3,031,376 United States Patent Office Patented Apr. 24, 1962 2 the performance all-out test, as well as on the naive stand 3,031376 ardized cardiovascular tests. CoMPOSITIONs coveristNG ocTAcosANOL, Despite the difficulty of improving the T-wave, during TRACONTANOL, TETRACOSANOL, OR HEXA COSANOL, AND METHODS EMPLOYING the twelve weeks of pretraining the T-wave improved SAME 1.12 mm., and during the six weeks of wheat germ oil Ezra Levin, 1109 W. University, Champaign, Ill., feeding improved 2.90 mm. The controls (the non-wheat Vernon Kirkpatrick Collins, Champaign, Dwight Steve germ oil group) averaged a loss of 0.13 mm. in the first Warner and John Darwin Mosser, Monticello, and twelve weeks, and a further loss of 1.54 mm. during the George Wolf, Urbana, Ill; said Collins, Varner, last six weeks. Mosser and Wolf assignors to said Levin O In 1953 studies were carried out on groups of middle No Drawing. Filed Oct. 11, 1956, Ser. No. 615,222 aged men. One group was on a dosage of wheat germ 10 Claims. (Cl. 167-65) oil and the other group took lard plus vitamin E equiva This invention relates as indicated to nutritional com lent of wheat germ oil. While men competed in the tests postions and their use, and more particularly to compo against their own scores, there was no competition against sitions which contain as an active component tetracosanol, 5 opponents. Furthermore, the work was carefully grad hexacosanol, octacosanol, and triacontanol, or their esters, uated to prevent the men from working too hard at first. either singly or in combinations thereof. This applica The group results showed significant advantage for the tion is related to co-pending case Serial 529,362, filed experimental subjects who took wheat germ oil over those August 18, 1955. who did not in both performance (willpower dominated) Within the recent past it has been learned that the 20 tests and in naive (non-willpower) tests. The physiolog value of wheat germ oil as a nutritional adjunct or sup ical advantage was shown in terms of running endurance plement goes beyond the attributes of vitamin E of which in "all out' treadmill runs, T-wave of the electrocardio it is a principal source. This conclusion is based upon gram, lower systolic blood pressure, the Schneider Index, various experiments carried on over the past years, some and the Illinois total body reaction-time tests in response of which have been noted in the co-pending application 25 to light, sound and combined signals. The next year the above identified. experiment was repeated with the subjects reversed. An early indication of this arose from an experiment Most of the men in the wheat germ oil group of the first carried out in 1949 at the University of Illinois on six year were given lard and vitamin E, and the men who groups of college students, three groups of eight, and were on lard in the previous year were given wheat germ three other groups of nine each, in basic swimming tests. 30 oil. The repeat experiment confirmed the results in the Significant changes in the T-wave (highest in lead first experiment, showing increased heart response and CRIvor v) of the electro-cardiogram were noted, as were physical endurance for groups taking wheat germ oil. improved responses in the Illinois vertical jump reaction Other tests have indicated the estrogenic, androgenic, time test, both of which favored raw unrefined wheat and gonadotrophic activity of wheat germ oil. Still other germ oil administered in capsules over the effect of vita 35 tests have suggested that wheat germ oil increased the min E (150 mg/week) encapsulated with refined corn glycogen in the testes or uterus. oil of equal calorific content. The synthetic vitamin E Because the tests with wheat germ oil show that it is (alpha tocopherol acetate) of the corn oil capsules was of value as a supplement to the diet, studies were insti calculated to equal the vitamin E of the wheat germ oil tuted to determine to what constituent or constituents capsules. The control groups were considered to be those 40 of wheat germ oil the physiological responses are on the vitamin E with refined corn oil. None of the sub attributable. jects knew what he was taking and the administration of The invention relates to the discovery of certain phys the capsules was carefully supervised. iologically active components in raw wheat germ oil to which these results are attributable, and among its ob Again in 1950-51, an advantage was shown for a group jects are the disclosure of such components heretofore of six men who were fed raw unrefined wheat germ oil in 45 unknown, to teach how they may be duplicated, or po dosages of twenty 3-minim capsules per day. The six tentiated, and to teach useful ways of applying these experimental subjects were put in hard physical training discoveries. for twelve weeks. During this interval, two were given It is a further object of the invention to provide com diet supplements of wheat germ oil, mentioned above. positions that may be relatively inexpensively synthesized At the end of this period, no further improvement on an 50 on a commercial scale which produce some of the same all-out bicycle ride test, nor on the brachial pulse wave physiological results as obtained with wheat germ oil. test or Schneider test was observed. A plateau had been It is another object of the invention to provide for reached in the physical response curve of each subject. the treatment of human beings and animals to obtain For the ensuing six weeks, two more of the subjects certain improved physiological responses and benefits were fed raw unrefined wheat germ oil capsules (as 55 therein. above), while the two remaining control subjects were To the foregoing and related ends, said invention com deprived of wheat germ oil. At the end of this period, prises the features hereinafter fully described and particu all groups were retested. The initial all-out bicycle test larly pointed out in the claims, the following description setting forth in detail certain illustrative embodiments riding times average of 247.8 seconds of the first group 60 of the invention, these being indicative, however, but of a improved after the twelve weeks of hard training to few of the various ways in which the principle of the 309.2 seconds, and improved again to 365.3 seconds dur invention may be employed. ing the six weeks wheat germ oil was fed. The control In accordance with the foregoing objects it has been subjects without wheat germ oil averaged 165.6 seconds discovered that some of the value that has been at times initially, 136.8 seconds at the end of the twelve weeks' 65 attributed to the vitamin E content of wheat germ oil is training period, and 172.8 seconds at the end of the next really due to some other factor. In the experiments six weeks' period, or a total improvement of only 3.74% conducted in the recent years showing the increased for the controls; whereas the subjects on wheat germ oil physical fitness in subjects who have taken what germ oil, improved 24.8% during the preliminary training period, the control groups have been administered vitamin E and 47.4% over the entire period. 70 without the same results that are attained with the use Favorable results for wheat germ oil were shown on of raw unrefined wheat germ oil. 8,031,876 3 4. The active component in wheat germ oil was dis ondary, alcohols, for example, 1-tetracosanoi, 1-hexa covered by the following chromatographic procedure: cosanol, 1-octacosanol, and 1-triacontanol. Esters which may be employed are 1-octacosyl acetate, 1-octacosyl Example 1 propionate, 1-triocontyl acetate, 1-tetracosyl acetate, 1 An unsaponifiable fraction of wheat germ oil was made hexacosyl acetate, 1-octacosyl phenoxyacetate, di-(1-tri in accordance with established methods by treating the ocontyl) phthalate, di-(1-octacosyl) succinate, and 1 oil with an alcoholic solution of potassium hydroxide at octacosyl phosphate. The foregoing compounds may be low temperatures and extracting the fraction from the used either singly or in admixture. Soap. The physiologically active compounds are associated Activated alumina was allowed to absorb moisture until 0. in the composition with a suitable compatible, non-toxic it reached about 4% moisture. This was made into a carrier. The carrier may be either a solid or liquid mate slurry with hexane and poured into a chromatographic rial, such as food or a pharmaceutical preparation. The tube with sintered glass bottom. Dimension of the compositions may take the form of tablets, powders, column of alumina in use was 76 mm. diameter by 330 capsules, or other dosage forms which are particularly mm. high. Before all the hexane had run down to 5 useful for oral ingestion. Also, they may be in suitable expose the alumina, the solution of unsaponifiable mat preparations for parenteral use. The compositions may ter in hexane was poured on, using about 20 grams of take the form of the above identified active compounds unsaponifiable matter per column. After the unsaponifi admixed with solid diluents or tableting adjuvants such able matter and hexane reached the top of the alumina, as corn starch, lactose, talc, stearic acid, magnesium a 50% mixture (by volume) of hexane-benzene was 20 stearate, gums, or the like; also, any of the other poured on the column without disturbing the unsaponifi tableting materials used in pharmaceutical practice or able portion on top of the column. No fractions were vitamins, hormones, and the like, where there is no iu collected until a bright orange band began to elute off compatibility with the active compounds. Alternatively the bottom of the column. This fraction required ap the active compounds may be placed in the usual capsule proximately 1 liter of 50% benzene-hexane. The 50% 25 such as a gelatin capsule and administered in that way. benzene-hexane level was then allowed to come down to In yet another embodiment the active compounds may the top of the alumina column, and benzene was added be put in powder packets. to fill the column to the top. A second fraction was The active compounds disclosed above are relatively obtained until the benzene just began to come out the insoluble in oil and water. Therefore, solubilizing and bottom of the column. Then a third fraction was col 30 dispersion agents are used. Examples of solubilizing lected having a volume of about 1 liter/column. agents for preparing oil solutions are the phosphatides, This third fraction was stripped over N under vacuum such as lecithin, and the sterols. Any solubilizing agent until free of benzene (by odor). A very few cc. of is suitable in the composition that will increase the solu petroleum ether was added to the flask and brought to bility of the active compound in the oil carrier, is com a boil. This was then quickly filtered while hot through 35 patible with the active compound, and is non-toxic. Any a coarse filter and the filtrate placed in the freezing of the well known available animal and vegetable oils, compartment of a refrigerator overnight. Crystalliza such as the refined vegetable oils, may be employed as tion occurred and the crystals were filtered off while cold. a liquid carrier, for example, refined cotton seed oil, They were light brown in color and slightly oily. They refined corn oil, and the like. were then recrystallized several times from hot acetone, 40 Dispersion agents may be used to form transparent giving a white waxy crystalline product which was aqueous emulsions. Suitable dispersion agents are the identified as a mixture of 1-octacosanol and 1-triacon surface active agents, such as a mixture of polyoxy tanol. These compounds proved to be physiologically ethylene ethers of mixed oleic esters of sorbitol anhy active in tests comparing them with wheat germ oil. drides, sold under the trademark "Tween 80' by the Further tests have shown that certain derivatives of 45 Atlas Powder Co. Alternatively, the active alcohols may 1-octacosanol and 1-triacontanol, and certain homologues, be rendered soluble by formation of chemical compounds are also active and produce the physiological improve thereof such as the phosphates and phosphoryl choline, ments characteristic of raw unrefined wheat germ oil. which are sufficiently polar to increase the solubility of Broadly stated, the present invention relates to a com the alcohol. position containing one or more physiologically active 50 The active compounds may be present in the compo compounds of the class consisting of tetracosanol, hexa sition in minimum amounts which range from traces to cosanol, octacosanol, triacontanol, and the esters there more than 0.15 percent by weight, the latter of which of. This invention also relates to the method of ad is greater than it appears in wheat germ oil. For ad ministering the foregoing composition to humans and ministering these compounds, the dosage for a human animals for reducing anoxia and/or improving physical 55 being may be as low as 0.05 mg. or as much as 150 mg. endurance and reducing fatigue, and/or stimulating or per day of the active compounds. No adverse effects improving heart response. have been noted if excess amounts are taken. How The physiologically active compounds which may be ever, good responses are obtained when dosage is main used in the composition of the invention have the tained at levels ranging from about 40 mg. to about formula: 60 80 mg. The alcohols, tetracosanol, hexacosanol, octacosanol, and triacontanol, and the esters thereof, prove to be wherein R is an alkyl radical having 24, 26, 28, or 30 physiologically active in the laboratory, as well as clini carbon atoms, such as tetracosyl, hexacosyl, octacosyl, cally. or trioconty; and X is either hydrogen or an acid radical. 65 One method of testing for physiological activity is a The acid radical may be the residue of an organic modification of Dorfman's androgenic assay, using the or inorganic acid. Examples of suitable organic acid chick-comb as a test object. Dorfman's method is given radicals include those of a monocarboxylic acid such in detail in Endocrinology 42, 1 (1948), and Endo as phenoxyacetic acid and benzoic acid or a short chain crinology 42, 7 (1948). In general it consists of apply 70 ing a small measured amount of an oily solution of the fatty acid such as acetic acid and propionic acid, or a substance to be tested directly to the chick-comb daily for dicarboxylic acid such as succinic acid and phthalic acid. a period of days, then sacrificing the birds and dissecting Examples of suitable inorganic acid radicals include off the combs and weighing them. The ratio of comb those of orthophosphoric acid. Alcohols of the fore weight to body weight is calculated for each bird, and going include the monohydric saturated, primary or sec 75 an average, with statistical analysis, is calculated for each 8,031,876 5 6 8. group. The difference between the treated and nega length of survival of rats on the various diets was as tive control groups is calculated, together with the sta follows: tistical significance. Tests employing Dorfman's andro genic assay and modifications of Dorfman's androgenic Average Change Dietary Group ength, of from assay with 1-octacosanol, 1-octacosyl acetate, 1-octa Survival, Basa cosyl propionate, 1-triacontanol, 1-hexacosanol, and Minutes 1-tetracosanol, demonstrated that these compounds were active in producing a response on chick-combs at ex Group -Cottonseed oil- 117------Group I-Wheat Gerra Oil- X - a - 86 59.0 tremely high dilutions in vegetable oil. Group II-Cottonseed oil and 1-octacosanol. 154 31.6 Gonadotrophic effect was also shown in an experiment O with rats. A purified diet was fed the animals. Supple Findings indicate that wheat germ oil and compositions mentary diets were fed two groups: the control group of the invention significantly increased the average length received refined cottonseed oil; the second group refined of survival of rats under conditions of anoxic anoxia as cottonseed oil containing 1-octacosanol. The second compared to that of animals fed a similar diet contain group showed that 1-octacosanol increased the size of the 5 ing cottonseed oil. The average increase of the wheat testes and seminal vesicles. germ oil group was 59% greater than the cottonseed In another test, female guinea pigs were selected at oil series. The compositions of the invention were 31.6% 200-250 gm. in body weight, divided into three groups greater than the cottonseed oil series. (12 animals per group) and all fed a basal ration with In a further test 22 men were divided into 2 very care each group receiving a supplementary diet as follows: 20 fully matched groups: Group A (12 men) received 1-octacosanol dissolved Group I-2.2 mg. of 1-octacosanol (in cottonseed oil) in cottonseed oil, each subject getting sixty minims of per kg. of diet of a basal ration. cottonseed oil daily. Group II. Wheat germ oil at a 2 percent level in the Group B (10 men) received the placebos which con diet of a basal ration. 25 tained cottonseed oil. These subjects received sixty Group -Cottonseed oil at a 2 percent level in the minims of cottonseed oil identical with group A but not diet of a basal ration. containing 1-octacosanol. The original measurements before any supplement was Food and water were provided ad libitum. Swim provided dealt with the mile run and the 14 lengths ming tests were conducted after 28 days of feeding at 30 swim, push-ups, squat jumps, and several other measure a water temperature of 37 C. A weight equal to 5% ments. These tests were carried out only twice-at the of the animal's body weight was attached to the hind beginning and the end of the experiment. The T-wave limbs (2%% on each hind leg). The length of time measurement on the electrocardiogram was also taken. that animals swam was determined for each guinea pig. Each day the 22 men were mustered out and the 12 men The swimming tests were discontinued after two hours 35 in group A were given the 1-octacosanol dissolved in for those animals still swimming at the end of this time cottonseed oil and the 10 men in group B were given and data were calculated on the basis of a swimming cottonseed oil. Direct observation was made to be sure time of 120 minutes for those animals still swimming that the proper individuals in each group were given the after this time. Findings clearly indicate that guinea supplement. The subjects did not know what the Sup pigs fed 1-octacosanol at a level of 2.2 mg. per kg. of 40 plements contained or the purpose of the study. diet had a significantly better swimming performance The following table summarizes the percentage differ than animals fed an identical ration with the alcohol omit ence (improvement) for various activities: ted. Seven out of twelve animals in group I fed the 1-octacosanol (58.3%) swam for over one hour in con Group A Group B trast to two out of nine animals (22.2%) in group II 45 fed the basal ration without 1-octacosanol. Guinea pigs '' Difference, 't' Difference, fed wheat germ oil at a 2% level in the diet also swam Percent Percent longer than those on the basal ration, although cotton Mile run------3.07 17 3.65 1. 50 466 yard swim 4.19 16 2.91 17.7 seed oil when fed at a comparable level was without Push-ups.--. 7.39 14 .0 9.5 protective effect. Squat jump--- 5.0 14.5 2.09 1. Vertical jump- 3.06 4.9 73 - In still another test female rats were selected at 21 Total Strength------2.9 7 1.24 5.0 to 23 days of age and between 38 and 50 gms, in body Strength?weight.--- 3,70 2 82 8.5 Step test Brouha 3.21. 18.0 2.32 15 weight. All the animals were fed a basal ration, and Breath holding- 2.55 40 ... 4 11.5 were divided into three groups (12 rats per group) for 55 ECG R. wave. 2.34 1,29 12.2 different supplementary diets, as follows: In a further experiment four matched groups of boys Group I-Basal ration and 2.5% cottonseed oil. were given different dietary supplements: Group II-Basal ration and 2.5% wheat germ oil. Group A-Cottonseed oil and 1-octacosanol Group III-Basal ration and cottonseed oil containing 60 Group B-Cottonseed oil placebos 1-octacosanol, equivalent to 0.25% wheat germ oil. Group C-Wheat germ Group D-Wheat germ oil After several months' feeding their resistance to anoxic anoxia was tested. The experimental procedure The time for the 600 yard run was measured at the be was as follows: Animals were placed in a series of de 65 ginning and the end of an eight week period. The re compression chambers and the oxygen pressure was ad sults are as follows: justed to simulate various altitudes. During the first half Average time in seconds hour animals were raised from sea level to a simulated Beginning End Gain, altitude of 30,000 feet. Thereafter they were gradually 70 Percent raised to 32,500 feet during the next 15 minutes. For the balance of the experiment the oxygen pressure gradu Group A------52.8 37.1 10.27 Group B.-- 156.0 149.7 4.04 ally changed to correspond to an increase of about 1,000 Group C- 156. 146,4 6.63 feet per hour. The experiment was terminated six hours Group D- O 53.5 39.7 8.99 after the start of oxygen decompression. The average s 8,031,876 7 8 The following examples illustrate a few preparations oxygen for the removal of the lactic acid, maintenance of that may be used as nutritional supplements, of which normal irritability, production of oxidative energy produc the active components are tetracosanol, hexacosanol, tion, and for the restoration of the anaerobic energy octacosanol, triacontanol, or the esters thereof. It is yielding systems. well understood that the inventions are not limited to At the outset of exercise, time is required for adjust these preparations, which are only given by way of ex ment of the circulation and respiration to meet the in ample, and may be modified by taking into considera creased oxygen demand. During this period of relative tion the purposes of the invention: oxygen insufficiency, lactic acid in the blood rises, in Exa, aple 2 dicating that the muscles are deriving energy from gly O colysis. Later the blood lactic acid may be unchanged A solid composition of a tablet dosage for human or fall. After exercise, the oxygen consumption does not consumption: return at once to the resting level. There is oxidative Gram recovery, associated with removal of lactic acid and 1-octacosanol------0.065 the refilling of the energy stores. Lactose ------0.170 15 It is believed that the compounds of the present inven Starch ------0.015 tion are effective during periods of anaerobic glycolysis Stearin ------0.005 when the lactic acid content ordinarily rises. They may Example 3 possibly aid in the removal of lactic acid by increasing the A solid composition of a tablet dosage for human con effectiveness of the tricarboxylic acid cycle which oper sumption: 20 ates on the oxidative, energy-yielding reactions or in the Gram electron-transporting phosphorylating reactions connecting 1-octacosyl acetate------0.101 the cycle to the oxygen supply. This may occur by in Lactose ------0.300 creasing oxygen uptake or by reducing the amount of Starch ------0.030 oxygen required. This may be accomplished either by 25 catalysis or in engaging in one or more of the many re Stearin ------0.004 actions or through changes in cell permeability. Example 4 Other modes of applying the principle of the invention A liquid composition for human consumption: may be employed, change being made as regards the de Grams tails described, provided the features stated in any of the Refined cottonseed oil------3 30 following claims or the equivalent of such be employed. Lecithin ------9 We, therefore, particularly point out and distinctly 1-octacosanol ------0.144 claim as our invention: 1. The method of increasing oxygen utilization in Example 5 human beings which comprises administering to human A liquid composition for human consumption: 35 beings a pharmaceutical composition comprising at least 0.15% by weight of a physiologically active non-toxic Grams compound selected from the class consisting of octa Refined cottonseed oil------3 cosanol, triacontanol, tetracosanol, and hexacosanol. Lecithin ------9 2. The method of claim 1 wherein said physiologically Phytosterols ------0.025 40 active compound is octacosanol. 1-triacontanol ------0.144 3. The method of claim 1 in which said pharmaceutical Example 6 composition contains a pharmaceutical acceptable oil An aqueous dispersion for human consumption was carrier. prepared as follows: 10 mg. octocosanol dissolved in 0.2 4. The method of claim 1 in which said pharmaceutical ml. "Tween 80' by heating to 80° C. Added, with 45 composition contains a refined oil and a dispersing agent stirring, 1 ml. water at 80 C. When completely mixed, for said physiologically active compound. added another 0.8 ml. water at 80 C., with stirring. 5. The method of claim 1 in which said pharmaceutical Stirred till mixing complete. This produces a clear trans composition contains a refined oil and a dispersing agent parent and stable emulsion. Dilutable with water to any for increasing the solubility of said physiologically active volume. 50 compound in said refined oil, and in which said physio The following theories for the physiological improve logically active compound is octacosanol. ment obtained with the compositions herein disclosed are 6. The method of claim 1 in which said pharmaceutical given for purposes of further explaining the invention. composition contains a pharmaceutically acceptable solid It is not intended that these theories should be limitations carrier. or restrictions on the scope of the invention. 55 7. The method of increasing stamina and physical en Muscular contraction and performance of work require durance which comprises administering to human beings energy which must be produced at the expense of sub a pharmaceutical composition comprising an effective stances within the cell, or brought to it by the blood amount from 0.05 mg. to 150 mg. per day of a physiologi stream. Muscle, like other tissues of the body, consumes cally active non-toxic compound selected from the class oxygen and produces carbon dioxide and water, both at 60 consisting of octacosanol, triacontanol, tetracosanol, and rest and in activity. Muscle may spring from rest into hexacosanol. instant, nearly maximal, activity and, thus, outrun its 8. The method of claim 7 wherein said physiologically blood-borne supplies of oxygen. During the time for active compound is octacosanol. the heart rate and the circulation of the blood to the 9. A composition consisting essentially of a refined oil muscle to increase, its energy requirements can be met 65 carrier, at least 0.15% by weight of a non-toxic physio by a rapid process of partial decomposition of carbohy logically active compound selected from the class con drate, a fermentation, which does not require oxygen. sisting of octacosanol, triacontanol, tetracosanol, and The carbohydrate required for this process of anaerobic hexacosanol, and an oil dispersing agent for increasing the glycolysis is stored in the muscle as the polysaccharide solubility of said physiologically active compound in said glycogen. The continuing energy requirements of a 70 refined oil. muscle in steady activity may be furnished for a limited time in the absence of oxygen by the anaerobic break 10. The composition of claim 9 wherein said dispersing down of the carbohydrate. agent is a mixture of polyoxyethylene ethers of mixed One source of this energy is the partial decomposition oleic esters of sorbitol anhydrides. of glycogen to lactic acid. Ultimately, muscle requires 75 (References on following page) 8,081,876 9 10 Referenees Cited in the file of this patent vol. II, 1950-1951, Eastman Kodak Company, pp. 72, 87 and 88, Abstracts 451, 577 and 589 respectively. UNITED STATES PATENTS Chemical Abstracts, vol. 28, page 3442, 1934. 1988,050 Rosenbusch et al. ------an. 15, 1935 Deuel: Lipids, vol. I, Chemistry, chapter IV, pages 305-312, esp. Table I, "Names and Properties of Saturated Monatomic Aliphatic Alcohols Obtained as Hydrolytic OTHER REFERENCES Products of Waxes or Prepared, Synthetically," on page Bunzell: Science, vol. 93, pp. 238-239, Mar. 7, 1941. 307, published 1951, by Interscience Publishers, Inc., New Harris et al.: Annotated Bibliography of Vitamin E, York, N.Y.