J. Physiol. (I938) 94, I2.352.i2:635.34

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1 249 J. Physiol. (I938) 94, I2.352.i2: EFFECTS OF CABBAGE EXTRACTS ON CARBOHYDRATE METABOLISM BY A. D. MACDONALD AND LEO WISLICKI From the Department of Pharmacology, The University of Manchester (Received 2 August 1938) SINCE COlliP [1923] described the insulin-like action of injected plant extracts in rabbits and dogs, many attempts have been made to produce substances which when administered by mouth would lower the blood sugar and increase the carbohydrate tolerance of diabetics [e.g. Eisler & Portheim, 1924; Allen, 1927; Leclerc, 1935; Clementi & Torrisi, 1935; Mark & Wagner, 1925; Sippola, 1927; Donard & Labbe, 1933; Laland & Havrevold, 1933]. The presence of a hypoglycaemic principle in plants is supported by a certain amount of clinical evidence. Thus Moscowitz [1937] has reported that diabetics tolerate carbohydrates in vegetables better than corresponding amounts in other foodstuffs. Mori & Seikeichi [1934] show that the hyperglycaemia after the intake of rice is much less when green vegetables are taken at the same time, and so on. The problem is complicated by the fact that the plant extracts often produce an initial rise in blood sugar. Donard & Labbe [1933] held that the presence of maltose in the extract was responsible for this hyperglycaemia, and Labbe [1936] reports that a potent hypoglycaemic extract was obtained from germinated barley after maltose and hordein had been removed. Laland & Havrevold [1933] obtained from onions a crystalline substance which lowered blood sugar in rabbits when mixed with allyl- or diallyl-disulphide, substances which in themselves have no insulin-like action. No extracts have, however, come into general use which can be relied upon when administered by mouth. Insulin modifications and synthetic preparations have proved similarly disappointing when thus exhibited, although certain guanidine derivatives are still extensively advertised as effective.

2 250 A. D. MACDONALD AND L. WISLICKI A preparation which would lower the blood sugar and not exhibit toxic effects when given by mouth would be of therapeutic value. We have, therefore, thought it worth while to reinvestigate the actions of vegetable extracts which have been reported to have such properties. An extract of cabbage has been used in the first instance. Our experiments have so far been limited by the large amount of material which has had to be subjected to extraction in order to yield a few doses of the active material. METHODS Several methods for the separation of the hypoglycaemia-producing substance have been evolved. We began our investigations with extracts of spring cabbage and savoy prepared according to the method of Dubin & Corbitt [1923]. The vegetables are minced, extracted with 70% alcohol, and the alcohol is evaporated in vacuo. To the remaining aqueous solution, after shaking with ether, four times its own volume of 95% alcohol is added. There separates from this a brown smear which yields a hyperglycaemia-producing extract. To the alcoholic liquor is added three times its own volume of 98% alcohol, and from the precipitate a picrate is prepared. This is decomposed with HCl-alcohol, and further precipitation with ether yields a small amount of a substance which, when administered per os, reduces the blood sugar. The extracts were administered to rabbits through a stomach tube. Blood sugar was estimated by the Hagedorn-Jensen method, sugar in the urine by Benedict's method. Glycogen in the liver was estimated by Pfliiger's method, slightly modified; all such estimations were made immediately after death. The rabbits were fed in the mornings on oats, water and greens, but obtained no food for 24 hr. before being used for experiments. The depancreatized dog on which the extract was also tested was given a measured diet-two meals daily of 200 g. minced meat, 30 g. pancreas, 250 c.c. milk, 7X5 g. sugar and a little meat extract. The cabbage preparation was given with this meat extract. At other times the dog was given 7-9 units of insulin hypodermically before each feed. RESULTS The hyperglyeaemic extract When doses of this extract equivalent to the yield from 1 kg. cabbage are given by mouth to fasting rabbits, their blood sugar rises rapidly. They begin to breathe heavily, and at the peak of the hyperglycaemia

3 CABBAGE AND CARBOHYDRATE METABOLISM 251 become weak, lie on their sides and often die in 5 or 6 hr. after the feeding. We have found up to 4 6% sugar in their urine, and the livers postmortem contained only very small amounts of glycogen. Tables I and II give typical results. TABLE I. Effect of "hyperglycaemic" extracts obtained from 1 kg. cabbage, given by mouth, on the blood sugar of rabbits No. 44 No. 46 No. 47 No. 60 Weight (kg.) Blood sugar (g./100 c.c.) Before After 1 hr ,, , 3,, * ,, ,, Dead Dead Dying TABLE II. Liver glycogen after feeding rabbits with "hyperglyeaemic" cabbage extract No. 44 No. 47 No. 60 Liver weight (g.) Glycogen total (g.) Glycogen (g./100 g.) *74 It appears that, under the action of the extract, the glycogen of the liver is broken down rapidly. When smaller doses (the yield from 500 g. cabbage) are given daily some sustained hyperglyeaemia develops. The morning blood sugar may rise to mg./100 c.c., as shown in Table III. TABLE III. Morning blood sugar of rabbits fed with "hyperglyeaemic" extract of 500 g. cabbage for five subsequent days Blood sugar Days g./100 c.c * *164 It was found that the extracts contained g./100 g. reducing sugar, calculated as dextrose. It seemed unlikely that such a carbohydrate content could produce the blood-sugar changes of Table I. When rabbits were fed with corresponding amounts of glucose, the blood sugar never rose above 212 mg./100 c.c., and had returned to normal in less than 3 hr. No symptoms of hyperglycaemia arose, hence it is concluded that the ingested carbohydrate is not the main cause of the action of the extract. Another possible factor is hydroquinone. Oettel [1936] has found that bilberry leaves contain considerable quantities of hydroquinone which, PH. XCrV. 17

4 252 A. D. MACDONALD AND L. WISLICKI according to Dietering [1938], causes an increase of blood sugar in the rabbit. Since hydroquinone is extracted from aqueous solutions by ether, and as our extracts had been shaken with ether several times, this also cannot explain our results. Some other hyperglycaemia-producing substance is present in cabbage which can be concentrated by suitable alcoholic extraction. The hypoglycaemic extract When this extract, in doses corresponding to the yield of 1 kg. cabbage, is given by mouth to fasting rabbits, the blood sugar falls. This fall varies a good deal in different animals. It usually reaches a turning point in 4 or 5 hr., and the effect may persist for 24 hr. Typical experiments are set out in Table IV. Over forty similar experiments TABLE IV. Effect of "hypoglyeaemic" extracts obtained from 1 kg. cabbage, given by mouth, on the blood sugar of fasting rabbits No. 11 No. 12 No. 17 No. 18 Weight (kg.) *4 2-1 Blood sugar (g./100 c.c.) Before After 1 hr. O 1O ,, 2,, ,, 3,, Pt 4,, ,, ,, TABLE V. Effect of "hypoglyeaemic" extract obtained from 1 kg. cabbage, when given by mouth and by hypodermic injection, on blood sugar in the same rabbit on different days. Weight of rabbit, 2-2 kg. Blood sugar (g./100 c.c.) Per os Hypodermically Before After I hr ,, 2,, ,, 3,, 0O ,, ,, 5,, ,, 24,, have been made. In none of these have convulsions been observed, nor have other untoward symptoms been detected. A series of experiments has been directed to a comparison of the effects of administration by mouth and by hypodermic injection. No marked difference has been found, nor have we seen any evidence of a reaction to the extract at the point of injection (Table V). For convenience we suggest that the name vegulin may be used for this substance which can be extracted from cabbage and causes hypoglyeaemia when given by mouth. The effects of graded doses of vegulin are shown in Fig. 1.

5 CABBAGE AND CARBOHYDRATE METABOLISM 253 We have made preliminary investigations to determine whether animals develop an early tolerance to the action of vegulin. Rabbits were fed with the extract for 5 days, each dose being the yield of 1 kg. cabbage. During the experimental period they were allowed their ordinary 150 Cs 100 ci, E Hours Fig. 1. Effect of different doses of vegulin, given by mouth, on the blood sugar of rabbits. The yield from 0-25 kg. cabbage has little effect; 1 kg. gives a well-marked response, and 3 kg. a profound and prolonged depression of the blood-sugar level.-* 0-25 kg. * 1kg. 3kg. diet, but no food was given in the morning till the sample for blood-sugar estimation had been taken. At the end of this period the response, as set out in Table VI, is not reduced. TABLE VI. Rabbits fed for 5 days with vegulin. Daily dose: yield from 1 kg. cabbage No. 21 No. 22 Weight (kg.) Blood sugar (g./100 c.c.) 1st day 5th day 1st day 5th day Before After 1 hr ,, 2,, ,, 3,,

6 254 A. D. MACDONALD AND L. WISLICKI In the depancreatized dog, kept almost sugar-free with insulin, blood sugar varied between 182 and 90 mg./100 c.c. at different hours of the day. When an attempt was made to replace the insulin by the vegulin yield from 3 kg. cabbage, blood sugar rose to 221 mg./100 c.c., but on doubling this dosage, it fell and remained between 154 and 205 mg./ 100 c.c. The glycosuria was no greater than during the insulin period. 1 Days Fig. 2. Action of vegulin on a depancreatized dog. Both blood sugar and glycosuria are well controlled by the vegulin by mouth. When this is discontinued, the diabetic state develops rapidly, but is controlled by resuming insulin on the eleventh day. On discontinuing the vegulin, blood sugar rose to 320 mg./100 c.c. and glycosuria to 49 g. per diem in 2 days. It appears, then, that the action of insulin in the depancreatized dog can be largely replaced by vegulin (see Fig. 2).

7 CABBAGE AND CARBOHYDRATE METABOLISM 255 The experiments are being continued with a view to purifying the extract and increasing the yield of active substance. It is also intended to investigate its nature and actions as well as its effect on man and in human diabetes mellitus. SUMMARY 1. Two fractions of cabbage have been prepared, both of which affect the blood-sugar level when given by mouth. 2. One causes an increase of the blood sugar in rabbits, accompanied by glycosuria and decrease of the glycogen content of the liver. 3. The other fraction produces Aypoglycaemia in the normal rabbit and can apparently replace insulin in the depancreatized dog. The name vegulin is proposed for this substance. This work was assisted by a grant from the Medical Research Council to one ofus (L. W.). REFERENCES Allen, F. M. [1927]. J. Amer. med. A88. 89, Clementi, A. & Torrisi, D. [1935]. Arch. Fi8iol. 34, 270. Collip, J. B. [1923]. J. biol. Chem. 56, 513; 57, 65. Dietering, H. [1938]. Arch. exp. Path. Pharmak. 188, 500. Donard, E. & Labb6, H. [1933]. C.R. Soc. Biol., Pari8, 112, Dubin, H. E. & Corbitt, H. B. [1923]. J. metab. Re8. 4, 89. Eisler, M. & Portheim, L. [1924]. Biochem. Z. 148, 566. Labb6, H. [1936]. J. Canad. med. A88. 34, 141. Laland, P. & Havrevold, 0. W. [1933]. Hoppe-Seyl. Z. 221, 180. Leclerc, H. [1935]. Progroe. m6d Mark, R. E. & Wagner, R. J. [1925]. Klin. W8chr. 4, Mori, S. & Seikeichi, K. [1934]. Quoted by Moscowitz [1937]. Moscowitz, E. [1937]. Z. klin. Med. 131, 648. Oettel, H. [1936]. Arch. exp. Path. Pharmak. 183, 319. Sippola, P. E. [1927]. Quoted by Laland & Havrevold [1933].

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