produces mild hypoglycemia. Preparations of the ripe fruit and the leaves but not the unripe fruit or seeds inhibited guanylate cyclase activity whereas adenylate cyclase activity was unaffected. The guanylate cyclase inhibitor (GCI) has been purified about 30-fold has an estimated molecular weight of 5000 to 50,000 and is acid stable and heat labile. GCI blocked the activation of guanylate cyclase by nitroso chemical carcinoges, and therefore, may be useful in studying mechanism carcinogenesis.

INTRODUCTION

The balsam pear (Momordica charantia abreviata) is a plant that grows wild throughout subtropical regions of the world inclufing the Gulf Coast and Florida. In folk-medicine the fruit and leaves of this plant have reportedly been used as hypoglycemic agents (1), purgatives (2), emetics (2), and abortifiacients (2). The plant is highly toxic and in Miami several instances of illness following ingestion of the ripe fruit have been described in children and domestic animals such as dogs (2). Because of the toxicity and the purported medicinal uses we examined the effects of the fruit and leaves on guanylate cyclase (E. C. 4.6.1.2.) and adenylate cyclase (E.C. 4.6.1.1.) to determine if any correlation could ultimately be derived for the observed in vivo phenomena and the intracellular concentrations of cyclic nucleotides.

The results show that the ripe fruit and leaves contain a guanylate cyclase inhibitor which has the ability to impair chemical carcinogen-induced increased in guanylate cyclase activity.

METHODS

Tissues used in these experiments were obtained from male Sprague-Dawley rats, weighing 150-200 grams that had been maintained ad libitum on Purina Laboratory chow. Alumina oxide, neutral activity I for column chromatography, was obtained from E. Merck, (Darmstadt, Germany). The alpha [³² p] GTP was from New England Nuclear Corporation (Boston, Mass.) and International Chemical and Nuclear Corporation (Irvine, Calif.).

The ripe fruit, unripe fruit, leaves, and seeds were homogenized in 0.03 M Tris, pH 7.6, filtered ythrough gauze and centrifuged at 12,000 g. These extracts had a protein concentration of about 8 g/ml and were added to the enzyme preparation described below at a ration approximating 0.4 g of extract protein to 400 g of enzyme protein.

Guanylate cyclase activity was measured as previously described (3-5) utilizing a modification of the original method of White and Zener (6). The various tissues were homogenized in cold 0.03 M Tris CHI, pH 7.6 and centrifuged at 37, 000 g in a Sorval refrigerated centrifuge at 40⁰ for 15 minutes. The supernatant was assayed at 37⁰ for 10 minutes for guanylate cyclase activity, using a reaction mixture consisting of 20 mm tris HCI, pH 7.6; 5 mm MnCI₂; 2.67 mM cyclic GMP (used to minimize destruction of [³² p]- GTP; a GTP regenerating system (5 mM creatine phosphate, 11.25U creatine phosphokinase); 100 g bovine serum albumin; 20 mM caffeine; [ a-³²p]-GTP approximately 5×10⁵ cpm; and the enzyme preparation having 0.2 to 0.6 mg protein. The reaction was terminated by the addition of 10 l of 0.1 M EDTA, pH 7.6, containing about 30,000 cpm of [³H]-cyclic GMP (to estimate recovery in the subsequent steps) and boiling for three minutes. After cooling in an ice bath, the [³²p]-cyclic GMP formed is isolated by sequential chromatography on Dowex-50-H⁺ and alumina using the modification of Krishna and Krishan (7). The reaction mixtures were diluted with 0.5 ml of distilled water and transferred to a Dowex-50-H⁺ column (10×75 mM). The column were then eluted with another 0.5 ml distilled H₂O and the eluates (1 ml) were discarded. The second 1 ml water fraction eleuted from Dowex-50-H⁼ column was allowed to directly pass through a column of dry neutral alumina (10×75mM). The alumina column were then eluted with 2 ml of 0.03 M Tris-HCI buffer, pH 7.6. The above three mls of elutant from the alumina column were collected directly into scintillation vials containing 15 mls of Bray’s solution. The eluates were then counted in a Packard Tri-Carb Liquid Scintillation spectrometer. The overall recovery of cyclic GMP after the two-stage chromatographic procedure was 85 to 95%. All of the [³²p] –containing material was identifiable as cyclic GMP as determined by thin layer chromatography on a cellulose PEI, Brickman) using 1 M LiCI as solvent and Chromar sheets (Mallinchrodt, St. Louis, Momordin.) developed with absolute alcohol and concentrated NH₄OH (5:v/v-insulin)/ Proteins was determined by the method of Lowry et al. (8). Adenylate cyclase was prepared and assayed as previously described from our laboratory (9).

RESULTS

The filtrates of the ripe fruit and the leaves produced virtually complete inhibition of rat hepatic guanlate cyclase activity from a control level of 280  12 pmoles accumulated/mg protein/10 min. to 5  2 pmoles for the ripe fruit and 10  4 pmoles for the leaves (Table I). The unripe fruit and seeds were without effect. The extract from the ripe fruit produced similar reductions in guanylate cyclase activity in other rat tissues including pancreas, heart, lung, stomach, colon and kidney (Table 2). In contrast to these findings with guanylate cyclase, the extracts had no measurable effects on adenylate cyclase activity.

The guanylate cyclase inhibitor (GCI) has been purified about 30-fold using, sequentially, a 12,000 g centrifugation of an aqueous extraction of the ripe fruit, Amicon-30 ultrafiltration, dialysis at pH 7.0 and DEASE Sephadex chromatography. The resultant purified material has a molecular weight of 5,000 to 50,000 as estimated by gel filtration and is acid stable and heat labile. GCI is probably not a lipid since the material remained in the aqueous phase after a double extraction with ether: ethanol 94:1) followed by an extraction with chloroform: methanol (2:1).

In the past year a number of nitroso chemical carcinogens (3-5, 10) including nitrosoamides, nitrosoamines, and hydrazine have been shown to activate guanylate cyclase, a finding which may be of considerable importance in understanding the mechanism of chemical carcinogenesis. We therefore examined the effect of GCI on the activation of guanylate cyclase by the nitrosoamides, streptozotocin and N-methyl-N-nitroso-N’-nitroguanidine (MNNG), which are among the most potent acrcinogen stimulators of guanylate cyclase.Table 3. demonstrate that GCI almost totally abolished the 20 to 25 –fold increases in guanylate cyclase activity produced by these carcinogens. The decreases in activity were highly significant (p<0.001).

DISCUSSION

The data in this report demonstrate that the balsam pear (Momordica charantia abr.) contains an inhibitor of guanylate cyclase. This inhibitor almost completely abolished guanylate cyclase activity in all rat tissues studied and was specific in so far as it did not alter adenlate cyclase activity. The inhibitory mateial is acid-stable and heat labile, has been partially purified (30-fold), and does not appear to be a lipid. When purified and identified it should be a useful probe to aid in further understanding the controversial, complex, anf intriguing role of cyclic GMP in cell biology (11).

A number of recent investigations have demonstrated that guanylate cyclase activity is increased by nitroso chemical carcinigens (3-5,10). In addition, cyclic GMP may play a role in malignant transformation (12) and cyclic GMP levels have been reported to be increased in some tumors (13,14) including an adenocarcinoma of the human colon (14). Therefore, it is interest and potentially great importance that GCI blocks the activation of guanylate cyclase by the nitroso chemical carcinogens, streptozotocin and MNNG. Whether or not GCI will abolish or prevent tumor-induction by these agents and what rhe effects of GCI are on other tumors formulates the basis for critical future experiments.

ACKNOWLEDGEMENTS

We thank Julia F. Morton who stimulated our interest in studying the balsam pear. This work was supported by NIH grant HL 13715-07 and USPHS AM-167-63-06.

Dr. Levey is an Investigator of Howard Hughes Medical Institute. Society, Chicago, Illinois, June 8,1977.

REFERENCES

Sharma, V. N., Sogani, R. K., and Arora, R. B. (1960) Ind. J. Med. Res., 48(4), 471-477.

Morton, J. F. (1967) Economic Botany, 21(2), 57-68.

Veseley, D. L., and Levey, G. S. (1977) Biochem. Biophys. Res. Commun., 74(2), 780-784.

Vesely, D. L., and Levey, G. S. (1977) Proc. Soc. Experimental. Biol. And Med., 155(3), 301-304.

Vesely, D. L., Rovere, L. E., and Levey, G. S. (1977) Cancer Res., 37, 28-31.

White, A. A., and Zenser, T. V. (1971) Anal Biochem., 41, 373-396.

Krishna, G., and Krishna, N. A. (1975) J. Cyclic Nucl. Res., 1, 293-302.

Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275.

Levey, G. S., and Eptein, S. E. (1969) J. Clinical . Invest., 48, 1663-1669.

DeRubertis, F. R., and Craven, P. A. (1976) Science 193, 897-899.

Goldberg, N. D., Haddox, M. K., Nicol, S. E., Glass, D. B., Sanford, C. H., Kuehl, F. A., Jr., and Estensen, R. (1975) Adverse. Cycl. Nucl. Res., 5, 307-338.

Kram, R., and Tomkins, G. M. (1973) Proc. Natl. Acad. Sci. USA 70, 1659-1663.

Kimura, H.,and Murad, F. (1975) Proc. Natl. Acad. Sci. USA 72, 1965-1969.

DeRubertis, F. R., Chayoth, R., and Field, J. B. (1976) J. Clinical . Invest. 57, 641-649.

Steroids an Hydrocarbons of the Leaves of Momordica charantia

filipinoweb — Thu, 10 Aug 2006 02:53:28 +0000

1979 Rev. latinoamer. Quim. 10, pp.171-173

by A. Ulubelen, U. Sankawa
Momordica charantia are well known for their pharmacological activities. The seeds of M. charantia have been used against diabetes mellitus…

Steroids an Hydrocarbons of the Leaves of Momordica charantia

Author: A. Ulubelen, U. Sankawa

Type of Publication: Pre-Clinical

Date of Publication: 1979

Publication: Rev. latinoamer. Quim. 10, pp.171-173, 1979

Organization: Faculty of Pharmacy, University of Istanbul, Faculty of Pharmaceutical Sciences, University of Tokyo

Abstract: The alcohol extract of the dried leaves of Momordica charantia yielded 7-stigmasten-Bol and 7,25-stigmastadien-3-ol as a mixture and 5,25-stigmastadien-3-ol and glucoside as well as n-octacosan (C₂₈ H₅₄), triacontanol (C₁₀ H₅₂ O) and a new phytosphingosin (C₃₃ H₅ O₆ n). The compounds were identified by spectral methods.

INTRODUCTION

Momordica charantia are well known for their pharmacological activities. The seeds of M. charantia have been used against diabetes mellitus in Puerto Rico (1,2), and an insulin-like hypoglycaemic action was shown to be prtesent in the seeds of the plant (3). This activity was attributed to a steroidal compound, cahrantin 94,5), which was later found to be a mixture of sitosteryl-3-D- glucoside and a new compound 5,25-stigmastadien-3-ol D-glucoside (6).

Tissue cultures of the unripe fruits of M. charantia yielded diosgenin and 7-stigmasten-ol (7) and 5-stigmasten-3, 25-diol (8), 5,25-stigmastadien-3-ol and 7,25-stigmastadien-3-ol were detected in the fruits themselves (9).

From the aqueous extract of the seeds of M. charantia a protein with oncostatic activity was obtained (10). Antihelmintic activity was also reported for the same plant (11,12). In 1936 Diaz mentioned alkaloid-like substances in the fruits of M. charantia (13). From the petroleum ether fractions of M. charantia and M. dioica a substance with alkaloid properties was later isolated (14). A non-quaternary alkaloid with antinicotinic and antimuscarinic activity was found in M. foetida (15). An aqueous extract of the plant showed abortive effects on pregnant rabbits (16). 5,25-Stimastadien-3-ol D-glucoside was also found in the same plant (17).

In the present study the alcoholic extract of the leaves of Momordica charantia was examined. Preliminary tests indicated the presence of a group of tertiary alkaloids. After separation of these alkaloids by 10% acetic acid extraction, the remaining extract was evaporated to dryness and subjected to column chromatography. The hydrocarbons n-octacosan, tricontanol, and the steroids, 7-stigmasten-3ol, 7,25-stigmastadien-3-ol and 5,24-stigmastadien-3-ol glucoside were isolated along with a new phytosphingosin (C₄₈ H₈₃ O₆N). The latter compounds is the first of this molecular-weight phytosphingo lipids are known from yeast molds and mushrooms (18) and a mixture of phthiocerols (C₃₆ H₇₆ O₃) and (C₃₂H₇₀O₃) was obtained from tubercle bacilli (19-21). The structures of the phthiocerols from tubercle bacilli were determined by mass spectral interpretation. In the determination of the partial structure of the new phytospingosin, the MS of the known phthiocerols and phytosphingosin were useful (21, 22).

Experimental

The spectra were recorded with the following instruments: IR, Jasco, DS 701: NMR, Joel 100 MHZ, Joel-Ol SG-2; GC, Hewlett-Parkard 402 A (3% OV-25, 270⁰, He 100 ml/min). Melting points were taken in a micro melting point apparatus and were not corrected.

The leaves of M. charantia (Cucurbitaceae) were collected from the Botanical Garden of the University of Istanbul. About 2 kg of the powdered leaves were macerated, then percolated with 95% ethanol. Upon evaporation under vacuum 160 g of a dark green residue was obtained. Preliminary tests (Dragendorff and Mayer’s) showed the presence of alkaloids. In order to separate the alkaloids the green residue was dissolved in chloroform, and the insoluble part was separated by centrifugation (50 g) (mainly inorganic compounds). The chloroform solution was extracted exhaustively with 10% aqueous acetic acid to remove the alkaloids (Mayer’s reagent). The acetic extract was made alkaline with ammonia and extracted with chloroform; about 1.2 g of a crude alkaloid mixture was obtained. The crude mixture showed two main and a few smaller alkaloid spots on TLC plates. (The determination of these alkaloids will be the subject of another study).

After removal of the alkaloids, the remaining solution was evaporated to dryness, yielding 20 g of residue. The material fractioned elutions yielded 147.8 mg of a hydrocarbon, crystallized from chloroform-methanol mixture (1:1) and determined to be n-octacosan by spectral methods, later fractions with ethyl acetate concentrations increasing to 70% yielded a hydrocarbon alcohol which was found to be triacontanol; 10% ethyl acetate yielded a steroidal mixture (A₃) which was later found to contain 7-stigmasten-3-ol and 7,25-stigmastadien-3-ol by GC-MS separation. 5% methanol in chloroform eluted the new phytosphingosin, and 10% methanol yielded stigmast-5,25-dien-3-ol D-glucoside.

n-OCTACOSAN (C₂₆ H₅₁): m.p. 59-60^o, Lit. 61-62⁰ (23, p.17). IR (in KBr) showed a long-chain hydrocarbon, NMR (in CDCI₃, TMS) corroborated this findings.

Analytical calculations: Found C, 84.97; H, 16.67

Calculated for C₂₆ H₅₈ C, 85.27; H, 16.72

TRIACONTANOL (C₁₀ H₆₂O): m.p. 84-86⁰, lit. 86-86.5⁰ (23, p. 49); IR (in KBr) and NMR (in CDCI₃ TMS) showed a long chain hydrocarbon alcohol.

Analytical calculations: Found C, 78.19; H, 13.67

Calculated for C₃₀ H₆₂ O. H₂O C, 78.60; H, 13.97

7-STIGMASTEN-3-OL and 7,25-STIGMASTADIEN-3-Ol (A₃). A₃ gave a single spot on TLC and argentized plates, m.p. 168-169⁰; IR (in KBr) showed an exocyclic methylene group (1635, 835 cm ^–1), NMR (in CDCI₃, TMS) 0.58 (CH₃ – 18,s) (indicated ∆-double bond), 0.80 (CH₃ – 19,s), 0.86 (CH₃ – 29, d,J= 7Hz), 1.00 (CH₃-21,d,J=6Hz), 3.5 (CH-O, m), 4.58 and 4.66 (1 H each, br. D, = CH₂); 5.08 (br.d, ∆ ⁷H) (integration of this signal indicated almost two protons). The mass fragments was similar to that given by Sucrow (9), except for a peak at 414 in addition to a molecular ion at 412. These data together with the d 5.08 signal in the NMR spectrum, suggested the presence of a mixture. Gas chromatographic separation showed the presence of two compounds (3:2 ratio ^a with retention times of 4.2 and 5.0 minutes respectively. MS of 7-stigmasten-3-ol, M 414, at m/z 399 (M-CH₃), 299, 271 (M-side chain-2H), 255, 246, 212 (base peak): MS of 7,25-stigmastadien-3-ol, M 412 at m/z 397 (M-CH₃), 328, 314, 299, 271, 254, 245, 211.

Direct comparison with standard samples confirmed the above given findings, namely that these compounds are 7-stigamasten-3-ol and 7,25-stigmastadien-3-ol. 5,25-STIGMASTADIEN-3-OL D-GLUCOSIDE: m.p. 275-280⁰ (degradation); IR (in KBr) showed exocyclic methylene (1635, 885 cm^-1) and the presence of sugar (broad band at 3600 and 1075, 1050, 1025 cm^-1); MS, M 576, at m/z 412 (aglycone), 396 (agly- H₂O) (base peak), 328, 314, 302, 254, 231.

Acetylation in pyridine anhydride at room temperature gave an acetate, m.p. 160-164⁰, IR (in KBr) showed that no free hydroxyl group was left. NMR (in CDCI₃, TMS) 0.72 (CH₃-C = ,s), 1.98, 2.00, 2.04, 2.08 (4 acetyl singlet), other peaks are at 3.6 (m), 4.2 (m), 4.6 (t), 4.9 (br. d), 5.3 (br. d) ppm.

MS, M^- 742, at m/z 421 (aglycone), 394 (agly-H₂O) other peaks are essentially the same as that of the glycoside.

Hydrolysis of the compound utilized 6N HCl under a reflux for 6 hr. The sugar was found to be glucose by comparison with a standard sample (TLC and PC). The aglycone was found to be 5,25-stigmastadien-3-ol on the basis of spectral findings and comparison to the literature values (6).

NEW PHYTOSPHINGOSIN (C₄₃ H₈₃ O₆N): m.p. 143-145⁰; IR (in KBr) 3350 ()H), 3200 (NH₂), 2910, 2845, 1615 (unsaturation), 1545, 1465, 1065, 1015 (C-O), 962 (N-H), 720 [(CH₃)n] cm^-1; NMR is taken at 50⁰ C (in CDCI₃, TMS) 0.92 (3H, br.s), 1.3 [(CH₂0 n,s], 2.55 (OH₁ D₂O exchange), 3.62 (OCH₃,s), 4.0 (br.t), 5.4 (vinylic protons, br.s. Acetylation of the compounds was performed in the usual manner, m.p. 58⁰ , IR (in KBr) 2920, 2850, 1745, 1735 (acetyl) 1660 (unsaturation), 1530, 1465, 1375, 1265, 1235, 1220 (acety C-O), 1140, 965, 720 [(CH₃)n] cm^-1 NMR (in CDCI₃, TMS) 0.9 (3H,s), 1.25 [(CH₂)n,s], 2.04, 2.06, 2.10, 2.18 (acetyl), 3.62 (OCH₃), 4.3 (m), 5.00 (m), 5.3 (m), UV (in MeOH)  max 209 nm (no conjugation).

Analytical calculations: Found C, 73.18; H, 12.31; N, 2.05

Calculated for C₄₃ H₆₃ O₆N C, 72.77; H, 11.70; N, 1.97

MS, at m/z 691 (M-H₂ O) this peak is characterized for hydrocarbon alcohols. The predeuteriomethyl derivatives of the compound gave a small molecular ion peak at m/z 792 ().3%) which indicated the perdeuteriomethylation of four hydroxyls and one amino group (5 X CD₃). These data together with NMR results, suggested the presence of a methoxyl group in the molecule.

When the mass spectral data were compared to those given in the literature for other phtiocerols (21), many similarities were observed; for example the strong peaks at m/z 337; 339 (for derivatized compound at m/z 354; 356) indicated that a part of the molecule should be: CH₃- (CH₂)₂₁-CH- Furthermore, the following peaks up to m/z

║

422 show that the following units at m/z 351, 365, 379, 393, 407, and 421 differ successively by 14 mass units, in accord with the cleavage of a (CH₂)n chain. Strong ions for C₁₀ H₃₈ and C₁₀ H₄₀, typical for phtiocerols 921), were also present at m/z 278; 280. The base peak at m/z 43 represents a-CH-CH-group, and a strong peak

║

at m/z 57 (80) indicates a – CH-Ch-CH₂ group. The ion at m/z 60

║

(30%) corresponds to the –CH-CH₂NH₂ group. In the derivatized

║

compounds corresponding peaks were found at m/z 61 and m/z 76. The base peak in ther derivatives is derived from the end group of the molecule, namely –CH-CH₃-N-CD₃ (a), m/z 61 ion being (a-NHCD₃)  

OCD₃H

And the latter corresponding (a-CD₃). The small peak at m/z 663 (M-H₂O-CHNH₂) in the underivatized compound is also indicative for the proposed end group. The double bonds are not conjugated as seen in the Uv spectrum of the molecule. The partial stucture of the molecule is given in Figure1.

CH₃ –(CH₂)₂₁-CH-(CH₂-[C₁₂H₁₈O₄] –CH-CH₂NH₂

 

OH OH

Figure 1

[ 3 double bonds 3 hydroxyl groups 1 methoxyl]

The complete of the new phytosphingosin will further investigation.

ACKNOWLEDGEMENTS

One of the authors (AU) would like to express her gratitude to JSPS for a three months grant to be able to work at the University of Tokyo.

We would like to thank Prof. W. Sucrow for some of the standard samples.

REFERENCES

G. Rivera, Am. J. Pharm., 13, 281 (1941).

J. A. Pons and D. S. Stevenson, Puerto Rico J. Pub. Health and Trop. Med., 19, 196 (1943); C. A. 38, 2117.

G. Rivera, Am. J. Pharm., 114, 72 (1942).

M. M. Lolitkar and M. R. Rajarama Rao, J. Univ. Bombay, 29, 223 (1962); C. A. 58, 9537d.

M. M. Lolitkar and M. R. Rajarama Rao, Indian J. Pharm., 28, 129 (1966).

W. Sucrow, Tetrahedron Lett. 26, 2217 (1965).

P. Khonna and S. Mohan, Indian J. Experimental. Biol., 11, 58 (1973): C. A. 79, 7588h.

W. Sucrow, Chem. Ber. 99, 2765 (1966).

W. Sucrow, ibid, 99, 3359 (1966).

T. Tun and Y. Rin, Japan Kokai 7.667.714 (C.I.A 61 K 35/78); C. A. 85P, 198150e.

F. M. Friese, Apoth, Ztg. 44, (1929); C.A. 24, 684.

J. Lal, S. Chandra, V. Raviprakash, M. Sabir, Ind. J. Phsiol., Pharmacol., 20, 64 (1976); C. A. 85, 104621.

L. R. Diaz, Puerto Rico J. Pub. Health and Trop. Med., 11, 812 (1936); C. A. 30, 6133.

J. W. Airan and N. D. Ghatge, Current Sci. (India)19, 19 (1950); C. A. 44, 5139.

A. A. Olaniyi and V. O. Marquis, J. Pharm (Yaba, Niger.) 6, 117 (1975); C. A. 84, 84360 n.

D .G. Steym, Onderstepoort, J. Vet. Sci. Animal Ind., 9, 107 (1937); C. A. 32, 4279.

A. A. Olaniyi, Lloydia, 38, 361 (1975).

H. E. Carter, W. D. Celmer, W. E. M. Lands, K. L. Mueller and H. H. Tomizawa, J. Biol Chem, 206, 613 (1954).

L. Ahquist, R. Ryhage, E. Stenhagen and E. Von Sydow, Arkiv for Kemi, 14, 211 (1959).

R. Ryhage, S. Stallberg-Stenhagen and E. Stenhagen, ibid, 14, 247 (1959).

R. Ryhage, S. Stallberg-Stenhagen and E. Stenhagen, Ibid, 14, 259 (1959).

J. H. Cardellina and R. E. Moore, Phytochem. 17, 554 (1978).

W. Karrer, Konstitution und Vorkmmen der Organischen Planzenstoffe, Brik Hauser Verlag Basel (1958).

Inhibition of Protein Synthesis in Vitro by a Lectin from Momordica charantia and by Other Haemagllutinins

filipinoweb — Wed, 09 Aug 2006 10:17:49 +0000

1979 Biochem J. 186, pp. 633-635

by Luigi Barbieri, Enzo Lorenzoni, Florenzo Stirpe
Protein synthesis by a rabbit reticulocyte lysate is inhibited by the heamagglunating lectins from Momordica charantia and Crotalaria juncea seeds…

Inhibition of Protein Synthesis in Vitro by a Lectin from Momordica charantia and by Other Haemagllutinins

Author: Luigi Barbieri, Enzo Lorenzoni, Florenzo Stirpe

Type of Publication: Pre-Clinical

Date of Publication: 1979

Publication: Biochem J. 186, pp. 633-635, 1979

Organization: Istituto di Patologia generale dell’ Universita di Bologna

Protein synthesis by a rabbit reticulocyte lysate is inhibited by the heamagglunating lectins from Momordica charantia and Crotalaria juncea seeds and from the role of Rutilus rutilus, and by a commercial preparation of the mitogenic lectin from Phytolacca Americana. The haemagglutinins from the seeds of Ricinus communis and of Vicia cracca aquired inhibitory activity after theor reduction with 2-mercapoethanol.

Lectins are protein or glycoproteins with binding sites for specific carbohydrates groups (Lis and Sharon, 1973; Liener, 1976; Brown & Hunt, 1978), which have interesting biological properties, in that many of them agglutinate erythrocytes or other cells, and some are mitogenic to lymphocytes. Among lectins there are three potent toxins from plants, namely ricin, brin (review by Olsnes & Pihl, 1977) and modeccin Refsnes et al., 1977; Stirpe et al., 1978), which are potent inhibitors of protein synthesis in cells and in cell-free systems.

Various plant material contain proteins which are non-toxic or scarcely toxic to animals and which inhibit protein stnthesis in cell-free systems but not in whole cells, presumably because they cannot enter into cells (Obrig et al., 1973; Irvin, 1975; Stirpe et al., 1977; Gasperi-Campani et al., 1977; Stewart et al., 1977; A. Gasperi-Campani, L. Barbieri, P. Morelli & F. Stirpe, unpublished work). Some these proteins were purified (Obrig et al., 1973; Irvin, 1975) or semi-prurified (Stirpe et al., 1976; Sperti et al., 1976), and these cases it was ascertained that they inhibit protein synthesis through the same mechanism as do ricin and the other toxins mentioned above, i.e. by inactivating enzymatically the 60S ribosomal subunit, and making it unable to bind the elongation factor 2.

Lin et al.(1978) reported two lectins (mol. wts. 32,000 and 24,000) from the seeds of a Cucurbitaceae, Momordica charantia (bitter pear melon), the latter inhibiting proteins synthesis by Ehrlich ascites cells at concentrations relatively high(from 100 g/ml) as compared with the toxic lectins.

We report that a haemagglutinating lectin from the seeds of Momordica charantia is a potent inhibitor of protein synthesis in acell-free system (a lysate a rabbit reticulocytes). Inhibition of protein snthesis in the same system was obtained also with lectins from other seeds from a fish roe.

Experimental

Materials

Seeds of Momordica charantia, originally from India, were obtained from Mr. F. G. Celo, Zweibrucken, West Germany, and the haemagglutinating lectin was purified by affinity chromatography on Sepaharose 4B as described by Tomita et al. (1972) with minor modifications. Ricinus communis agglutinin was prepared as described by Nicolson & Blaustein (1972), and other lectins were obtained from the sources listed in Table1. Other chemicals were from the same sources as described by Gasperi-Campani et al. (1978).

Methods

Protein synthesis was determined as described by Gasperi-Campani et al. (1978) with a lysate of rabbit reticulocytes prepared as described by Allen & Schweet (1962) or with Yoshida AH-130 ascites cells. Protein was determined by the method of Lowry et al. (1951) or spectrophotometrically (Kalb & Bernlohr, 1977).

Results and Discussion

The lectin purified by affinity chromatography from Momordica charantia seeds a single band on a polyacrylamide-gel electrophoresis in either the presence or the absence of sodium dodecyl sulphate, had mol. wt. 115000, and agglutinated human erythrocytes (group ), the lowest active concentration being 24 ng/ml.

This agglutinin inhibited protein synthesis by a lysate of rabbit reticulocytes: the ID₅₀(concentration giving 50% inhibition) was 1.74 g/ml, i.e. similar to those of the toxic lectins mentioned above (e.g. modeccin; see Stirpe et al., 1978). The inhibition was unchanged in the presence of 100 mM-galactose, which inhibits haemagllutination by the lectin, as shown by Tomita et al. (1970) and confirmed with our preparation. This indicates that the inhibitory effect on protein synthesis is independent of the haemagglutinating property of the lectin.

The lectin at 100 g/ml inhibited by 30% protein synthsesis by Yoshida ascites cells: 50 g/ml had no effect. The fact that relatively high concentrations, as compared with the toxic lectins, are required to affect protein synthesis by wgole cells suggests that Momordica charantia lectin enters with difficulty into cells, and this in turn could account for its low toxicity to animals: it did not cause any apparent harm to rats when injected intraperitoneally at the dose of 1 mg/mg 100 g body wt.

Momordica charantia lectin is the second example of non-toxic lectin inhibiting protein synthesis in vitro, after the Ricinus communis agglutinin (distinct from ricin), as shown by Saltedt (1976) and by Cawley et al. (1978). This led us to the hypothesis that other lectins could have same property. Therefore 27 lectins were examined, before and after reduction with 2-mercaptoethanol, a treatment which greatly enhances the inhibitory effects of ricn and abrin (Olsnes & Phil, 1972) and of modeccin (Refsnes et al., 1077; Gasperi-Campani et al., 1978), lectins were inhibitory (Table 1), and among the already known agglutinin from Ricinus communis the Momordica charantia lectin prepared by a recent method (Horejsi & Kocourek, 1978) in and laboratory, and the pokeweed mitogen. The inhibitory actitivity of the latter was observed before (Gasperi-Campani et al., 1977), and was attributed to tamination by the powerful pokeweed and peptide (Irvin, 1975). It was comfirmed that mercial preparations of pokeweed mitogen 0.3% of pokeweed antiviral peptide, which is eficeint to account5 for the inhibition(J. D. personal communication). It is noteworthy the lectin from the roe of the fish Rutilus rutilus was inhibitory. Reduction with 2-mercaptoethanol hanced greatly the effects of the lectins from Ricinus communis and from Vicia cracca, and abolished effect of the agglutinin from Ruticulus ruticulus.

It is not known whether all these lectins protein synthesis through the same mechanism. However, our results demonstrate that the capable og inhibiting protein synthesis is common to lectins, presumably to many othersa besides identified by the present experiments, and thus show considered another general property of lectins, the mitogenic activity.

Acknowledgements

We thank Dr. B. Ersson, Dr. A. Falasca, Dr. L. G. Dr. J. Kocourek, Dr. T. Kurokawa and Dr. F. Cessi for generous gifts of lectins. The research supported by a grant from the Consiglio Nazionale Ricerche, Rome, within the Progetto finalizzato trollo della crescitaneoplastica, and by the Pallotti’s for cancer research.

References

MISSING TEXT

Lin, J. Y., Hou, M. J. & Chen, Y. C. (1978) Toxicon 16, 653, 660.

Lis, H. & Sharon, N. (1973) Annu. Rev. Biochem. 42, 541, 574.

Lowry, O. H., Rosebrough, N. J., farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265, 275.

Nicolson, G. L. & Blaustein, J. (1972) Biochim. Biophys. Acta 266, 543-547.

Obrig, T. G., Irvin, J. D. & Hardesty, B. (1973) Arch. Biochem. Biophys. 155, 278-289.

Olsnes, S. & Pihl, A. (1972) FEBS Lett. 28, 48-50.

Olsnes, S. & Pihl, A. (1972) in Receptors and Recognition, Series B, vol. 1 (Cuatrecasaa, P., ed.), pp. 129-173, Champman and Hall, London.

Refsnes, K., Haylett, T., Sandvig, K. & Olsnes, S. (1977) Biochem. Biophys. Res. Commun. 79, 1176-1183.

Saltvedt, E. (1976) Biochim Biophys. Acta 451, 536-548.

Serafini-Cessi, F., Franceschi, C. & Sperti, S. (1979) Biochem. J. in the press.

Sperti, S., Montanaro, L., Mattioli, A., Testoni, G. & Stirpe, F. (1976) Biochem. J. 156, 7-13.

Stewart, T. S., hruby, D. E., Sharma, O. K. & Roberts, W. K. (1977) Biochim Biophys. Acta 479, 31-38.

Stirpe, F., Pession-Brizzi, A., Lorenzoni, E., Strocchi, P., Montanaro, l. & Sperti, S. (1976) Biochem. J. 156, 1-6.

Stirpe, F., Gasperi-Campani, A., Barbieri, L., Lorenzoni, E., Montanaro, L., Sperti, S. & Bonetti, E. (1978) FEBS Lett. 85, 65-67.

Tomita, M., Osawa, T., Sakurai, Y. & Ukita, T. (1970) Int. J. Cancer 6, 283-289.

Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N., Osawa, T. & Ukita, T. (1972) Experientia 28, 84-85.

Studies on the Constituents of Momordica charantia L. I. Isolation and Characterization of Momordicosides A and B, Glycosides of a Pentahydroxyl-Cucurbitane Triterpene

filipinoweb — Wed, 09 Aug 2006 10:02:22 +0000

1980 Chem. Pharm. Bull. Vol.28, 2753-2762

by Hikaru Okabe, Yumi Miyahara, Tatsuo Yamauchi, Kazumoto Miyahara, Toshio Kawasaki
The fruit of Momordica charantia L.…

Studies on the Constituents of Momordica charantia L. I. Isolation and Characterization of Momordicosides A and B, Glycosides of a Pentahydroxyl-Cucurbitane Triterpene

Author: Hikaru Okabe, Yumi Miyahara, Tatsuo Yamauchi, Kazumoto Miyahara, Toshio Kawasaki

Type of Publication: Pre-Clinical

Date of Publication: 1980

Publication: Chem. Pharm. Bull. Vol.28, 2753-2762, 1980

Organization: Fukuoka University and Kyushi University

Abstract: Two triterpene glycosides, momodicosides A and B, were isolated from the seeds of Momordica charantia L. (Cucurbitaceae). Their stucture were determined on the basis of spectral and chemical evidence and by X-ray analysis as the 3-O--gentiobioside and 3-O--D-xylopyranosyal (1-4)-[ -D-glucopyranosyl(1-6)] --D-glucopyranoside, respectively, of cucurbit-5-ene-3-, 22(S), 23(R), 24(R), 25-pentaol

The fruit of Momordica charantia L. (cucurbitaceae) (Niga-uri or Tsuru-reishi in Japanese) has been used as bitter stomachic southern Japan, and as a laxative and an anthelmintic for children in India.³⁾ An antidiabetic effect of its alcohol extract was also reported.⁴⁾As a first in chemical studies of the bitter principles of the fruit, the seed was investigated. This paper deals with the isolation and stucture elucidation of two kinds of oligoglycosides of a cucurbitane derivative, named momordicosides A (I) and B (II).

The defatted methanol extractive of the seeds was partitioned between butanol and water and the latter layer, containing vicine (III),⁵⁾ was discarded. The butanol layer was subjected to successive column chromatographies and two compounds, I, C₄₂H₇₂O₁₅2H₂O_, mp 181-187 (dec.), []_D + 1.05⁰, and II, C₄₇H₈₀O₁₉. 3/2H₂O, mp 238-242⁰ (dec.), []_D + 6.15⁰, were isolated.

On mild methanolysis, I gave methyl glucoside and an aglycone (IV), while II furnished a product identical with I, methyl xyloside and glucoside, and IV, indicating II to be a xyloside of I. IV was assumed to retain its stucture in glucoside I and II, sine IV was also obtained on enzymatic hydrolysis of I, and the carbon –13 nuclear magnetic resonance (CMR) spectrum of IV was almost identical with those of I and II, except for the signals due to sugar moieties and those of the aglycone carbons affected by the glycoside linkage.

IV was formulated as C₃₀H₅₂O₅based on the elementary analysis results and the molecular ion peak (M+) observed at m/z 492 in the field desorption mass spectrum (FD-MS). The proton nuclear magnetic resonance (PMR) of IV exhibited signals of seven tertiary (0.96 (x2), 0.94, 1.12, 1.40, 1.67 and 1.75 ppm) and one secondary (1:43 ppm) methyl groups along with four methines bearing hydroxyl groups (J= ca 4 Hz). The presence of four secondary and one tertiary hydroxyl groups was seen in the CMR spectrum of IV as five peaks between 70 and 76 ppm, four of which were transformed to doublets by off-resonance decoupling measurement, while one remained as a singlet. In the rgion from 34.8 to 49.2 ppm, four quarternary carbons signals were observed, and olfinic carbons were found at 143.0 (singlet) and 119.1 ppm (doublet).

Its molecular formula and the presence of one double bond, seven tertiary methyl group and four C-C-bonded and one hydroxylated quaternary carbons, as described above, suggested IV to be a tetracyclic triterpenoid. The PMR and CMR patterns due to a proton and carbon-of a double bond, respectively, were similar to those of cucurbit-5-ene derivatives,⁶ and the EI-MS spectrum of IV showed peaks at m/z 152 and 340 which could be considered to be provided by the typical retro-Diels-Adler fragmentation of ring B of a cucurbit-5-ene derivative having hydroxyl group in ring A.^6a,7)

IV was acetylated with Ac₂O and pyridine at room temperature to give a tetraacetate (V) and at 90⁰ to give a pentaacetate (VI). The hudroxyl group acylable only on heating was tentatively located at C-25 of the cucurbitane nucleus, and the two singlets at 1.67 and 1.75 ppm in the PMR spectrum of IV were assigned to the 26-and-27-methyl groups.

The finding that IV afforded mono-(VII) and di- (VIII) acetonides, and the coupling patterns ( 3.83 (d, J = 9 Hz),  4.10 (dd, J = 7, 9 Hz) and  4.36 (d, J = 7 hz) of three protons on three carbinyl carbons of VIII, suggested that two glycol system were adjacent to each other. When the monoacetonide (VII) was subjected to periodate oxidation, an aldehyde IX) was obtained. An FD-MS spectrum of IX showed the (M + H)^ion peak at m/z 472, indicating that bond fission in VII took place between C-24 and 25. On the other hand, when IV was treated with periodic acid in methanol, the bond between C-22 and –23 was cleaved ti give mixture of an aldehyde (X) and its dimethyl acetal (IX) which retained one secondary hydroxyl group. Therefore, four hydroxyl groups were thought to be located at C-22, -23, 24 and –25.

The remaining one hydroxyl group was assumed to be at C-3, and this was confirmed by converting XI to an oxo derivative (XII), and further to the corresponding 2-hydroxy-1-n-3-one (XIII); the PMR spectrum of the latter showed the proton at C-1 at 6.12 ppm as a doublet (J = 3 Hz), also supporting the presence of a proton at C-10.

Consequently, IV was presumed to be a cucurbit-5-ene-3, 22, 23, 24, 25-pentaol. In order to confirm the cucurbitane framework and to determine the configurations of substituents, X-ray analysis of IV was carried out by the direct method. The final atomic parameters, bond lengths and bond agles for nonhydrogen atoms are given in Tables I, II and III, respectively, and an ORTEP drawing is shown in Fig.1.

The aglycone was thus identified as cucurbit-5-ene-3,22(S), 23(R), 24(R), 25(R)-pentaol (IV) or its enantiomer (IV’).

The CD spectrum of XII showed a negative Cotton curve ([0]₂₉₄ – 8030⁰) indicating the presence of a 10-hydrogen atom and a 5,6-double bond.⁸⁰

Thus, the absolute structure was established as IV.

Momordicoside A (I) showed the (M + Na)^- peak at m/z 839 in the FD-MS spectrum, and the anomeric carbon signals at 105.2 and 106.9 ppm in the CMR spectrum. The EI-MS spectrum of its acetate (XIV) showed a fragment peak (m/z 619) due to an acetylated biose moiety. These data suggest that the sugar moiety of I is composed of two moles of glucose. The signals in the CMR spectrum ascribed to the sugar carbons were quite similar to those of pregnenolone gentioside.¹⁰⁾

When I was methylatede in tetrahydrofuran with NaH and CH₃I, two methylates (XV and XVI) were obtained. Methanolysis of their XV or XVI furnished equal amounts of methyl pyranosides of 2, 3, 4, 6-tetra-O-methyl glucose and 2, 3, 4-tri-O-methyl glucose. The linkage between the two glucose units as well as that to the aglycone were regarded as  in view of the coupling constants (ca. 7 Hz) of the two anomeric protons in the PMR spectrum of XV> The aglycones provided by methanolysis of XV and XVI were 25, 26-anhydro- (XVII), 22, 25-anhydro (XVIII) and 22, 23, 24, 25-tetra-o-methyl (XIX) derivative from the former, and XVII, XVIII and 2, 23, 24,-tri-o-methyl ether (XX) from the latter. Thus, XV is the permethylate (missing text), while XVI has a free hydroxyl group at C-25.

Sugar linkage with the hydroxyl group at C-3 in I was confirmed by its CMR spectrum in which a downfield shift of the C-3 signal compared with that in IV was observed, and by the result that XIX was affordable by methanolysis of XV.

Accordingly, I was concluded to be the 3-O--gentiobioside of IV.

On comparison of the FD-MS spectra and the molecular formulae of I and II, the sugar moiety of the latter appeared to be composed of two moles of glucose and one of xylose. The EI-Ms spectrum of II-acetate (XXI) showed peaks at m/z 331 and 259, corresponding to the pyronium ions originated from the terminal acetylated glucose and xylase residues, indicating a branched chain trisaccharide structure. II-Permethylate (XXIIa) was subsequently methanolyzed and the resulting methylated monosaccharide were identified by GLC as methyl pyranosides of 2, 3,4-tri-O-methyl xylose, 2, 3, 4, 6-tetra-O-methyl glucose and 2, 3,-di-O-methyl glucose. Since I was formed by partial hydrolysis of II, the xylose unit II should be linked to the hydroxyl group at C-4 of the glucose combined with the aglycone. The mode of linkage of the xylose unit was determined as  judging from the coupling constant (6 Hz) of its anomeric proton signal in the PMR spectrum of XXIIa.

Thus, II is the 3-O--D-xylopyranosyl (1-4)-[-D-glucopyranosyl (1-6)]- -D-glucopyranoside of IV.

More than thirty cucurbitane derivatives have so far been isolated from plants, mainly of Cucurbitaceae and also of Primulaceae,¹¹⁾ Cruciferae,¹²⁾ Begoniaceae¹³⁾ and Datiscaceae.¹⁴ They are, in general, highly oxygenated and the oxygen functions are distributed in the nucleus and side chain (C-3 and C-11 being invariably oxygenated).

The aglycone (IV of momordicosides A (I) and B (II) is also typical cucurbitane derivative, but is unusual in that four of the five hydroxyl groups are located in the side chain and in that there is no oxygen function at C-11.

Experimental¹⁵⁾

Extraction and Isolation of I, II and III – Seeds (3.34 kg) of Momordica charantia L. were crushed and percolated successively with MeOH (201) and MeOH-H₂O (1:1) (201). The MeOH solution was concentrated under reduced pressure to 1/10 volume, repeatedly extracted with hexane to remove lipids. The MeOH solution was concentrated to dryness, suspended in water and extracted with BuOH. The BuOH solution was concentrated to dryness to give a light brown powder (55 g). This was repeatedly chromatographed on silica gel (50-100 times the weight of material) using CHCI₃-MeOH-H₂O (70:25:3) as an eluent to give (4.3 g) and II (0.3 g).

I: colorless needles from MeOH, mp 181-187⁰ (dec.), []_D²⁰ + 1.05⁰ (c=0.96, CHCI₃-MeOH (2:1), FD-MS m/z: 839 (M + Na) ^, 855 (M + K)⁺. Anal. Calcd for C₄₂H_72O15_.2H₂O: C, 59.13; H, 8.98. Found: 58.95; h, 8.85. CMr: olefinic carbons: 14.2 (s), 118.66(d) , 72.41.63(d), 71.19, 62.72(t); quaternary carbons: 49.12, 4678, 41.66, 34.70.

II: colorless needles from MeOH-CHCI₃, mp 238-242⁰ (dec.), [a] _D²⁰ + 6.15⁰ (c= 0.98, CHCI₃-MeOH 2:1). FD-MS m/z: 971 (M + Na)⁺. Anal Cald for C₄₇H₈₀O₁₉. 3/2H₂O: C, 57.83; H, 8.57. Found: C, 57.60; H, 8.43. CMR: olefinic carbons: 142.85(s), 118.48(d); oxygen-bearing carbons: 106.58(d), 104.82(d), 87.19(d), 79.98(d), 79.51(d), 76.11. 74.94, 74.71, 74.24(s), 72.25, 71.25, 71.48, 71.48, 71.07, 71.07, 70.66,68.83, 67.38, 62.64(t); quaternary carbons: 49.04, 46.70, 41.60, 34.63.

The aqueous layer from the BuOH-H₂O extraction of the defatted NeOH extractives and the MeOH H₂O extract were combined and concentrated. On standing at room temperature colorless needles (11 g) were separated out, and these were recrystallized from water to give III: colorless, mp 250-255⁰ (decrease.), [_D²⁵ – 13.5⁰ (c= 2.15, 0.1% NaOH). Anal. Cald for C₁₀H₁₆N₄.1/2H₂O: C, 38.33; H, 5.43; N, 17.89. Found: C, 38.66; H, 5.47; N, 17.95. UV _max^{0.5 NHCI} nm (): 273 (15800), _max^H2O nm (): 275 (12700), 210 (20400), _max^{0.1 NnaOH} nm (): 268 (9700), 235 (5624), 217 98500). CMR (1_N KOH): base moiety, 170.3, 160.9, 158.8, 118.6; sugar moiety, 108.0 (1’), 78.6 (3’), 77.9 (5’), 74.7 (2’), 71.5 (4’), 62.6 (4’), 62.6 (6’). IR (KBr): superimposable on the spectrum of vicine¹⁶ isolated from faba beans beans (Vicia faba L.) according to the method at Lin et a.¹⁷

Methanolysis of I, Identification of the Component Sugar and Isolation of the Aglycone (IV) – A solution :1 (610 mg) in 1_N HCI-MeOH (20 ml) was stirred for 5 days at room temperature then neutralized with Ag₂CO_3.The precipitates were filtered off and the filtrate was evaporated to dryness under reduced pressure. The residue was chromatographed on silica gel (160 g). Elution with 3% MeOH-CHI₃ gave an aglycone 223 mg), which was crystallized from MeOH to give colorless needles (IV): mp 193-195⁰ []_D²⁰ + 48.1⁰ 0.94, CHCI₃-MeOH (2:1). FD-MS m/z 492 (M^). Anal Cald for C₃₀H₅₂O₅.1/2H₂O: C, 71.86; H, 19.58. Found: C, 72.04; H, 10.79. PMR: .90 (6H, s, C-CH₃x2), 0.94 (3H,s, C-CH₃), 1.40 (3H,s, C-CH₃), 1.43 (3H, d, J= 6Hz, >CH-CH₃), 1.67, 1.75 (3H each, s, HO-C(CH₃)₂), 3.74 (h, br, s,>CH-OH), 4.07 (H, d, J= 9 Hz, -CH (OH)-CH(OH)-), 4.37 (H, d, J= 9 Hz, - CH(OH)-CH-H)-), 4.57 (H, d, J= 4 Hz,>CH-OH), 5.61(H, br d, J= 4 Hz, >C=CH-). CMR: olefinic carbons: 143.02 s, 119.12(d); oxygen-bearing carbons: 75.94(d), 75.23(d), 74.30(s), 72.30(d), 71.13(d); quartenary carbons: 19.16, 46.82, 41.60. EI-MS m/z: 492 (M^), 474 (M^-H₂O), 340, 322, 177, 159, 163, 152, 134, 59.

Elution with 25% MeOH-CHI₃gave resinous glycoside (160 mg) ([] _D²⁵ + 93.1⁰ (c= 1.23, MeOh)). Its PMR spectrum exhibited two methoxyl proton signals at 3.46 and 3.61 in ration of 2:1 and anomeric proton signals at 4.73 (d, J= 7 Hz) and 5.17 ppm (d, J= 4 Hz) (methoxyl proton signals: methyl -_D-glucoside, 3.46: -anomer, 3.61 ppm. Anomeric proton signals: methyl -_D-glucopyranoside, 5.17 (d, J = 4 Hz) ; -anomer, 4.73 ppm (d, J= & Hz)). The methyl glycoside was acetylated in the usual manner and examined by GLC.¹⁸⁾ It gave two peaks with retention times (4.6 and 5.2 min) identical with those of methyl 2,3,4,6tetra-O-acetyl--_D-glucopyranoside and its

- anomer.

Enzymatic Hydrolysis of I- I (80 mg) was suspended in water (8 ml) and cellulose (Type I, Sigma Chemical Co.) ( 80 mg) was added. The mixture was shaken for a week at 38⁰. The reaction mixture was extracted with CHCI₃ and then with BuOH. Both extracts were combined and chromatorgraph on silica gel (15 g). The fraction eluted witg 3% MeOH-CHCI₃ gave the aglycone (33 mg) as colorless needles (crystallized from MeOH), identical with IV (IR and mixed mp).

Methanolysis of II, Identification of the Component Sugars and isolation of Momordicoside A (I) and the Aglycone (IV) – II (660 mg) was dissolved in 1 N HCI-MeOH (20 ml) and stirred fo 5 hour at room temperature. The reaction mixture was neutralized with Ag₂CO₃ and worked up in the same way as for I. The product was treated with 15% acetone- H₂O and the insoluble material (550 mg) was chromatographed on silica gel (70 g), using CHCI₃-MeOH-H₂O (70:25:3) as an eluent to give Fr. 1 (143 mg), Fr.2 (170 mg) and Fr. (170 mg). Fr. 1 was further chromatographed on silica gel (30 g) (eluent, 3% MeOH-CHCI₃) to provide a thin- layer-chromatographically homogeneous compound, which was crystallized from MeOH to give colorless needles (46 mg): mp 192-195⁰. The IR spectrum was superimposable on that of IV and the melting point was not depressed on admixture with IV. Fr. 2 was crystallized from MeOH-H₂O to give colorless needles (94 mg): mp 180-188⁰ (dec.). This product gave the same IR and CMR spectral as I. Fr. 3 gave II on recystallization. The 15% acetone-soluble substance 9122 mg) showed two spots on TLC and their Rf values were identical with those of methyl -_D-xylopyranoside (Rf: 0.46, CHCI₃-MeOH-H₂O (70:35:5) and methyl -_D-glucopyranoside (Rf: 0.32)_.This mixture was subjected to column chromatography (silica gel, 11 g; CHCI₃-MeOH-H₂O (70:25:3) to give two methyl glycoside. The less polar one (27 mg) showed [] _D²⁵⁰ + 127.6⁰ (c= 1.34, MeOH)¹⁹⁾ and its PMR spectrum exhibited methoxyl proton signals at 3.49 and 3.58 ppm (ration 3:1). It was acetylated and subjected to GLC, ¹⁸⁾ giving two peaks with t_R 1.60 and 1.80 min (methyl -_D-xylopyranoside triacetate, t_R 1.55; -anomer, 1.80). In the same way, the polar methyl glycoside was identified as a mixture of methyl -_D-glycopyranoside and its -anomer.

Acetylation of IV- i) IV (20 mg) was dissolved in 0.6 ml of Ac₂O-pyridine (1:1) and the solution was stirred for 15 hour at room temperature. The solution was then evaporated to dryness and the residue was chromatographed on silica gel (6 g) (4% acetone-benzene) to give a tetraacetate (V) (7 mg): fine needles (from acetone- benzene), mp 178-182⁰. PMR: 1.48 (6H, s, HO-C (CH₃)₂), 2.00 (3H, s, -Oac), 2.12 (6H, s, -OAc x2), 2.18 (3H, s, -OAc), 4.91 (H, perturbed t, >CH-OAc), 5.46 (H, d, J= 7 Hz, > CH-OAc), 5.49 (H, m, >C=CH-), 5.71 (H, br s, >CH-OAc), 6.12 (H, d, J= 7 Hz, >CH-OAc). CMR: olefinic carbons: 141.95(s), 119.44(d); oxygen-bearing carbons: 78.85(d), 76.65(d), 73.29(d), 71.58(s), 69.15(d); quaternary carbons: 49.12, 46.93, 39.91, 34.74; acetyl carbonyl carbons: 170.65, 170.65, 170.56, 170.36, 170.17. EI-MS m/z: 660 (M^), 600 (M^-AcOH), 466, 406, 194, 163, 134. ii) IV (50 mg) in 1 ml of Ac₂O-pyridine (1:1) was heated at 90⁰ for 6 hour. The solvent was removed and the residue was chromatograhed on silica gel 920 g) (2.5% acetone-benzene) to give a pentaacetate (VI) (31 mg): colorless needles (from MEOH), mp 104-107⁰. Anal. Calcd for C₄₀H₆₂O₁₀.H₂O: C, 66.62; H, 8.95. Found: C, 66.50; H, 8.73. PMR: 1.65, 1.69 (3H each, s, AcO-C(CH₃)₂), 2.04 (6H, s, -OAc x2 ), 2.16 (3H, s, -OAc), 2.18 (3H, s, -OAc), 2.21 (3H, s, -OAc), 4.92 (br s, >CH-OAc, partially overlapping with an H₂O signal),5.54 (2H, br s, >C-CH-and>CH-OAc), 5.81 (H, d, J= 8 Hz,> CH-OAc), 6.01 (H, d, J= 8 Hz,>CH_OAc). CMR: olefinic carbons 141.68(s), 119.18(d); oxygen-bearing carbons: 81.97(s), 78.63(d), 73.42(d), 72.719d), 68.61(d); quartenary carbons: 49.04, 46.87, 39.79, 34.69; acetyl carbonyl carbons: 170.16, 169.98, 169.51. EI-MS m/z: 702 (M^), 641 (M^), 642 (M^-AcOH), 508, 448, 163, 134.

Formation of Mono- (VII) and Diacetonides (VIII)- A mixture of IV (60 mg) and anhydrous CuSO₄ (160 mg) in anhydrous acetone (2.3 ml) was stirred at room temperature for 19 hour. The mixture diluted with CHCI₃ and filtered, and the filtrate was concentrated. The residue (53 mg) was repeatedly chromatographed on silica gel (200 times the weight of material), using 1% MeOH-CHCI₃ as an eluent, to give diacetonide (VIII) (7 mg) and monoacetonide (VII) (41 mg). VIII: colorless needles (from MeOH), mp 208-209⁰. PMR: 3.75 (H, br s, >CH-OH), 3.83 (h, d, J= 9 Hz), 4.10 (H, dd, J= 7, 9 Hz), 4.38 (H, d, J = 7Hz), 5.69 (br d, J= 5 Hz)>C=CH-). EI-MS m/z: 572 (M^), 557 (M^-CH₃), 554 (M^-H₂O), 443, 420 (M^-152), 405 (M^-CH₃-152), 163, 152, 134. VII: colorless needles (acetone-benzene), mp 176-178⁰. Anal. Calcd fro C₃₃H₅₆O₅.3/2H₂O: C, 70.80; H, 10.62. Found: C, 70.68; H, 10.25. PMR: 3.74 (H, br s. >CH-OH), 3.83 (H,d, j= 9 Hz), 4.32 (H, dd, J= 6,9 Hz), 4.61(H, d, J= 6 Hz), 5.61(H, d, J=4 Hz). CMR: olefinic carbons: 142.93(s), 119.00(d); oxygen-bearing carbons: 107.62(s), 84.77(d), 79.03(d), 77.47(d), 75.82(d), 73.05(s); quartenary carbons: 49.22, 46.69, 41.53, 34.72. EI-MS m/z: 532 (M^), 517 (M^-CH₃), 443, 380 (M^-152), 362 (380-H₂O), 163, 152, 134.

Periodate Oxidation of VII- VII (1.8 mg) was dissolved in o.1 ml of MeOH, and 10% NaIO₄ aqueous solution (10 l) was added. The mixture was stirred in the dark for 40 hour, then diluted with water. The precipitate were collected by centrifugation and recrystallized from MeOH to give colorless needles (IX: mp 109-115⁰. FD-MS m/z: 473 (M+H)^. Calcd for C₃₀H₄₈O₄: 472.

Periodic Acid Oxidation of IV- IV (261 mg) in MeOH (16 ml) was mixed 25% HIO₄ aqueous solution (1.6 ml) and the whole was stirred in the dark for 2 days. Precipitates were centrifuged off and the supernatant was diluted with water then extracted with CHCI₃. The precipitates and CHCI₃ extract were combined and chromatographed on a silica gel (100 g) column, eluting 12% AcOEt-hexane, to give an aldehyde (X) (32 mg) and a dimethyl acetal (XI) (150 mg). X: colorless needles (MeOH), mp 156-159⁰. PMR: 3.74 (h, br s,>CH-OH), 5.64 (H, br s, J= 6 Hz,>C=CH-), 9.70 (H, D, J= 3 Hz, >CH-CHO. CMR: an aldehyde carbon: 204.49(d); olefinic carbons: 142.81(s), 118.95(d); oxygen-bearing carbon: 75.82 (d), quartenary carbons: 48.87, 46.93, 41.45, 34.86. EI-MS m/z: 372 (M^), 357 (M^-CH₃), 354 (M^-H₂O), 339, (354-H₂O), 220 (M^-152), 205 (220-CH₃), 152, 134. XI: colorless needles (MeOH), mp 155-157⁰. Anal. Calcd fro C₂₇H₄₆O₃.H₂O: C, 74.26; H, 11.08. Found: C, 74.35; H, 11.03. PMR: 3.37, 3.45 (3H each, s, -CH (OCH₃)₂), 3.72 (H, br s,>CH-OH), 4.22 (H, d, J= 1.5 Hz,>CH-CH(OCH₃)₂), 5.64 (H, br d, J=5 Hz.>C=CH-). CMR: olefinic carbons: 143.09(s), 119.00(d); oxygen-bearing carbons: 109.34(d), 75.88(d), 56.72(q), 55.61(q); quartenary carbons: 49.10, 46.58, 41.60, 34.80. EI-MS m/z: 418 (M^), 386 (M^-MeOH), 368 (386-H₂O), 266 ( M^-152), 234 (266-MeOH), 152, 134, 75 (-CH(Ome)₂^).

Oxidation of Dimethyl Acetal (XI) with CrO₃- XI (160 mg) was added to 3% Cro₃ pyridine solution (5.6 ml) and the whole was stirred at room temperature for 20 hour. CHCI₃ (24 ml) was then added to the reaction mixture, and precipitates were collected by filtration and washed with CHCI₃. The filtrate and washing were combined and shaken with water. The CHCI₃ layer was dried with anhydrous Na₂SO₄ and concentrated. The residue was chromatographed on silica gel (90 g), using CHCI₃ as an eluent, to give a ketone 980 mg), which was crystallized from MeOH to afford colorless plates (XII): mp 158-160⁰. PMR (missing text) 3.48 (3H each, s, -CH(Ome)₂), 4.24 (H, d, J= 3 Hz, -CH(Ome)₂), 5.64 (H, d, J= 6 Hz,>C=CH-). CMR: carbonyl carbon: 212.40; olefinic carbons: 142.91(s), 119.88; dimethyl acetal carbons: 109.22(d), (missing text), 55.61(q); quartenary carbons: 50.86, 48.93, 46.46, 46.46, 35.04. EI-MS m/z: 416 (M^), 384 (M^-MeOH), (missing text) 384-OCH₃), 313, 266, 234, 219, 163, 150, 75. CD (c= 0.43 x 10^-3 g.ml, dioxane) [0]²⁰⁰ (nm): 0⁰ (326), 4530⁰ (313), -7740⁰ (303), -8030⁰ (294), -1930⁰ (265), 0⁰ (244).

Air Oxidation of XII, Formation of Diosphenol (XIII) – XII (30 mg) was dissolved in 1 _N tert-BuOK, tert-BuOH (2 ml), and oxygen gas was bubbled through the solution. The mixture was stirred at room temperature until the spot of XII disappeared on TLC. After neutralization with 1 _NHCI-MeOH, and evaporation to dryness in vacuo, the residue was treated with CHCI₃. The soluble was chromatographed on silica gel (8 g) (eluent, 10% AcOEt-hexane) to give a diosphenol (XIII) (6.5 mg), which was crystallized from hexane containing s mall amount of acetone to give colorless needles, mp 173-176⁰. CHCI₃ test in EtOH: brown. PMR (CDCI₃): 3.39, 3.44 (3H each, s, -CH (Ome)₂), 4.14 (H, d, J= 2 Hz, -CH-Ome)₂), 5.70 (H, m,>C=CH-), 5.86 (H, s, echangeable with D₂O, C₂-OH), (H, d, J= 3 Hz, C₁-H). EI-MS m/z: 430 (M^), 398 (M^-MeOH), 366 (398-MeOH), 266, 234 (266-MeOH), 164, 163, 75. UV_max^DIOXANEnm (): 212 (6290), 271 97320). CD (c=0.17 x 10^-3 g/ml, dioxane) [0]²¹⁰(nm): 0⁰ (370), -2300⁰ (360), -8100⁰ (350), -21000⁰ (330), -25300⁰ (321), -22300⁰ (313), 0⁰ (289).

X-Ray Analysis of IV- Crystal data: C₃₀O₅.H₂O (M.W.=510.762), monoclinic (from CH₃CN-H₂O), space group P2₁, =14.697(3) A, b= 7.742(2) A, c= 13.053(3) A, =99.41(2)⁰, V= 1465.3(6) A³, Z=2, D_calcd=1.16 g/cm³.

A crystal with approximate dimensions of 0.1×0.2×0.35 mm was mounted on a SYNTEX P₁ fully automated four-circle diffractometer, and the lattice constants were derived by a least-squares fitting of 15 reflections. The intensities of all 3621 unique reflections having 20 ≤ 55⁰ were measured using graphite-monochromated Mo K radiation (=0.71069 A) and the 20-0 scan technique. After correction by the usual borentz and polarization factors, the intensities were converted to normalized structure factors. The phases were assigned to the 270 largest E-valuye (E 1.7) by a multisolution, weighted tangent formula approach.²⁰⁰ The E-synthesis from the phase set having the least R-value showed 33 plausible nonhydrogen atoms. The complete structure (les hydrogens and with one molecule of water) was obtained by successive D-Fourier synthesis. Hydrogen atoms other than five hydroxyl protons were located on the basis of geometrical considerations.

Block-diagonal least refinement with anisotropic temperature factors for nonhydrogen atoms and isotropic temperature factors for hydrogen reduced the R-factor to the final value of 0.059 for the 2180 observed reflections (I>2.3 _I).

An ORTEP drawing of the structure, the final atomic parameters, bond lengths, and bond angles for nonhydrogen atoms are shown in Fig.1 and Tables I, II and III, respectively.

All the calculations were performed on a FACOM M-190 computer at the Computer Center of Kyushu University using the UNICS II²¹⁾ MUNTAN²⁰⁾programs.

Acetylation of I- A solution of I (104 mg) in Ac₂O-pyridine (1:1) (2 ml) was heated on a boiling water bath for 16 hour. The mixture was then evaporated down under an air stream , and the residue (138 mg) was chromatographed on a silica gel (40 g) column, eluting with benzene-acetone mixture (4% acetone20%), to give two compounds. The less polar one (55 mg) was crystallized from EtOH to give colorless needles XIV): mp 148-152⁰. CMR: olfinic carbons: 142.09(s), 118.83(d); oxygen-bearing carbons: 101.98(d), 191.02(d), 86.66(d), 81.97(s), 73.77, 73.42, 72.71, 72.07, 71.78, 69.67, 68.91, 68.61, 62.61(t); quaternary carbons: 49.04, 46.87, 41.19, 34.80; acetyl carbonyl carbons were omitted. EI-MS m/z: 619, 331, 271 (331-AcOH), 582, 448, 134.

Methylation of I – A mixture of I (600 mg) in freshly distilled anhydrous tetrahydrofuran (7 ml) and sodium hydride (350 mg) was sonicated for 10 min. CH₃I (7 ml) was added to the mixture amd the whole altrate and washing were combined and concentrated under reduced pressure to give a thick syrup (818 mg). Chromatography on silica gel 9125 g) using 15% acetone-benzene gave two compounds: XV (196 mg) and methyl groups at C-25 (XV, 1.30 ppm; XVI, 1.46 ppm) and an additional OCH₃ at 3.26 ppm in XV.

Methanolysis of XV and XVI, Identification of Methylated Sugars and Isolation of XVII, XVII, XIX and XX – Xv 9208 mg) was dissolved in 1 _NHCI-MeOH (3 ml) and refluxed for 5 hour. After neutralization with Ag₂CO₃, the precipitates were filtered off. The filtrate was concentrated in vacuo and the residue was chromatographed on silica gel (hexane-AcOEt (5:1)) to give two fractions (Fr. 1, 61 mg; Fr. 2, 30 mg). Further elution with AcOEt gave a mixture of methylated sugars, which was examined by GLC. The results are summarized in Table IV. Fr. 2 (XIX) showed a single spot on TLC, but could be crystallized. The PMR spectrum showed signals of a hydroxymethine group at  3.74 (C₃-H, br. s), four OCH₃ (3.27, 2.42, 3.48 and 3.58 ppm) and two groups at C-25 at 1.30 ppm (6H, s).

XVI (171 mg) was methanolyzed and worked up in the manner described above to give two fractions (Fr. 1’, 22 mg; Fr. 2’, 38 mg) and a mixture of methylated sugars, which gave a GLC chromatogram identical with that of the methylated sugars from XV. Fr. 2’ was crystallized from MeOH to give colorless nedles (XX) (15 mg): mp 164-166⁰. The PMR spectrum exhibited signals of three OCH₃ groups (3.42, 3.58 and 3.64 ppm) and a HO-C(CH₃)₂ group at 1.49 ppm (6H, s). Fr. 1’ showed the same IR spectrum as Fr. 1, and both fractions were combined and chromatographed on silica gel, using 10% AcOEt-hexane as an eluent, to give two thin-layer-chromatographed homogeneous compounds, XVII (28 mg) and XVIII (22.7 mg). XVII was crystallized from MeOH to provide colorless prisms, mp 198-200⁰. PMR: 1.84 (3H, s, CH₃-C-CH₂), 3.26, 3.43, 3.47, (3h each, s, -OCH₃), 5.17 (2H, perturbed d, CH₃-C=CH₂). CMR: olefinic carbons: 143.56(s), 143.31(s), 119.29(d), 116.08(t); oxygen-bearing carbons: 84.41(d), 81.28(d), 80.80(d), 75.97(d), 58.57(q), 57.70(q), 55.70(q); quartenary carbons: 49.17, 47.07, 41.62, 34.84. XVIII was crystallized from MeOH to give colorless needles, mp 190-193.5⁰. PMR: 3.44, 3.58 (3H each, s, -OCH₃). CMR: olefinic carbons: 143.31(s), 119.39(d); oxygen-bearing carbons: 90.64(d), 81.72(d), 81.38(d), 79.48(s), 76.02(d), 59.94(q), 59.11(q); quaternary carbons: 48.93, 47.41, 41.71, 34.89. EI-MS m/z: 502 (M^-CH₃) 350 (M^-152), 152, 134.

Acetylation of II – A solution of II (19 mg) in Ac₂O-pyridine (1:1) (0.5 ml) was heated on a boiling water bath for 25 hour. The reaction mixture was poured into ice-water, then the precipitates were collected by filtration, dried, and recrystallized from EtOH to give colorless fine needles (XXI), mp 133-140⁰. EI- Ms m/z: 331, 271 (331-AcOH), 259, 199 (259-AcOH), 582, 508, 448, 134.

Methylation of II – II (320 mg) was methylated in the manner described for the methylated of I to give two methylates XXIIa (94 mg), olorless needles (MeOH), mp 178-180⁰ and XXIIb (67 mg), colorless needles (MeOH), mp 167-168.5⁰. They exhibited PMR spectra similar (differing only in the number of methoxyl signals) to those of XV and XVI, respectively.

Methanolysis of XXIIa, Identification of Methylated Sugars – A solution of XXIIa (3 mg) in 1 _N HCI-MeOH (0.1 ml) was refluxed for 1 hour. The reaction mixture was worked up as usual. The aglycone was identified as a mixture of XVII, XVIII and XIX (TLC). The methylated sugars obtained were examined by GLC, and the results are shown in Table IV.

Acknowledgements. The authors are grateful to Professor I. Ueda and Associate Professor S. Kono, College of General Education, and Associate Professor T. Komori, Faculty of Pharmaceutical Sciences, Kyushu University, for providing computer programs and helpful discussions. They are also indebted to Dr. T. Nohara of Tokushima University and Mr. K. Mihashi of Fukuoka University for valuable suggestions and discussions. Thanks are also due to Mr. Y. Tanaka and Miss M. Kawamura of Kyushu University, to Mr. M. Nishi of Fukuoka University for PMR, CMR and MS measurements, and to Mr. S. Hara of Fukuoka University for elementary analyses.

References

A part of this work was presented at the 99^th Annual Meeting of the Pharmaceutical Society of Japan, Saporo, August 1979.
Location: a) Nanakuma, Nishi-ku, Fuluoka; b) 3-1-1 Maedashi Higashi-ku, Fukuoka.
K. R. Kritikar and B. D. Basu, “Indian Medicinal Plants,” The Indian Press, Allbabad, 1918, p.590.
M. M. Lolitkar and M. R. Rajarama Rao, J. Univ. Bombay, 29, 223 (1960-61) [C.A., 58, 9537d (1963)]; S.S. Gupta, Indian J. Med. Res., 51, 716 (1963) [C. A., 60, 6094e(1964)]; W. Sucrow, Tetrachedron Lett., 1965, 2217; V. S. Baldwa, C. M. Bahndari, A. Pangaria, and R. K. Goyal, Upsala J. Med. Sci., 82, 39 (1977) [Planta Medica, 34, 280 (1978)].
A. Bendich and G. C. Clements, Bioche. Biophys. Acta, 12, 462 (1953).
a) R. Tschesche, G. Biernoth and G. Snatzke, Ann., 674, 196 (1964); b) Y. Yamada, K. Hagiwara K. Iguchi, and Y. Takahashi, Chemistry Letter, 1978, 319.
P. Tunmann, W. Gerner and G. Stapel, Ann., 694, 162 (1966).
Alnusenone, a pentacyclic triterpene having a 10 -hydrogen,  5, 6-double bond and a 3-keto group gives a negative CD curve [0]₂₉₅-3630⁰), while 4,4-dimethycholest-5-en-3-one and 17-acetoxy-4,4-dimethyl-19-norlanost-5-en-3-one show positive CD curves ([0]₂₉₄ + 5148⁰ and [0]₃₀₇+ 5752⁰, respectively).⁹⁾ These CD data were referred to in the determination of the cucurbit-5-ene nucleus of gratiogenin.^6a
P. Witz, H. Hermann, J-M. Lehn and G. Ourisson, Bull. Soc. Chim. France, 1963, 1101.
T. Yamauchi, M. Hara and K. Mihashi, Phytochemistry, 11,3345 (1972).
Y. Yamada, K. Hagiwara, K. Iguchi and S. Suzuki, Tetrahedron Lett., 1977, 2099.
P. J. Curtis and P. M. Meade, Phytochemistry, 10, 3081 (1971).
R. W. Doskotch and C. D. Hufford, Can. J. Chem. 48, 1787 (1970).
R. J. Restivo, R. F. Bryan and S. M. Kupchan, J. Chem. Soc., 1973, 692.
Instruments and materials used in this work were as follows: Yanaco micromelting point apparatus (melting points), Shimadzu UV-200S double beam spectrophotometer (UV spectra), Hitachi gratin-infrared spectrophometer, model EPI-G3 (IR spectra), JASCO DIP-4 digital polarimeter (specin.rotations), JASCO J-20 sutomatic recording spectropolarimeter (CD spectra), JEOL JNM PS-100 (100 MHz) and Hitachi R-22 (90 MHz) spectrometer (PMR spectra), JEOL JMN FX-100 (25 MHz) spectrometer (CMR spectra), Shimadzu GC-3BF gas chromatograph (GLC), JEOL JMS-01SG mass spectrometer (EI-MS spectra), JEOL D-300 FD mass spectrometer (FD-MS spectra), Kieselgel 60 (70-230 mest) (E. Mecrk) (column chromatography), precoated Kieselgel 60 F₂₅₄ plates (e. Merck) (TLC). Melting points are uncorrected. PMR and CMR spectra were measured in pyridine-d₅ unless otherwise stated and chemical shifts are expressed in the -scale using tetramethylsilane as an internal standard singlet; br, broad; d, doublet; dd, doublet; t, triplet; q, quartet; m, multiplet).
Reported for vicine: mp 243-244⁰ (dec.), []_D²⁶⁰ – 11.7⁰, UV _max^{0.1 NHCI}nm (): 274 (16400). Merck Index.)
J. Y. Lin and K.H. Ling, T’ai-wan I Hsueh Hui Tsa Chih, 61, 484 (1962) [C.A., 65, 4143 g (1966)].
5⁰ 1,4-butanediol succinate on Shimalite W; 2.1 m x 3 mm; N₂ carrier gas at 1 kg/cm²; 220⁰.
Reported for methyl -_D-xylopyranoside: + 153.9⁰ (H₂O) [J. Am. Chem. Soc., 47, 265 (1925)].
G. Germain, P. Main and M. M. Woolfson, Acta crystallogr., A27, 368 (1971).
T. Sakurai, H. Iwasaki, Y. Watanabe, K. Kobayashi, Y. Bando and Y. Nakamichi, Rikagaku Kenkyusho Hookoku, 50, 75 (1974).

Purification and Partial Characterization of Two Lectins from Momordica charantia

filipinoweb — Wed, 09 Aug 2006 09:57:41 +0000

1980 Experentia 36, pp.525-527

by S.S., L. Li
Summary. 2 different lectin have been prurified from the seeds of Momordica charantia by gel-filtration and ion-exchange chromatography. These 2…

Purification and Partial Characterization of Two Lectins from Momordica charantia

Author: S.S., L. Li

Type of Publication: Pre-Clinical

Date of Publication: 1980

Publication: Experentia 36, pp.525-527, 1980

Organization: Department of Microbiology, Mount Sinai School of Medicine, New York, Environmental Mutagenesis, NIEHS, NIH Research Triangle Park

Summary. 2 different lectin have been prurified from the seeds of Momordica charantia by gel-filtration and ion-exchange chromatography. These 2 lectins appear to be composed of 2 subunits of 26,000 daltons. Protein fraction I, but not showed agglutinating activity toward human type-O red blood cells. The amino acid composition and amino-terminal sequences of these homologous proteins are quite different.

The fruit of Momordica charantia is widely used in the orient, although the seeds are not eaten. The D-galactose-binding agglutinin from Momordica charantia has been shown to agglutinate human-O red blood cells, but not Yoshida sarcoma cells. Recently, toxic momordin and non-toxic momordica agglutinin have also been separated by Cm-cellulose chromatography, and the momordin inhibits protein biosynthesis of Ehrrlich ascites tumor cells. In this report, 2 lectins have been purified from the seeds of Momordica charantia, and their molecular weights, amino acid composition and aminoterminal sequence of 27 residues have been determined.

Materials and methods. The seeds of Momordica charantia were obtained from the Chan Man Hop Seed Co., Hong Kong. The proteins were isolated as previously described. Haemagglutination assays were performed in microtiter plates with human type-O red blood cells. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed on 12.5% slab gel in Tris-glycine buffer, pH 8.3. The gels were stained for protein with Coomassie brilliant blue and for carbohydrate with periodic acid-Schift reagent.

The proteins were hydrolyzed in 6 HCI at 110^o C for 24, 48 and 72 h, and the hydrolysates were analyzed with automatic amino acid analyzer (Beckman 121). Cystein and/ or half-cysteine were determined as cyteic acid after performic acid oxidation. Although Edman degradation were performed with the Beckman protein sequencer using N, N’-dimethylallylamine buffer and single acid cleayage. Phenylthiohydantoin-amino acids were identified by GLC, TLC, and/ or amino acid analysis after back hydrolysis with 6 N HCI or 56% HI. Phenylthiohydantoin arginine was also identified by the phenanthrene quinone spot test.

Results. The crude protein extract was chromatographed of a column of DEAE-Sephadex (Fig.1a) followed by gel filtration on a Sephadex G-150 column (Fig.1b). The proteins under peak G1 were further separated into fractions I and II on CM-cellulose using a linear gradient of sodium phosphate buffer (Fig.1c). The proteins under peak G2 were shown to be low-molecular-weight storage proteins, and have been described previously.

The proteins from DEAE-A50 and fraction G1 on G-150 had hemagglutinating titers of 1280 and 320, relatively, toward human type-O red blood cells. The titled protein fraction I agglutinated human type-O red blood cells at the concentration of about 2-4 g/ml, while protein fraction II did not show agllutination even at 2-4 g/ml. This hemagglutination could be inhibited by 10 mm D-galactose.

The reduced samples of proteins were run on SDS-polyarcylamide gel electrophoresis, and the gels were stained protein and carbohydrate. Both protein fractions I and showed single protein band corresponding mol. wt. of 1000 daltons (Fig.2). Both proteins were also periodic Schiff positive, indicating the presence of carbohydrate (data are not presented). The mol. wt of proteins after peak G1 (Fig.1b) was estimated to be approximately 49,000 daltons by gel-filtration on a calibrated Sephadex G-150 column. These results indicate that both proteins consist of 2 subunits of approximately 26,000 daltons.

Amino acid composition of both proteins (Table 1) was calculated from the molar ration and the assumed mol. wt of 15,000 daltons . These 2 proteins contain quite different amino acid composition, although they have very simlar mol. wts. The amino acid-terminal sequences of 27 residue of these 2 proteins were deduced from 2 runs of automatic many degradations. The residue identified are summarized in Table 2 and both sequences are compared among the 27 residues compared.

The protein fractions I and II showed very similar nearly absorption spectra with a typical maximum around 80 mm and the ratio of A₂₈₀ to A₂₆₀ being 2.0. These spectra are quite different from those of momordica storage proteins reported previously.

Discussion. 2 D-galactose-binding lectins were purified from Momordica charantia, and only protein fraction I, not hemagglutinated human red blood cells. On the basis of hemagglutinating activities and the behavior on CM-cellulose chromatography, the protein fractions I and II appear to correspond to the previously reported momordica agglutinin and toxic momrdin, respectively. However, the mol. wts and amino acid composition of both momordin proteins, I and II, determined in this study differ from published data. The larger mol. wt. and higher glutanin content of the previously isolated momordica agglutinin might be due to the contaminated momordica storage protein. Momoridca storage protein was eluted from cellulose column at similar position as the case of momordica agglutinin and it has an apparent mol. wt of 55000 daltons and very high content (32-34 moles%) of glutanin acid. It may be noted that both protein fractions I and like the D-galactose binding sophora agglutinin and E, did not bind to sepharose 4B column.

The partial amino acid sequences of both proteins and with their amino acid composition indicated that 2 homologous proteins have very different primary structures. 5 threonine residues are present at position nos. and 24 of protein fraction II while theronine is in the first 27 residues of protein fraction I. It will be interest to define the exact correlation of the structural sequences with their dinstinct agglutinating and toxic properties.

Acknowledgements. The skillful assistance of Ms. M. Diane Forde is greatly acknowledged. Part of this work was supported by NIH grants CA18621 and A109810 when the author was the Mount Sinai, School of Medicine.

References

Tomita, T. Kurokawa, K. Onozaki, N. Ichiki, T. Osawa and T. Unika. Experientia 28, 84 (1972).

Y. Lin, M-J. Hou and C, Chen, Toxicon 16, 653 (1978).

S. L. Li, Experientia 33, 895 (1977).

Kaplan, S. S. L. Li and J. M. Kehoe, Biochemistry 16, 4297 (1977).

Weber and M. Osborn, J. Biol Chem. 244, 4406 (1969).

R. M. Zacharius, T. E. Zell, J. H. Morrison and J. J. Woodlock, Analyt. Biochem., 30, 148 (1969).

D .H. W. Hirs, J. biol. Chem. 219, 611 (1956).

P. Edman and G. Begg, Eur. J. Biochem. 1, 80 (1967).

S. S.-L. Li, J. Hanlon and C. Yanofsky, Biochemistry 13, 1736 (1974).

J. Pisano, T. J. Bronzert and H. B. Brewer, Jr, Analyt. Biuochem 45, 43 (1972).

M. R. Summers, G. W. Smythers and S. Oroszlan, Analyt. Biochem. 53, 624 (1973).

O. Smithes, D Gibson, E. M. Fanning, R. M. Goodfliesh, J. G. Gilman and D. L. Ballantype, Biochemistry 10, 4912 (1971).

S. Yamada and H. A. Itano, Biochim. Biophys. Acta 130, 538 (1966).

T. Mise, G. Funatzu, M. Ishiguro and M. Funatzu, Agric. Biol. Chem. 41, 2041 (1977).

IUPAC-IUB Commission on Biochemical Nomenclature, J. biol. Chem. 241, 2491 (1966).

Inhibition of Protein Synthesis in Vitro by Proteins from the Seeds of Momordica charantia (Bitter Pear Melon)

filipinoweb — Wed, 09 Aug 2006 09:49:07 +0000

1980 Biochem. J. 186, pp.443-452

by Luigi Barbieri, MariaChristina Zamboni, Enzo Lorenzoni, Lucio Montanaro, Simonetta Sperti, Fiorenzo Stirpe
A haegmagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated…

Inhibition of Protein Synthesis in Vitro by Proteins from the Seeds of Momordica charantia (Bitter Pear Melon)

Author: Luigi Barbieri, MariaChristina Zamboni, Enzo Lorenzoni, Lucio Montanaro, Simonetta Sperti, Fiorenzo Stirpe

Type of Publication: Pre-Clinical

Date of Publication: 1980

Publication: Biochem. J. 186, pp.443-452, 1980

Organization: Instituto di Patologia dell’ Universitia di Bologna

A haegmagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol. wt. 115000 and consists of four subunits, of mol. wts. 30500, 29000, 28500 and 27000. The lectin inhibits proteins synthesis by a rabbit reticulocyte lysate with an ID₅₀ (concentration giving 50% inhibition) of approx. 5 g/ml. Protein synthesis by Yoshida ascites is partially inhibited by the lectin at a concentration of 100 g/ml. From the same seeds another protein was purified which has mol. wt. 23000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID₅₀ of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haegmagglutinating properties. Artenia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The protein seem to act catalytically , since they inactivate a molar excess of ribosomes. The lectin and the inhibitor are somewhat toxic to mice, the LD₅₀ being 316 and 340 g/100 g body wt. respectively.

Several proteins of plant origin are powerful inhibitors of protein synthesis, and can be divided into two categories. The first includes three highly toxic lectins, ricin, abrin (review by Olsness & Pihl, 1977) and modeccin (Refness et al., 1977; Stirpe et al., 1978), which inhibit protein synthesis in intact cells and in cell-free systems. The second category includes several proteins scarely toxic to animals, which inhibit protein synthesis in cell-free systems, but have little or no effect on whole cells. These are a ‘Phytolacca Americana peptide’ (Obrig et al., 1973), crotins and curcins (Stirpe et al., 1976), a protein from wheat germ (Stewart et al., 1977), and a number of unidentified proteins from seeds (Gasperi-Campani et al., 1977, 1980). It is well established that the toxic inhibitors act by inactivating irreversibly and in a catalytic manner (i.e.enzymically) the 60S ribosomal subunits, which becomes unable to bind elongation factor 2 (Sperti et al., 1973; Montanaro et al., 1978; Olsnes & Abraham, 1979). The non-toxic proteins when purified (Irvin, 1975) or semipurified (Sperti et al., 1976) act in the same way, except for the inhibitor from wheat germ, whose action is ATP-dependent (Stewart et al., 1977).

Some non-toxic haegtmagglutinating lectins also have an inhibitory effect on protein synthesis. Salvedt (1976) reported that the haegmagglutinating from Ricinus communis seeds (dinstinct from ricin) inhibits protein synthesis in HeLa cells, although at higher concentrations than ricin. This effect was due to an A’ subunit of the lectin, which was a potent inhibitor of protein synthesis in a cell-free system. Lin et al. (1978) purified two lectins from the seeds of Momordica charantia (bitter pear melon, a cucurbit): one of these, called Momordica charantia agglutinin, was a potent haemagglutinating whereas the other one, called momordin, had less haemagglutinating power and was a moderate inhibitor of protein synthesis by Ehrlich ascites-tumour cells. Barbieri et al. (1979) observed a marked inhibition of protein synthesis in a lysate of rabbit reticulocytes with a haemagglutinin from the seeds of Momordica charantia and with other haemagglutinating lectins from seeds and from a fish roe.

We report now that the inhjibitory activity on protein synthesis of the extracts from Momordica charantia seeds (Gasperi-Campani et al., 1980) is due to the haemagglutinating lectin (referred to here as Momordica charantia lectin, or the lectin) and to a much more potent protein, referred to here as Momordica charantia inhibitor, somewhat simlar to momordin (Lin et al., 1978), although devoid of haemagglutinating activity. Some properties and the mechanism of action of these two proteins were studied, and it was ascertained that they act on ribosomes in a catalytic manner, the lectin having little effect and the inhibitor no effect on protein synthesis by Yoshida ascites-tumour cells. Both the lectin and the inhibitor are some 100-fold less toxic to animals than ricin and related toxins, although they have, respectively, a comparable or a greater effect on protein synthesis in cell-free systems.

Experimental

Materials

Momordica charantia seeds, originally from India, were obtained from Mr. G. G. Celo, Zweibrucken, West Germany. L-[¹⁴C]Leucine (specific radioactivity 343mCi/mmol) and [methyl³H]-thymidine (specific radioactivity 20Ci/mmol) were purchased from The Radiochemical Centre, Amersham, Bucks., U. K. L-[¹⁴C]PhenylalanyltRNA (mixed charged tRNA containing 0.260 Ci of L-[¹⁴C]phenylalanine/mg of tRNA; 414 mCi/mmol of L-phenylalanine; percentage of L-phenylalanine-acceptor tRNA bound to L-phenylalanine, 36.9) was from New England Nuclear Corp., Boston, MA. U.S.A.

Bovine serum albumin and trehalose were from Sigma Chemical Co., St Louis, MOMORDIN, U.S.A.; all other sugars were from the same sources as described by Falasca et al. (1979). Neuraminidase was from Behringwerke, Marburg/Lahn, West Germany (one unit is defined as the amount of enzyme required to release 1 g of N-acetylneuraminic acid from human a₁-acid glycoprotein in 15 min at 37^o C); reference proteins for molecular-weight determinations and poly(U) were from Boehringer Mannheim G.m.b.H., Mannheim, West Germany.

Sepaharose 4B, Sepharose 6B, Sepahdex G-150 and CM-Sephadex C-50 were from Pharmacia Fine Chemical, Uppsala, Sweden. Acid-treated Sepharose 6B was prepared as described by Ersson et al. (1973), with a 3 h acid hydolysis.

All other chemicals were from the same sources as in previous work (Gasperi-Campani et al., 1978; Montanaro et al., 1978).

Toxicity experiments

The toxicity of the purified proteins was evaluated in Swiss mice of both sexes, weighing 28-30g (males) and 20-22g (females). Animals received food and water ad libitum. The proteins dissolved in 0.9% NaCl, were injected intraperitoneally, at five or six different concentrations, into groups of six animals for each dose. Concentrations ranged from 10 g to 1 mg/100g body wt., with a ratio between doses of 3.162 for the lectin and from 100 g to 1.78 mg/100g body wt., with ratio between doses of 1.778 for the inhibitor. The lectin was also given at a single dose (1 mg/100 g body wt.) to male Wistar rats weighing 100-120 g. LD₅₀ was evaluated by the method of Spearman-Karber as described by Finney (1964).

Assay of haemagglutinating and mitogenic activity

Erythrocytes were collected and washed described by Falasca et al. (1979), and were treated with formalin as described by Butler (1963). Haemagglutination tests were performed in Greiner microtitre plates, U-shaped (Microcult M29; Pool Bioanalysis Italiana, Milan, Italy). Each well contained in a final volume of 100 l: serial dilutions (by doubling, starting from 50 g/wee) of the preparations to be tested, 2.5 units of neuraminidase (when present), the sugars assayed for inhibitory activity, and 50 l of a suspension of fresh (1.2%,v/v-insulin) or formalin-treated (2%, v/v-insulin) erythrocytes. All solutions and suspensions were in 0.14M-NaCl containing 20 mM-sodium phosphate buffer, pH 7.2, and bovine serum albumin (15 g/ml). Comparable results were obtained with fresh or formalin-treated erythrocytes. Haemagglutination was evaluated visually after at least 1 h at room temparature (20^oC). A haemagglutinating units is defined as the lowest concentrations, in g/ml, giving visaible agglutination.

Mitogenic activity was assayed from the incorporation of [methyl-³H]thymidine into human peripheral-blood lymphocytes in culture as described by Falasca et al. (1979).

Protein synthesis

Protein synthesis was determined from the incorporation of [¹⁴ C]leucine as described by Gasperi Campani et al. (1978), with Yoshida AH-130 ascites-tomour cells, transplanted in Wistar rats and collected 6-8 days after inoculum, or with a lysate of rabbit reticulocytes prepared as described by Allen & Schweet (1962).

Poly (U)-dierected polymerization of phenylalanine was assayed with KCI-washed Artemia salina ribosomes prepared as described by Sierre et al. (1974). The reaction mixtures (Montanaro et al., 1978) contained, in a final volume of 0.25 ml: 80 mM-Tris/HCI buffer, pH 7.4, 120 mM-KCI, 7 mM-magnesium acetate, 2mM-dithiothreitol, 2 mM-GTP, 25 pmol of [¹⁴C]phenylalanyl-tRNA, 200 g of poly (U), 250 g of ‘S-105 supernatant’ (Sierra et al., 1974), and ribosomes. Incubation was at 24^oC for 30 min. The reaction was arrested with 0.25 ml of 10% (w/v-insulin) trichloacetic acid, and the samples were treated as described by Montanaro et al. (1978) for the determination of hot-acid insoluble radio activity.

Electrophoresis and determination of molecular weight and of isoelectric point

Polyacrylamide-gel elctrophoresis was performed at pH 4.5 (Reisfeld et al., 1962), at pH 7.0 (Williams & Reisfeld, 1964) and at pH 9.5 (Liao et al., 1969). Gels were stained with Coomassie Blue and destained as described by Weber & Osborn (1969).

For molecular-weight determinations, discontinous sodium dodecyl sulphate/polyacrylamide-gel elctrophoresis was carried out in slabs (1.5 mm x 10 cm x14 cm) in the model 220 Vertical Slab Electrophoresis Cell (Bio-Rad Laboartories, Richmond, CA, U. S. A.) with the Laemmli (1970) system as described in the instruction manual. Before being placed in the wells of the stacking gel, samples (10-30 g of protein) in 50 mM_tris/HCI buffer, pH 6.8, were heated for 3 min at 90^oC in the presence of 1 % sodium dodecyl sulphate, 1% glycerol and 0.0025% Bromophenol Blue; 2-mercaptoethanol (5%, v/v-insulin) was either absent or present. Electrophoresis was for 6 h at 25 m A per slab. Soya bean trypsin inhibitor (mol.wt. 21500), bovine serum albumin (mol.wt. 68000) and RNA polymerase (n-chain, mol.wt. 39000; -chain, mol. wt. 155000; -chain, mol.wt. 165000) were used as calibration proteins.

Gel isoelectric focusing was carried out essentially as described by Catsimpoolas (1968) in glass tubes (3 mm x 130 mm) filed with 0.75 ml of gel-protein medium (10-50 g of protein) prepared by mixing 0.3 vol. Of a solution containing 25% (w/v0 acrylamide and 1% (w/v) NN’-methylenebisacrylamide, 0.075 vol. 7% (w/v) ammonium persulphate, o.075 vol. Of NNN’N-tetramethylethylenediamine diluted 1:50 with water, and 0.9 vol. of protein sample. After polymerization isoelectric focusing was performed for 30 min at 250 v-insulin and overnight at 150 v. Gels were stained with 0.1% Coomassie Blue in 50% (w/v) trichloroacetic acid for 20 min, destained with several changes of 40% (v/v) methanol at 50^o C, and were finally stored in 7.5% acetic acid. The pH gradient was measured in each run on unstained gels ad described by Wrigley (1968).

Other determinations

Proetin was determined by the method of Lowry et al. (1951), with bovine serum albumin as a standard, or spectrophotometrically (Kalb & bernlohr, 1977).

Total neutral-sugar content was determined by the anthrone method (Spiro, 1966) with D-galactose as a standard, after extensive dialysis of the proteins against water. The sugar content was determined also after denaturation of the lectin with 6M-guanidinium chloride, to avoid the possibility of contamination with residual galactose. For this the lectin solution was dialsed against 0.1 M-Tris/HCI buffer, pH 8.5, then made 6 M with guanidium chloride, was boiled for 3 min in a water, bath, kept at 37^o C for 2 h, and finally was dialysed against 1000 vol. of water.

Radioactivity was measured with a Packard TriCarb liquid-scintllation spectrometer with an external standard, with a counting efficiency of approx. 80% for ¹⁴C and 40% for ³H.

Results

Purification of the lectin and of the inhibitor

Seeds were shelled and were extracted six to eight times with diethyl ether in a blender, and the resulting powder was dried in the air. All subsequent operations were carried out at 2-4^o C. The powder was extracted with 10 vol. of 0.2 M-NaCl containing 0.005 M-sodium phosphate buffer, pH 7.2. The suspension was left overnight at 11000 g for 20 min. To this crude extract (NH₄)₂SO₄ was added slowly under constant stirring: the precipitates obtained at 30, 60 and 100% saturation of the salt were collected by centrifugation, dissolved in NaCl/sodium phosphate buffer and dialysed against the same solution. Most of the heamagglutinating and inhibitiory activity was recovered in the 30-60%-saltd,0 (NH₄)₂SO₄ precipitate. From this, purification of the heamagglutinating lectin was attempted by affinity chromatography on a Sepharose 4 B column, as described by Tomita et al. (1972). Part of the agglutinating activity was retained on the column, from which it could be eluted with 0.1 M-galactose, (Fig.1a). However, a considerable haemagglutinating activity was found in the effluent before elution with galactose: this activity could be absorbed completely on a column of acid-treated Sepharose 6B, from which it was eluted with 0.2 M-galactose (Fig.1b). The pufirificaton of the lectin is summarized in Table 1.

Lectin purified in this way inhibited protein synthesis by a lysate of rabbit reticulocytes, as discussed below. However, the non-haegmagglutinating material, which was not retained by Sepharose 6B, also showed a strong inhibitory activity in the lysate system. This material was concentrated by dialysis against solid sucrose, and then was dialysed over night against 5mM-sodium phosphate buffer, pH 6.5,

After further concentration in a Minicon B15 concentrator (Amicon Corp., Lexington, MA, U.S.A.) and further dialysis against 5mM-sodium phosphate buffer, pH 6.5, the solution was applied on a Sephadex G-150 (superfine grade) column. From this column three peaks emerged, with the inhibitory activity entirely in the second peak (Fig.2a). The active frctions were pooled, and were applied to a CM-Sephadex C-50 column, which was sodium phosphate buffer, pH 6.5. The inhibitory activity was eluted with 0.1 M-buffer as a sharp peak followed by a shoulder (Fig.2b). The purification of the inhibitor is summarized in Table 1.

Electrophoresis, molecular weight and isoelectric point

Both the lectin and the inhibitor gave a single band on polyacrylamide-gel electrophoresis at ph 4.5, 7.0 and 9.5 (results not shown). Prior incubation at 37⁰ C for 1h in the presence of 1% 2-mercaptoethanol did not modify these electrophoretic patterns.

A single bands was observed when the freshly prepared lectin and inhibitor were subjected to eletrophoresis in the presence of sodium dodecyl sulphate. Only with lectin preparations kept for some time at 2-4^o C some minor bands appeared, corresponding to subunits and possibly to their aggregates (Fig.3). The lectin preincubated with 2-mercaptoethanol showed four different subunits on gel elctrophoresis in the presence of sodium dodecyl sulphate (Fig.3), whereas the inhibitor was not modified.

The molecular weight of the lectin, as estimated by sodium dodecyl sulphate/polyacrylamide-gel elctrophoresis, was 115000, and those of the lectin subunits were 30500, 29000, 28500 and 27000 that of the inhibitor was 23000.

The isoelectric point, as estimated by isoelectric focusing were 6.0 for the lectin and 8.6 for the inhibitor.

Sugar content

The total neutral-sugar content of the lectin, expressed as galactose, was 116 g/mg of protein (average of three determinations), and did not change after denaturation of the lectin with guanidinium chloride. No neutral sugar was detected in the inhibitor.

Toxicity

Both the lectin and the inhibitor were somewhat toxic to mice: the LD₅₀ by the intraperitoneal route was 316 ± 1.4 g/100 g body wt. (95% confidence limits 172-580) for the lectin, and 430 ± 1.3 g/100 g body wt. (95% confidence limits 262-705) for the inhibitor. Deaths occurred between 12 and 72 h of poisoning, depending on the dose administered. No apparent effects were observed when the lectin was given intraperitoneally to rats at dose of 1 mg/ 100 g body wt.

Haemagglutinating and mitogenic activity

The agglutinating activity of Momordica charantia lectin on erythrocytes from various species is shown in Table 2. Maximal activity was observed with human erythrocytes. In all cases except with rat erythrocytes the agglutinating activity was enhanced in the presence of neurominidase.

Agglutination of human erythrocytes (group O) was prevented by the following sugars, all tested at 50 mm concentration, with the lectin at 100 ng/ml: D-galactose, D-galactosamine, N-acetyl-D-galactosamine, D-fucose, 1-0-methyl a-D-glucopyranoside and riffinose. The following sugars had no effect, under the same experimental conditions: D-arabinose, D-ribose, D-xylose, D-fructose, D-glucose, D- glucosamine, N-acetyl-D-glucosamine, cellobiose, maltose, sucrose, trethalose and melezitose.

The purified inhibitor at concentrations up to 250 g/ml did not agglutinate the erythrocytes of any of the species reported in Table 2, either in the presence or in the absence of neuraminidase.

Both the lectin and the inhibitor were not mitogenic to human peripheral blood lymphocytes cultured in vitro, at concentrations from 0.01 to 10 g/ml (lectin) and from 0.1 ng to 50g/ml (inhibitory).

Inhibition of protein synthesis

Cells. Momordica charantia lectin had a partial inhibitory effect on protein synthesis by Yoshida ascites cells at 100 g/ml, but not a 10 g/ml (Fig.4). The inhibition was not modified in the presence of neuraminidase (25 units/ml; results not shown).

Reticulocyte lysate. Momordica charantia lectin was a strong inhibitor of protein synthesis by a rabbit reticulocyte lysate (Fig.5). The ID₅₀ (concentration giving 50% inhibition) of the freshly prepared lectin varied from one preparation to another, ranging from 0.6 to 45 g/ml, more usually from 1.5 to 15 g/ml (measured on ten preparation from the same seeds). On storage at 2-4^o C the activity of the more-active preparation decreased, and that of the less-active ones increased, so that within a few days the ID₅₀ of all preparations reached a stable value around 5 g/ml (average of ten preparations). Inhibition of protein synthesis by the less-active preparations was increased at 37^o C for 2 h in the presence of 2-mercaptoethanol, whereas more-active and ‘stablized’ preparations were not affected by the thiol. The inhibition was not affected by the presence in the reaction mixture of galactose at a concentration (0.1 M) higher that inhibiting agllutination. Once stabilized, the activity of the lectin was unchanged on storage at 2-4^o C for several days, or at –25^o C for a longer time, although in the latter case it was considerably increased after a few thawings, The lectin could freeze-dried without loss of activity, although it became rather difficult to redissolve.

The purified inhibitor was over 1000-fold more active than the lectin inhibiting protein synthesis by the lysate, the ID₅₀being 1.8 ng/ml (Fig.6). This effect of the inhibitor was not affected by pretreatment with 2-mercaptoethanol. The activity of the inhibitor decreased slowly on storage at 2-4^o C.

Ribosomes. Artemia salina ribosomes preincubated with the lectin or with the inhibitor and washed by centrifugation through 5% sucrose had a greatly decreased capacity to perform poly (U) directed polymerization of phenylalanine (Table 3). Both proteins were effective at concentration less than equimolar with ribosomes.

Protein synthesis by untreated ribosomes was unaffected on addition of ribosomes preincubated with the lectin, but was decrerased when ribosomes pre-treated with inhibitor were added. It cannot be excluded that this is due to traces of the inhibitor remaining attached to preincubated ribosomes.

Discussion

The seeds of Momordica charantia contain two proteins which are potent inhibitors of protein synthesis in cell-free systems, but have little or no effect on intact cells, thus accounting for the effect of seed extracts (gasperi-Campani et al., 1980).

One of these proteins is a haemagglutinating lectin inhibited by galactose, galactose-containing sugars and by  and -methylglucose; part is retained by Sepharose 4B (Tomita et al., 1972) and the rest by acid-treated Sepharose 6B. The materials eluted from the two columns have identical properties; and thus it can be assumed they are a single protein, with higher affinity for Sepharose 6B than for Sepharose 4B. Hence, the passing through Sepharose 4B can be eliminated from the purification procedure, as it was confirmed by the complete retention of the haemagglutinating activity on acid-treated Sepharose 6B obtained in subsequent preparations. The inhibitory activity of the freshly prepared lectin is rather variable, and is modified on storage at 0^o C. This could be due to a rearrangement or to other modification(s) of the lectin molecule, such as the liberation of subunits and the formation of different aggregates, as suggested by the electrophoretic pattern (Fig.3).

The second inhibitory protein (Momordica charantia) has no haemagglutinating properties, and is one of the most potent inhibitors of protein synthesis hitherto described, more potent than the A chains of ricin, abrin and modeccin, and second only to the Phytolacca Americana peptide (Irvin, 1975) which in our system had ID₅₀ <1 ng/ml.

The recovery of the lectin and especially of the inhibitor was higher than 100% (see Table 1). A similar phenomenon was observed by Ersson et al. (1973) and by Ercson (1977) during the purification of the lectin from Crotalaria juncea, and was attributed to removal of interfering substances present in crude extracts. The possibility must also be considered that the proteins could be modified and rendered more active by the purification procedure.

Both proteins act by altering ribosomes, and making them unable to perform protein synthesis. As with ricin (Montanaro et al., 1973) and modeccin (Montanaro et al., 1978), no cofactors are necessary for the inactivation of ribosomes. This effect of the proteins seems to be catalytic, i.e enzymic, since they inactivate more than one molar equivalent of ribosomes. Thus these proteins seems to act in a similar way to ricin and related toxins, from which, however, they differ in being much less toxic to animals or to whole cells. The toxicity of ricin and of the other toxic lectins results from the combined action of both their constituent A and B subunits: the B subunits binds to cells allowing the penetration of the A subunit, which acts on ribosomes (Olsnes & Pihl, 1977). Momordica charantia lectin consists of four different subunits, of which at least one should have the A function and inhibits protein synthesis, where as at least one should behave like a B subunit capable of binding to cells, as demonstrated by the haemagglutinating activity. It is likely that the low toxicity of Momordica charantia lectin is because the properties of its B subunit differ from those of the B chain of other more toxic lectins, or because of a peculiar binding of the B and other subunits with the A chain, which does not allow the latter to enter cells. Considerations of the same kind lead us to suppose that the inhibitor should be similar to an A chain and this would account (i) for the lack of effect on whole cells, and consequently for the low toxicity to animals, and (ii) for the lack of haemagglutinating activity. It shoul be considered also that the inhibitor cannot be a subunit of the lectin, since its molecular weight is lower than that of any subunit. It is possible, however, that it is precursor or a derivatives of the active of thesubunit of the lectin.

Lin et al. (1978) isolated from the seeds of Momordica charantia two lectins, called Momordica charantia agglutinin and momordin. Both agglutinated erythrocytes and momordin inhibited protein synthesis by Ehrlich ascites cells, at relatively high concentrations. The molecular weights of these lectins are 32000 (agglutinin) and 24000 (momordin), and it is possible that they are subunits of the lectin that we isolated, separated during the purification procedure. Momordin has practically the same molecular weight as the inhibitor; the latter., however, has no heamagglutinating activity and does not inhibit protein synthesis by intact cells.

The property of inhibiting protein synthesis is common to several lectins and proteins from plants (see the introduction). For the inhibitory proteins purified from Momordica charantia seeds, as in all other cases studied so far, the inhibitory activity is due to ribosomal damage, which seems to be brought about enzymically.

Acknowledgements. This research was supported by a contract from the Consiglio Nazionale delle Richerche, Rome, within the Progetto finalizzato ‘Controllo delle crescita neoplastica’ and by the Pallotti’s legacy for cancer research.

References

Allen, E. H. & Schweet, R. S. (1962) J. Biol Chem 237, 760-767.

Barbieri, L. Lorenzoni, E. & Stirpe, F. (1979). Biochem. J. 182, 633-636.

Butler, W. T. (1963) J. Immunol. 90, 663,-671.

Castimpoolas, N. (1968) anal. Biochem, 26, 480-482.

Ersson, B. (1977) Biochim. Biophys. Acta 494, 51-60.

Ersson, B., Aspberg, K. & Porath, J. (1973) Biochim. Biophys. Acta 310, 446-452.

Falasca, A., Frenceschi, C., Rossi, C. A. & Stirpe, F. (1979) Biochim. Biophys. Acta 577, 71-81.

Finney, D. J. (1964) Statistical Methods in Biological Assay, pp.524-530, Griffin, London.

Gasperi-Campani, A., Barbieri, L., Lorenzoni, E. & Stirpe, F. (1977) FEBS Lett. 76, 173-176.

Gasperi-Campani, A., Barbieri, L., Lorenzoni, E., Montanaro, L., Sperti, S., Bonetti, E. & Stirpe, F. (1978) Biochem J. 174, 491-496.

Gasperi-Campani, A., Barbieri, L., Morelli, P. & Stirpe, F. (1980) Biochem. J. 186, 439-441.

Irvin, J. D. (1975) Arch. Biochem. Biphys. 169, 522-528.

Kalb, V. F., Jr. & Bernlohr, R. W. (1977) Anal. Biochem. 82, 362-371.

Laemmli, U. K. (1970) Nature (London) 227, 680-685.

Liao, T. H., Hennen, G., Howard. S. M., Shome. B. & Pierce, J. D. (1969) J. Biol Chem. 244, 6458-6467.

Lin, J-Y., Hou, M. J. & Chen, Y. C. (1978) Toxicon 16, 653-660.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem 193, 265-275.

Montanaro, L., Sperti, F. (1973) Biochem. J. 136, 677-683.

Montanaro, L., Sperti, S., Zamboni, M., Denaro, M., Testoni, G., Gasperi-Campani, A. & Stirpe, F. (1978) Biochem. J. 176, 371-379.

Obrig, T. G., Irvin, J. D. & Hardesty. B. (1973) Arch. Biochem. Biophys. 155, 278-289.

Olsnes, S. & Pihl, A. (1977) in Receptors and Recognition, Series B, vol. 1 (Cuatrecasas. P., ed) pp. 129-173, Chapman and Hall, London.

Olsnes, S. & Abraham, A. K. (1979) Eur. J. Biochem. 93, 447-452.

Puck, T. T., Ceciura, S. J. & Fisher, H. W. (1957) J. Experimental. Med. 106, 145-157.

Refsnes, K., Haylett, T., Sandvig, K. & Olsnes, S. (1977) Biochem. Biophys. Res. Commun. 79, 1176-1183.

Reisfeld, R. A., Lewis, U. J. & Williams, D. E. (1962) Nature (London) 195, 281-283.

Saltvedt, E. (1976) Biochim . Biophys. Acta 451, 536-548.

Sierra, J. M., Meier, D. & Ochoa, S. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2693-2697.

Sperti, S., Montanaro, L., Mattioli, A. & Stirpe, F. (1973) Biochem, J. 136, 813-815.

Sperti, S., Montanaro, L., Mattioli A., Testoni, G. & Stirpe, F. (1976) Biochem. J. 156, 7-13.

Spiro, R. G. (1966) methods Enzymol. 8, 3-26.

Stewart, T. S., Hruby, D. E., Sharma, O. K. & W. K. (1977) Biochim. Biophys. Acta 479, 31.

Stirpe, F., Gasperi-Campani, A., Barbieri, L., I. E., Montanaro, L., Sperti, S. & Bonetti, FEBS Lett. 85, 65-67.

Tomita, M., Kurokawa, T., Onozaki, K., I, Osawa, T. & Ukita, T. (1972) Experientia 28.

Weber, K. & Osborn, M. (1969) J. Biol Clin 4406-4412.

Williams, D. E. & Reisfeld, R. A. (1964) Ann. N. Sci. 121, 373-381.

Wrigley, C. W. (1968) j. Chromatogr. 36, 362.

Hypoglycemic Activity of Polypeptide-p from A plant Source

filipinoweb — Wed, 09 Aug 2006 09:43:01 +0000

Nov-Dec 1981 Journal of Natural Products Vol.44, No.6, pp.65

by Pesupa Khanna, S. C. Jain, A. Pangaria, V. P. Dixit
Insulin used in the treatment of diabetes mellitus has usually been obtained in very low yield from animal pancreas, i.e…

Hypoglycemic Activity of Polypeptide-p from A plant Source

Author: Pesupa Khanna, S. C. Jain, A. Pangaria, V. P. Dixit

Type of Publication: Pre-Clinical

Date of Publication: Nov-Dec 1981

Publication: Journal of Natural Products Vol.44, No.6, pp.65, Nov-Dec 1981

Organization: University of Rajasthan

Abstract: A hypoglycemic peptide, Ploypeptide-p, has been isolated from fruit, seeds, and tissue of Momordica charantia Linn (bitter gourd). Amino acid analysis indicates a minimum molecular weight of approximately 11,000 (166 residues). Polypeptide-p is a very effective hypoglycemic agent when administered subcutaneously to gerbils, langurs, and humans.

Insulin used in the treatment of diabetes mellitus has usually been obtained in very low yield from animal pancreas, i.e., one pound of pure insulin per 10,000 animals. Side effects of the animal insulin are well known. Recently, insulin has been synthesized by genetic manipulation in Escherichia coli, which is a significant scientific achievement.

A number of indedigenous drugs have been tried in the past for the treatment of diabetes mellitus. In the tropical world, fruits of Momordica charantia (biiter gourd) have been successfully used by diabetic patients; crude extracts have shown hypoglycaemic activity in rabbits (1-3). Khanna et al. (4,50 were able to isolate an active principle earlier called p-insulin of v-insulin from fruits, seeds and tissue culture of this plant species (6,7). When administered subcutaneously to human patients, v-insulin showed a significant blood sugar lowering effect (6).

MATERIALS AND METHODS

TISSUE CULTURE

Seed coats of Momordica charantia were removed, and the seeds were inoculated on revised Murashinge and Skoog’s medium (8,9) supplemented with 1 ppm of 2,4-dichlorophenoxyacetic acid (2,4-1) and 1% agar. The seeds took 5-6 days to germinate and form seedlings. Organized tissue was established (10) from the seedlings and maintained for 12-18 months by frequent subculturing of 6-8 weeks in fresh RT medium. This tissue was harvested after the transfer age of 6 weeks and extracted for its polypeptide-p content.

Fruits, soaked seeds, and tissue samples (100 g each) were crushed separately and then frozen. Each of the frozen samples was dissolved in 10 ml of distilled water, 45 ml of 955 ethanol and 3.6 ml of sulfuric acid 99.5%). The mixture was stirred vigorously (5,11) for 15-20 min at 25-28⁰ and then homogenized by the addition of 60 ml distilled water and 250 ml of 95% ethanol separately. After each of the mixtures was filtered enough ammonium hydroxide (28%) was added to adjust the pH of the filtrate to 3. To each of the filtrates, 1.5 liters of acetone was added till a while flocculent precipitate was formed. These mixtures were kept at 5⁰ for 8-10 hour.

The supernatant from each of the containers was decanted off, and the precipitate was dialyzed in a dialysis membrane (36 DIABETES MELLITUS, Union Carbide Corporation, Chicago, U.S.A.; molecular weight cut-off was 6000): distilled water was usd to removed the last traces of salt and other dialyzable impurities until the outside water gave a negative test with barium carbonate. The non-dialyzed fraction was collected, dried and crystallized in a 0.0001% solution of zinc acetate in water (12). The excess was removed by washing with ethylenediame tetracetic acid (EDTA) solution.

Crsytallized metrial was applied to silica gel coated and activated glass plates along with a standard sample of bovine insulin. The plates were deloped in a solvent mixture of n-butanol acetic acid-water 12:5:2. When the plates were sprayed with ninhydrin (0.25% in acetone) and heated, a single yellow spot (Rf 0.19), which nearly coincided with that of the standard sample of bovine insulin, was observed.

Disc electrophoresis was carried out (10% SDS Biophore Gel, run in tris buffer, operating pH 6.1; 3% acetic acid in lower cell; 90 V, 2.5 per tube; Bromophenol blue tracking dye). Samples of the crystallized isolate and bovine insulin were separately with SDS biophore buffer containing dithiothreitol EDTA, injected, and run for 7 hour. Gels collected from the tubes were stained (0.05% Coomassie Brilliant Blue R-250 in 7% aqueous acetic acid) and washed with 10% acetic acid.

The isolate (25 mg) from each of the samples was hydrolyzed with 6N HCI at 100⁰ for 24 hour and filtered. The filtrate was dried and the residue taken up in 50% ethanol. Two dimensional tlc was carried out (silica gel C; solvent system first: n-butanol-acetic acid-water, 5:1:1; secondly phenol saturated with water; 0.25% ninhydrin in acetone as spraying reagent), and seventeen amino acids were resolved. The isolates were also run in an automatic amino acid analyzer separately (Table 1).

A derivative of the crystallized material (polypeptide-p ZnCI₂) was prepared in the same manner (12) as bovine insulin-ZnCI₂. Doses of polypertide-p and polypeptide-p ZNCI₂ inn 0.9% NaCI as the vehicle were prepared (1.8 mg/ml equivalent to 40 units) as used in the case of bovine insulin. Immunoassays also carried out.

PHARMACOLOGICAL TRIALS

Pharmacological studies involved Meriones Hurianne Jerdon (gerbils), males and females, and Presbytis entillus entillus Dufsne (male langurs). 105 gerbils weighing 63-7 g were used in the present work. The animals were divided into groups of five each and all were tested for 12 hour before the beginning of the experiment. These animals were provided with water and libitum. Polypeptide-p-ZnCI₂ (0.5 unit/kg in 0.9% NaCI) was administered subcutaneously. Thirty-five animals were injected with an equal amount og 0.9% saline vehicle. The blood samples were obtained, through eardias, and the total blood sugar was estimated (13) at different time intervals (0, ½,1,2,4,8,12 hour). These results were compared with those of the vehicle-treated controls and statistically analyzed (14; Table 2).

A total of six healthy adult male langurs of different age groups with large eannines, a well developed pinkish oedematous band, and the sexual skin on the rump were used as experimental non-human primate models. The animals were fed with wheat chapaty (unleavened bread), banana, onion, carrot, potatoes, and soaked Bengal grams were provide with water ad libitum. Continous veterinary supervision was maintained.

Polypeptide-p-ZnCI₂ (0.5 unit/kg in saline) was administered subcutaneously. Fasting blood sugar samples of each of the animals were taken before any dose of drug was given. Blood samples were taken at different time intervals. As shown in Table 3. Food was given after 4 hour blood samples were taken. An equal number of male langurs were kept fasting and injected with saline (0.9% NaCI in water); their blood sugar samples were taken according to the schedule in Table 3.

CLINICAL TRIALS

A total of nineteen patients (15 males and 4 females) suffering from primary idiopathic 915) diabetes mellitus (15-56 hour age group) for a period of three months to eight years were selected for clinical trials. Out of the nineteen patients selected 11 cases were of juvenile diabetes and 8 were of maturity onset diabetes. Diabetic patients suffering from ketoacidosis, cerebrovascular accidents, acute myocardial infarction and renal failure were excluded from this study.

All patients were admitted to medical wards of S.m.S. Hospital, Jaipur, 4-5 days prior to the commencement of the study. Long-lasting insulin was withdrawn from patients 72 hour prior to the test, and plain insulin was withdrawn 12-18 hour before the test. Oral hypoglycemics were withdrawn 48 hour preceding the study. A blood sugar sample after the overnight fast was taken at 7 a.m. Polypeptide-p- preparations in saline solution was administered subcutaneously in a dose depending on the severity of diabetes mellitus (less than 180 mg/100 ml blood sugar level, 10 units; 180 250 mg/ml blood sugar level, 20 units; 250 mg/ml of blood sugar or above, 30 units).

After administration of the polypeptide-p preparation, the first three samples were taken at half-hour intervals to record the onset of the hypoglycemic effect. Subsequent samples were taken at different time interval, as shown in Table 4, to show the peak effect and duration of the action of this polypeptide. The blood samples were withdrawn from the medial cubital vein. The subjects were kept fasting during the study: only plain lemon water was given, if desired by the patients. Supervision was maintained for administration of glucose upon development of hypoglycemic symptoms. Blood sugar determination were performed by the method of Nelson-Somogyi (16).

The control group consisted of eight of the original nineteen patients with diabetes mellitus. Control blood samples were withdrawn at the same time intervals without the polypeptide-p being administered (Table 4). Polypeptide-p-ZnCI₂ was administered s.c. to three juvenile patients. These patients required smaller doses of this drug than on bovine insulin.

RESULTS

A single electrophoretic band of dialyzed and crystallized substance (Rf 0.41) was observed which, however, did not coincide (Rf 0.47) with that of bovine insulin (Fig.1). On scanning, a single main peak (Rf 0.41) of pure polypeptide-p was observed (Fig.2).

Two-dimensional tlc and the amino acid analysis (automatic amino acid analyzer) of the polypeptide-p hydroyzate showed 17 amino acids with a total of 166 residues and a minimum molecular weight of approximately 11000 (Table 1). Methionine was the extra amino acid observed in the unknown samples when compared with that of the known bovine insulin. Bio-immunoassays of this polypeptide were found to be negative against bovine insulin.

The pharmacological study revealed that the polypeptide-p-ZnCI₂ was long acting in gerbis and langurs and showed a significant blood-sugar-lowering effect (Table 2,3).

Clinical trials showed a hypoglycemic effect of polypeptide-p in juvenile and maturity-onset diabetic patients (Table 4). The peak effect in the juvenile diabetic may be between 4-8 hour as compared with 2 hour for crystalline bovine insulin. The peak response in maturity-onset diabetics as readily determined as in juvenile diabetics (Table 4).

No complaints of any side effects followed administration of polypeptide-p-ZnCl₂ to the three juvenile patients. One juvenile patients who expressed frequent heaviness of the head, a swollen face, apin in the stomch, and recurrent episodes of hypoglycemia when kept on crystalline bovine insulin was free of the these side effects when maintained continuously on polypeptide-p-ZnCI₂ for a period of five months. Immunoassays did not show cross reaction wheb tested with bovine insulin.

DISCUSSION

No apparent side effects were observed when the p-insulin was screened in diabetic patients. Bovine insulin so far is the only remedy against diabetes mellitus. With these new data, a new horizon in the treatment of diabetes mellitus. With these new data, a new horizon in the treatment of diabetes mellitus amy have been opened. Since the active principles is from a plant source, it is likely to be antigenic. More clinical trials of action, antigenecity, and various effects of intermediary metabolism in human beings are in progress and shall be reported later.

ACKNOWLEDGEMENTS

We gratefully acknowledge the financial support from the Indian Council of Medical Research, New Delhi, and the Department of Science and Technology, New Delhi, for carrying of Eli Lilly & Company, Indianapolis, Indiana, U.S.A., for the help extended for further confirmation of this polypeptide.

LITERATURE CITED

G., Rivera, Amer. J. Pahrm., 113, 281 (1941).
G., Rivera, Amer. J. Pahrm., 114, 72 (1942).
V. N. Sharma, R. K. Sogani and R. B. Arora, Indian J. Med. Res., 48, 471 (1960).
P. Khanna, T. N. Nag, S. C. Jain and S. Mohan, 3^rd Intl. Congr. On Plant Tissue and Cell Culture, Leicester, England (1974).
P. Khanna, T. N. Nag, S. C. Jain and S. Mohan, Indian Patent No. 136565 (1974).
V. S. Baldwa, C. M. Bhandri, A. Pangariya and R. K. Goyal, Upsala. J. Med. Sci., 82, 39 (1977).
A. Pangaria, Annual. Aonf. (XXIV) of Physiologists and Pharmacologists, Jaipur, India (1978).
B. Kaul and E. J. Staba, Lloydia, 31, 171 (1968).
T. Murashige and F. Skoog, Physiol. Pl., 15, 473 (1962).
P. Khanna, S. Mohan, T. N. Nag and S. C. Jain, Indian J. Pl. Physiol., 14, 34 (1971).
C. S. Vestling, Biochem. Preps., 6, 28, (1958).
C .O. Wilson, O. Glsvold and R. F. Doerge, ‘Text Books of Organic Medicinal and Phamaceutical Chemistry”, J. B. Lipponeott Co., Philadelphia, 1971.
Wu, Folin, “Hawk’s Physiological Chemistry”, B. L. Oser, ed. McGraw IiiII Ltd., New York, 1965.
R. A. Fisher, “Statistical Methods for Research Workers”, 11^th Edn/. Oliver and Boyd, Edinburgh, 1959.
J. Steinke and W. Thorn Geoge, Diabetes Mellitus, In “Harrison’s Principles of Internal Medicine”, 7^th Edn., McGraw Hill Inc., New York, 1974.
E. J. King and I. D. P. Wooton, “Microanalysis in Medical Biochemistry”, J. A. Churchill Ltd., London, 1956.