INSULIN-LIKE MATERIALS from PLANT TISSUES CHARLES E. BRAUN AND FRANCIS M. REES University of Vermont, Burlington, Vermont
The literature i n thefield of insulin-like materials from have been unable to duplicate their own observations. the more common plant tissues has been reviewed, and the The results of the authors' researches have been Hediscrepancies and erratic results have been presented from sented briefly to corroborate the conclusion of previous a somewhat critical and comparative point of tiew. A n commentators that the results of many investigations i n the attempt has been made to cite some of the experimental field of hypoglycemic plant extracts cannot be accepted at dificnlties encountered i n afield where many investigators their face value.
yeast mown on a medium rich in vitamin D and subcutaneksly administered produced, in several inHE DISCOVERY of insulin in the mammalian stances, a lowering of the blood sugar to the extent of pancreas by Banting and Best in 1922 ( I ) , and 3040% in 3 to 4 hours after the injection. the development of the extraction methods by The preliminary hyperglycemia of yeast extracts and which it could be obtained on a commercial scale by their delayed action as noted by Funk and Corbitt (8) Collip (2) were most significant advances in modern had also been observed by Collip (4, 6 ) , who suggested therapeutics and resulted in lengthened lives for thon- that yeast extracts and insulin might not be identical. sands suffering from diabetes mellitus. It is not surFunk and Corbitt (8) postulated that the material from prising, therefore, that the work in this field has been yeast might slowly undergo transformation into insulin extended. Continued investigation has been along two and thus account for the delayed action. Collip (4, 6 ) . distinct lines, namely, the chemical study of inblin on the other hand, believed that the yeast extract was with a view toward its synthesis, and the preparation another hormone and on this assumption assigned to i t from a readily accessible source of a substance which, the name "glucokinin." when administered orally, would have a physiological Winter and Smith (9), and Hutchinson, Smith, and action analogous to that of insulin. Winter (10) obtained from baker:s and brewer's yeast The results of the chemical investigation of insulin a substance which possessed insulin-like activity, and recently have been summarized in a very complete showed that increased yields of the active material, review by Jensen and Evans (3). which they called glucokinin after Collip's nomenclaI t is the purpose of this paper to present a resume of ture, could be obtained by incubating the yeast for 24 the attempts to obtain an insulin-like substance from hours a t 26' in contact with disodium phosphate and sources other than the pancreas, and to point out some boiled white potatoes before extraction. However, of the literature discrepancies and experimental diffi- despite preliminary incubation, the extracts from variculties encountered in this field. 1ous yeast cultures showed considerable variation in activity, some extracts being completely inactive. YEAST While the convulsions which sometimes followed the inOf the many materials which have been investigated jection of these yeast extracts could be relieved by adas possible sources of insulin, yeast probably has re- ministration of glucose, and therefore suggested a simiceived most attention. This may be attributed to larity to insulin, the yeast extracts always showed a Collip's prediction (4) that, since glycogen formation is delayed action compared to that of insulin. definitely a function of insulin, the presence of glycogen Brugsch and Horsters ( l l ) ,Alzona and Orlandi (12), might be indicative of the presence of insulin or some Simola ( I 3 ) , Shikinami ( I d ) , von Euler (15),and Maesimilar hormone. hara (16) all reported the preparation of active extracts In 1922 Warner, Dixon, and Dixon ( 5 ) obtained a from yeast, including baker's, brewer's, and wine yeasts. substance from yeast which decreased the blood sugar Alzona and Orlandi concluded that the active molecule and diminished the glycosuria in depancreatized dogs. in these extracts must he small since the extracts reCollip ( 6 ) ,following up his glycogen formation hypothe- tained their activity after filtration through a Berkefeld sis, succeeded in obtaining from yeast a substance which candle. Simola suggested that the activity might be lowered the blood-sugar level in dogs. A similar ob- due to a guanidine derivative, althoughinsufficient maservation was made by Fetzer (7). Funk and Corbitt terial prevented a chemical investigation. Collip (6), (8) reported that the injection of sterilized fresh whole had previously considered the possibility of a relationyeast cells produced a hyperglyremir effect, but that ship between insulin, plant extracts, and guanidine. 453 INTRODUCTION
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The suggestion of Funk and Corbitt (8) that there might be some relationship between active yeast extracts and vitamins was revived by the work of Bufano (17) who concluded that the activity of his yeast extracts might have been due to their high vitamin content. Kaufmann (18) observed that extracts possessing insulin-like properties could be prepared from yeast and yeast nucleic acids by extraction with normal bydrochloric acid. From his work with these extracts, Kaufmann concluded that no true enzyme was involved in their physiological activity. Boivin (19),working with three strains of yeast, was unable to produce extracts of high hypoglycemic activity. With solubility and isoelectric-point data as his basis, Boivin concluded that the hypoglycemic substance in yeast is not identical with insulin. Nagai (20) developed a simplified and convenient method for the preparation of active extracts from yeast. The raw yeast was shaken with a saturated solution of picric acid, and alcohol was added to a concentration of less than 60%, whereupon the active principle went into solution as a picrate. It was then precipitated by increasing the alcoholic concentration to over 70%. A hygroscopic, physiologically active substance was obtained when the picrate was treated with sodium phosphate. In 1933, Binet, Fabre, and Bargeton (21)announced the preparation from beer yeast of a substance which had hypoglycemic properties. However, these workers, like Hutchinson, Smith, and Winter (lo),reported that extracts from different batches of yeast varied greatly in physiological activity. Bickel and Nigmann (22) observed that in general the ingestion of 5 grams of dried yeast not only caused a fall in blood sugar but also produced a definite increase in liver glycogen in fasting rabbits. The maximum hypoglycemia was observed 4 to 6 hours after feeding. The yeast preparation was a special yeast powder called "lewrinose." Costa (23),on the other hand, although able to produce only a slight hypoglyceihia in normal rabbits by administration of 5 grams of beer yeast, noted that the hypoglycemic peak, obtained when glucose is injected into rabbits, was retarded. Very recently Costa (23) observed that beer yeast in general exerted a hyperglycemic action, a result contrary to his earlier findings. G b e r and Halpern (24) reported the preparation from boiled yeast-press juice of a substance which they considered to be an insulin activator. The boiled juice itself showed no hypoglycemic effects. The socalled activation took place only in a slightly alkaline medium (pH 7.8). These workers postulated that part of the insulin in regular laboratory preparations is inactivated, therefore cannot exert a physiological effect. A somewhat similar view was held by Kaufmann (18) who was inclined to believe that the active substance in yeast extracts was not produced from the yeast as such but from the yeast nucleic acids. He advanced the idea that an active substance might be expected
wherever carbohydrate development is rapid, and that insulin and insulin substitutes, such as yeast extracts, which he implied might be enzymes of a sort, or activators, show a similarity which can be explained only on the basis that they are derived from a common parent material. BACTERIA
It has been noted that certain investigators working with yeast extracts often obtained wide variations in the hypoglycemic properties of their extracts, some of which were completely inactive. Hutchinson, Smith, and Winter (25),in an attempt to account for the varying activities of their different yeast extracts, advanced the opinion that these differences were probably not due to any variations in the nutritional state of the yeast but possibly were caused by the presence of certain microorganisms, not yeasts, which produced the insulin-like activity of the active yeast extracts. Testing out this theory, they plated a portion of a moderately active yeast on nutrient agar and succeeded in isolating a pure culture of a bacillus. The latter was grown in a peptone-glucose-phosphate medium a t 37'. Extracts of this bacillus in doses of 0.06 milligram per gram of body weight produced a marked lowering of the blood sugar in rabbits, and in some cases caused convulsions which could be relieved by the injection of glucose. Since extracts of the sterile medium were entirely inactive, it was concluded that the hypoglycemic material, which they called glucokinin, had been formed by the action of the bacillus. The active material produced in this way and that obtained from yeast were similar in that both were slower acting than insulm, the hypoglycemic convulsions usually occurring about six hours after injectiofi. Hutchinson, Smith, and Winter, although unable to identify the organism employed in their experiments, described it as being a short coli-form bacillus which produced acid and gas from glucose and lactose in twelve hours but did not ,liquefy gelatin. They were unable to obtain an active substance from a strain of Bacillus coli. Following this lead, Little, Levine, and Best (26), Levine and Kolars (27), Simola (13), and Shikinami (14) reported the production of a hypoglycemic substance from bacteria, living and dead, including Bacillus coli, Bacillus subtilis, and Streptococcus hewzolyticus. Menteti and Kipp (28) observed that, in large doses, paratyphoid B toxin produced in normal dogs a hyperglycemia which was followed by a fatal hypoglycemia. In depancreatized dogs, large doses produced an early and progressive hypoglycemia endmg in death. Linton (29), investigating Streptococcus hemolyticus, reported that in rabbits, after intravenous injection, the blood sugar dropped at a constant rate but never reached a true hypoglycemic level. Delafield (30) was able to lower the blood sugar of fasting rabbits by as much as 50-milligram per cent. by injecting suspensions of dead Bacterium aertrycke. When the suspension was autoclaved prior to injection, the hypoglycemic effect was diminished. The hypo-
glycemia which followed the injections of suspensions of dead Bacterium aertrycke, or of the bacterial filtrate was preceded by a definite hyperglycemia, the increase in blwd.sugar being as great as 70-milligram per cent. in some cases. Delafield further demonstrated that dead Gram-positive organisms had no effect upon the blood sugar but that Gram-negative organisms have a definite effect upon the blood sugar and blood inorganic phosphorus. The usual result following injections of dead Gram-negative organisms was a hyperglycemia accompanied by diminished phosphorus content 2 hours after injection, followed by hypoglycemia and a high phosphorus content 24 hours after injection. The demonstration of the production of a hypoglycemic material through a bacillus, and the hypoglycemic properties of certain bacteria, living and dead, suggest that the insulin-like properties of yeast extracts might be due not to some material within the yeast cell itself, but to the presence of some bacillus or organism associated with the yeast, and thus might account for the variations observed in the physiological activity of different yeast extracts and of extracts from different strains of yeast. In view of these findings it is rather difficult to say definitely that yeast tissue itself contains a material which possesses insulin-like activity. HIGHER PLANTS
Many plant tissues other than yeast have been investigated as possible sources of insulin or a material having insulin-like properties. Collip (4, 6) in 1923 reported the preparation of active extracts from onion tops and onion roots. Janot and Laurin (31) in 1930, and Laurin (32) in 1931 obtained active extracts from the bulbs of common onions (Allium cepa Z.). Laland and Havrevold (33)* in 1933 reported the results of their intensive investigation on onion (garlic, Allium sativum) extracts which they found produced marked hypoglycemia in normal rabbits and .depancreatized dogs by either oral or intravenous administration. The maximum blood-sugar lowering occurred bet$een 1and 5 hours after administration, showing a physiological action much slower than that of insulin. They succeeded in isolating from their active extract a crystalline compound, volatile with steam and ether-soluble, which melted a t 174O, contained no sulfur, andgave the characteristic color and precipitation reactions of alkaloids. This compound was active physiologically only when combined with the sulfides commonly present in onions. It should he pointed out that many of the extracts of Laland and Havrevold were inactive, and even the active preparations lost their activity upon standing in an ice chest from 1to 10 days despite the addition of preservatives. The most active extracts were ohtained from onions (garlic) which had been stored for six months.
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* In the bibliography of
the paper by Laland and Havrevold The name Sippala has heen used instead of Sirnola, both in th? biblioma~hvand throughout the text (33), reference No. 2 is recorded incorrectly.
Best, Jephcott, and Scott (34), using a method developed for the preparation of insulin from pancreas, were unable to obtain any physiologically active extracts from onions. In this laboratory, applying the method of Laland and Havrevold (33), the authors failed to obtain any active extracts from stored onions, garlic, onion tops, or bulbs and roots of growing onion sets. Thalhimer and Perry (35) in 1923 observed a hypoglycemic effect following the injection of raw potato juice. Best and Scott (36), Simola (13), and Laland and Havrevold (33) reported the preparation of active extracts from potatoes. Contrary to these findings, Hutchinson, Smith, and Winter (10) prepared extracts from raw potatoes which were physiologically inactive. Dubin and Corbitt (37) and Best and Scott (36) in 1923 reported the preparation of hypoglycemic extracts from celery. Best and Scott (36) and Sammartino (38) obtained active preparations from heet roots and beets (Beta oulgaris), respectively. Very recently Rychlik (39) reported that alcoholic extracts of dried beets produced marked hypoglycemia in dogs following suhcutaneous administration. On the other hand, Dubin and Corbitt (37) and Best, Jephcott, and Scott (34) were unable to obtain any hypoglycemic material from not dried beets. Franke, Malczynski, Giedosz, and Onysymow (40). bv fractionatinz the oetroleum ether extract of vacuumdked carrots, :btaiied an amorphous yellow mate% which, in almond oil, produced definite hypoglycemia when subcutaneously injected into men, rabbits, and dogs. Dubin and Corbitt (37), using a differentmethod of extraction, failed to obtain any active material from carrots. Extracts possessing hypoglyceniic properties were reported to have been prepared from lettuce by Collip (4, 6), and by Dubin and Corbitt (37). The presence of an insulin-like material has been reported in various green leaves other than lettuce. Collip (4, 6) obtained an active extra'ct from green wheat leaves and ordinary lawn grass. Simola (13) reported the preparation of a hypoglycemic principle from green rhubarb leaves. Sammartino (38) obtained a similar material from blue grass (Poa firatensis) and endive (Cichorium endiwia). Dubin and Corbitt (37) and Sammartino (38) prepared hypoglycemic extracts from spinach (Spimcia oleracea). Dubin and Corbitt (37) in 1923, and Erderer in 1927 (dl), reported the preparation of extracts from cabbage which produced hypoglycemia. However, as in the case of most plant extracts, the hypoglycemia was preceded generally by a hyperglycemia. I t was mentioned previously that Collip (4, G), who first noticed these two effects of plant extracts, concluded that they were produced not by insulin, but by a new plant hormone which he named "glucokinin." In their investigation of plant extracts, Duhii and Corbitt (37) succeeded in separating the active material into two fractions, one hyperglycemic and the other hypoglycemic. The latter was reported to be very
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similar to insulin in its physiological effects and produced no preliminary hyperglycemia. The fractionation depended upon changing the alcoholic concentration from 80y0, where the hyperglycemic principle was precipitated, to 93%, which caused the precipitation of the hypoglycemic principle. The latter was further purified by precipitating i t as a picrate or dinitrosalicylate and then decomposing the precipitate with alcoholic hydrochloric acid. The purified hydrochloride, after precipitation from the alcoholic solution with ether, was injected into normal rabbits for physiological assay. The hypoglycemic material could also be separated from the hyperglycemic fraction by adsorption upon charcoal or kaolin (37, 41). Therefore, it would appear from the work of Duhin and Corhitt that the "glucokinin" of Collip is not a new plant hormone but is a complex material containing two antagonistic factors, one hyperglycemic and the other hypoglycemic, which can he separated by fractional precipitation with alcohol. The authors, in this laboratory, employing the extraction methods of Collip (6) and also of Dubin and Corbitt (37), were unable to obtain any hypoglycemic extracts from cabbage in twelve attempts. Furthermore, it was found that the 93Y0 alcohol precipitate, which Dubin and Corhitt (37) described as heing a "grayish-white" material, turned almost immediately to a brown viscous simp upon the slightest exposure to the air. This material, protein-like in chemical behavior, appeared to he exceedingly hygroscopic. The same observation was made when working with the socalled hydrochloride of the hypoglycemic principle. The SOYo alcohol precipitate, obtained as described by Duhin and Corbitt (37) as a brown, sirupy mass, contrary to the observations of these investigators, failed to produce a marked hyperglycemia when administered subcutaneously to normal fasting rahhits in doses as high as 2 grams per kilo. Chemical investigation of this material showed it to consist chiefly of carbohydrate. It reduced Fehling?s solution, gave a positive Molisch reaction, a positive Bar$oed reaction, and formed an orange-yellow phenylosazone which melted a t 205.5O after recrystallization. A mixed melting-point determination with phenylglucosazone (m. p. 204-205O) gave 205'. Sodium fusion of the 80% alcohol precipitate showed the presence of sulfur and phosphorus and the absence of nitrogen. An examination of the ash revealed the presence of sulfate and phosphate. From its general chemical behavior, its solubility in water and insolubility in alcohol, and from the result of the mixed melting-point determinations, the authors concluded that the 80% alcohol precipitate from cabbage extracts consists chiefly of glucose. Cereals and grains have been investigated as possible sources of insulin. Collip (4, 6 ) announced the preparation of a hypoglycemic material from barley roots and sprouted grain. Best and Scott (36) and Kaufmann (42) obtained an insulin-like action from wheat extracts. Simola (13) and Kaufmann (42) reported an active principle in oats and oat hulls, and
Kaufmann (42) noted similar results with rye extracts. In a subsequent investigation, Kaufmann (43) showed that the hypoglycemic activity of these cereal extracts increased in the order of rye, wheat, and oats. Best andScott (36),in 1923, reported the preparation of a hypoglycemic substance by extracting rice with 80y0 alcohol made 0.35Yo acid with hydrochloric acid. Funk and Corbitt (8) obtained only a hyperglycemic effectfrom crude extracts of rice polishings. In this laboratory the authors, using polished rice, the whole kernel and also pulverized whole rice, and employing the extraction technic of Best and Scott (36), failed in eleven experiments to obtain a single extract which showed hypoglycemic properties when subcutaneously administered to normal fasting rahhits. Since the failure to obtain an active material from rice has been substantiated by work done in Dr. Best's laboratory (44),it appears that rice is not a source of an insulin-like material despite the report in the literature to the contrary. The legumes have been reported to be possible sources of insulin-like materials. Collip (4, 6 ) prepared active extracts from bean tops. Brugsch and Horsters ( l l ) , Eisler and Portheim (45), and Kanfmann (42, 46) reported the preparation of hypoglycemic extracts from lentils, peas, beans (Phaseolus vulgaris), bean pods, and bean husks. Among the miscellaneous plant sources which have been claimed to be productive of insulin-like materials are the following: turnips, mushrooms, and horseradish were reported to yield a hypoglycemic principle by Glaser and Wittner (47). Fisher and McKinley (48) obtained insulin-like substances from the juice, pulp, and rind of citrous fruits (oranges, grapefruit, and lemons). Marx and Alder (49) showed that the administration of an extract of nettles (Herba urtica dioic?) to rabbits with an adrenaline or alimentary hyperglycemia produced a lowering of the blood sugar. Kaufmann (50) prepared hypoglycemic extracts from jamhul bark. Solanaceous plants, ' Solanum indicum and Solanum sanitwongsei, when taken orally were reported to reduce the glycosuria in diabetics (51). However, subsequent investigations by Long and Bischoff (52), by Bulger (53), and by Kleiner (54) failed to show the presence of a hypoglycemic substance in these plants. MYRTILLIN
A plant extract of unknown constitution under the name of "myrtillin" has received considerable attention as a therapeutic agent in the treatment of diabetes. Myrtilliu is the hypoglycemic fraction of an alcoholic hydrochloric acid extract of the dried green leaves of the whortleherry or the blueberry, and was first prepared from whortleberry leaves by Mark and Wagner (55). The name myrtillin was assigned on the basis that the whortleherry and the blueberry were members of the myrtle family. This nomenclature is unfortunate for two reasons: First, neither plant is a myrtle, the whortleberry heing Vaccinium uliginosum L. and the hlne-
berry an Ericacre. Secondly, myrtillin was the name cal activity. Sometimes the plant juice was pressed given by Willstatter (56) to the anthocyanin obtained out and treated directly with picric acid or ammonium from the skins of whortleberries, and to which he as- sulfate to precipitate the active principle. The picrate signed the formula C.22H21012C1 for myrtillin chloride. then was usually converted into the hydrochloride with From the latter he prepared the anthocyanidin myr- alcoholic hydrochloric acid. Very few attempts were tillidin chloride, C,eHuOrC1, which has the following made to purify the active material other than to prestructure: cipitate it as its picrate or dinitrosalicylate or through preferential adsorption. In most cases the physiologiOCI OH cal assay was not made upon a dry-weight basis but upon the effect produced by an extract from a given weight of starting material of unknown moisture content. In this connection it seems of interest to mention the statement made by Dubin and Corbitt (37) that OH "one kilo of vegetable yields approximately 0.100 g. Myrtillidin Chloride of the crude uavish-white nreci~itate"which in turn produces "ab&t'0.010 g. of the very potent hydrochloMyrtillin was reported to reduce alimentary hyper- ride." Thus, despite not only the seasonal variations glycemia in normal dogs after oral administration, an in water content of any plant but also the difference in observation confirmed by Eppinger, Mark, and Wagner water content of different plants, i t is reported that the (57), Allen (58), Shpiner (59), andothers. Totally de- yield of hypoglycemic material per given weight of pancreatized dogs could be kept alive for long periods "fresh vegetable" tissue is approximately constant. by the administration of myrtillin. It even has been Very little work has been done on the chemical naused clinically on diabetic patients with some degree of ture of these plant extracts. For the most part invessuccess in mild cases. tigators report that insufficient material prevented a Several rather abnormal properties have been as- chemical examination. From the reports available, sociated with myrtillin. Its hypoglycemic action is the active principle appears to act like a protein. I t is variable and weak compared to that of insulin, which nitrogenous, shows certain typical protein reactions, makes it useless in acidosis and coma. It does not and has been precipitated from the plant juices, in some appreciably change the normal fasting blood sugar and cases, by ammonium sulfate and picric acid. However, does not produce a hypoglycemia in normal dogs. It these chemical characteristics are not accepted by all is reported by Allen (58) to be ineffective in reducing workers in this field. The only chemical individual hyperglycemia in rats and rabbits. I t is active by oral which has been isolated from plant extracts claimed to administration but only in relatively large doses, and possess hypoglycemic properties is the physiologically exerts a prolonged effect sometimes lasting for several inactive, crystalline compound melting a t 174' isolated days. No satisfactory explanation has been offered from onion extracts by Laland and Havrevold (33), for the generally observed variable physiological ac- and reported by these investigators to be alkaloidal in tivity of myrtilliu. No claims are made that it is simi- nature. lar to insulin or a substitute for it. Mvrtilliu merelv CONCLUSION a ~ ~ e ato r sbe an aid in carbohydrate metabolism and tiirefore might be of value in the treatment of diabetes. From the foregoing discussion it. is apparent that I t was mentioned above that myrtillin was said to be there are many discrepancies and cbnflicting results ineffective in rabbits with alimentary hyperglycemia, recorded in the literature covering the field of insulinbut no explanation for this has been advanced. In like materials in plants. Best, Jephcott, and Scott (34) this laboratory the authors were able to show that the in 1932 considered this situation and advanced the alimentary hyperglycemia produced in normal fasting idea that in many cases the physiological assay had been rabbits by the ingestion of 10 grams of glucose per kilo interpreted incorrectly and a hypoglycemic action reis depressed distinctly but very variably by the oral ported where none actually existed. This argument readministration of one gram of myrtillin* per kilo of ceives support from the work of Scott (60) who showed body weight. from 1625 observations on 215 rabbits that the blood sugar of fed normal rabbits varies appreciably and has PREPARATION AND CHEMISTRY OF PLANT EXTRACTS a mean value of 123 milligrams (glucose) per 100 cc. The preparation of the various plant extracts re- of blood with amean deviation of 13. Best, Jephcott, ferred to above was accomplished in general by first and Scott also consider contamination with insulin as treating the plant tissue with steam or freezing it with being responsible for many reports in the literature that carbon dioxide snow and then extracting with either plant extracts produced hypoglycemic effects, and acidified alcohol or water. For the most part the ex- point out that in several instances, subsequent work tracts were concentrated in vacuum a t low tempera- by the same investigators failed to con& their pretures, and the concentrate tested directly for physiologi- vious results. As a third explanation for the erratic * The myrtillin used in this work was obtained from the Cole reports in the literature, Best, Jephcott, and Scott cousider the possibility of contamination of the plant exChemical Co., St. Louis, Mo.
