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The Unidentified Vitamins Of Grasses And Alfalfa: In the development of nutritional knowledge, a new vitamin is usually discovered by observing the development of symptoms in animals fed a diet which is complete in available amino acids, minerals, carbohydrates and known vitamins. If the symptoms can be prevented or cured by adding some particular food or concentrate to the diet, it is tentatively concluded that the corrective food contains a new nutritional factor or unidentified vitamin. This is an oversimplification of the process since a great deal of painstaking and tedious laboratory work must be done to unequivocally rule out the effects of known minerals and vitamins, energy effects, and protein digestibility, as well as interactions of these factors. The initial work is usually carried out on some laboratory animal such as the rat, guinea pig, chick or hamster. In recent years bacteria and protozoa have been used effectively. After the existence of a new factor is established, the difficult task of isolation is undertaken. Once having isolated a pure biologically active compound from the natural source, chemical studies are undertaken to establish the chemical structure. When this is known, chemical synthesis is attempted. Along with the chemical work which follows the initial discovery, biological studies are usually carried on to determine the mode of physiological action and the requirements of various species for the (unidentified growth factor). Once the pure compound has been isolated and a reasonable number of its physical and biological properties are known, the new factor, or vitamin, is no longer referred to as "unidentified." Even though its complete chemical structure may not be known. In most cases it has been found that the unidentified vitamin is a group of related chemical compounds all of which have similar biological activity. Rapid Progress The rate of progress in this difficult but tremendously important field of research has been relatively fast. In the last few years folic acid (also known as eluate factor, L. caseii factor, vitamin M), which has been known by its biological effects for over 20 years, has been isolated characterized and synthesized. The related vitamin, folinic acid (citrovorum factor, tetrahydro formyl folic acid)has also reached the synthesis stage, as has thioctic acid (Protogen, acetate factor, alfalfa lipoic acid, pyruvate oxidation factor). Also, vitamin B12 has been isolated in pure form and is commercially available even though its chemical structure is not yet known. Typical analyses of dehydrated grass and alfalfa for identified nutrients are shown in Table 1. TYPICAL ANALYSES OF DEHYDRATED UNJOINTED GRASS AND ALFALFA Unjointed Cereal Grass Alfalfa Solids 95% 95.0% Protein 20% 17.5% Ether extractives 8% 6.0% Mineral(ash) 12% 12.0% Fiber 17% 25% Vitamin A (carotene) IU/lb. 300,000 150,000 Vitamin K mg./lb. 70 35 Vitamin E mg./lb. 150 110 Riboflavin 11 7 Niacin mg./lb. 35 18 Pantothenic acid mg./lb. 11 16 Folic Acid mg. /lb. 5 4 Folinc Acid mg./lb. (2)* ?*** Thiamine mg./lb 5 3 Pyridoxine mg./lb. 6 6 Ascorbic acid mg./lb. 1600 700 Choline mg/lb. 470 500 Inositol mg./lb. 300 950 Betaine mg./lb. ?*** 2000 Vitamin B-12 mcg./lb. 20 20 Biotin mcg./lb. 500 150 Thioctic acid mcg./lb. 1100 275** * This figure does not represent dehydrated product but is a calculated figure based on an analysis on juice. Conventional dehydration destroys most of this factor ** Leaf meal *** No data available In terms of animal or human requirements or compared with other natural sources, green leaf products are very rich in vitamin A activity (carotene), ascorbic acid, vitamin K, vitamin E and thioctic acid. They are also good sources of riboflavin, folic acid and folinic acid even though the latter appears to be largely lost during dehydration. In addition to the "identified" vitamins, leafy green vegetation is an excellent source of a variety of unidentified vitamins. Some of these described below. Unidentified Growth Factors in Grass The "grass juice factor" for rats, guinea pigs and rabbits is a water soluble growth factor which is different from all identified vitamins. A number of the chemical properties have been determined. The symptoms of deficiency in rats and guinea pigs are principally poor growth, reduced activity and dull hair coat. The grass juice factor is found in the milk of pasture-fed cattle, while "winter milk" is a poor source, especially if poor quality hay and silage are fed. Acid-preserved silage is a better source than is fermented silage. Whole vacuum-dried liver is a good source of the factor although some commercially available liver extracts and by-products are not. Most samples of yeast are relatively poor sources of the factor as are grains and grain by-products, meats and eggs. Peas and other green vegetables are good sources.(1-21) A water-soluble growth factor in grass has been found to be required for optimum growth of chicks, turkeys and geese. This factor is similar to the guinea pig "grass juice factor" in many of its properties and has been found to be destroyed to a variable degree by dehydration. Some samples of suncured hay have been shown to contain a high degree of potency. The most consistently potent products have been obtained by suitable juicing procedures. It is of practical significance that the grass factor for poultry usually gives a response on practical type rations. Antibiotics have been found to partially replace the growth factor but addition of potent juice preparations gives a further response in most experimental groups. Purified diets have been devised which allow improved response to potent leaf preparations (22-31). Several workers have shown that dehydrated alfalfa contains a growth factor for pigs (32-33).. Some evidence that an unidentified factor in grass is required for optimum human nutrition has been presented by von Wendt (34-35). He found that when milk from grass-fed cows is fed to nursing mothers, the children developed more rapidly than when milk from cows on dry rations was fed. The grass factor was preserved in acid silage but was lost from hay during storage. Growth factors in grass have been reported for insects including corn borers (36-38) and Tenebrio molitor (vitamin B-T—now known to be carnitine) (39). Planarian worms require a growth factor found in grass (40-41). A microbiological growth factor in grass has been reported for Leuconostoc mesenteroides (42). Studies in the author’s laboratory have shown that T. geleii, L. helveticus and L. leichmaneii require unidentified growth factors found in grass and alfalfa. Unidentified Factors Related to Reproduction. Studies with poultry have shown that grass and alfalfa contain unidentified factors which improve egg production and/or hatchibility of eggs. Practical type rations were used in these studies. Cereal grains, meat meal, fish meal and whey are not good sources of the factor. Dried pork liver and grass were good sources (43-45). Hogan and his coworkers (46-49)found that green forage contains unidentified nutrients essential to normal reproduction of swine. Part of the activity originally observed was found to be due to vitamin K. A water soluble factor was also involved, however. Similar results have been reported by Vestal, et al (50-52). The pigs from gilts receiving the basal ration were unthrifty and showed high mortality before weaning. The factor from grass was passed through the milk to the pigs and although the milk to the pigs and allowed normal livability and development during the suckling period. Somewhat similar effects have been observed in rabbits and sheep (46, 53-55). The feeding of immature grasses to dairy cattle has been shown to produce increases in milk production (56). A considerable amount of research has been done on an unidentified water soluble factor in cereal grass juice which is effective in producing ovulation of rabbits sensitized with estrogen. The factor apparently exerts its effect by causing release of gonadotropic hormone of the pituitary. While the factor is concentrated by the Katzman-Doisy benzoic acid method designed for extraction of gonadotropic material from pregnancy urine. Its stability and other properties are different from the animal hormone. A great deal of variability was observed in different lots of material which could not be correlated with any environmental factors or stage of growth of the plants (55-63). An unidentified factor in grass has been reported which produces early vaginal opening and stimulates early ovarian activity in immature rats. This material is orally active. It is water soluble and was concentrated by alcohol precipitation of grass juice. It was referred to as the "sex maturity factor" (64-65). Recent work on sheep, mice and guinea pigs has shown that some fresh leaf materials contain estrogenic materials. Several pure biologically active flavone derivatives have been isolated from fresh "subterranean clover" but it is not yet known whether these compounds occur in other forage crops. There is some indication that the active principle(s) is unstable to drying (66-69). Miscellaneous Unidentified Factors in Grass or Alfalfa. A factor has been reported which is effective in preventing the development of histamine induced ulcers of the stomach and duodenum of guinea pigs. The factor, called vitamin U or the antipeptic-ulcer dietary factor, is found in a variety of green vegetables. Clinical studies with patients appear promising (70-71). During the course of a search for an antimetabolite for ascorbic acid, it was found that feeding glucoascorbic acid to mice produced a scurvy-like condition. Strangely, though, the toxicity of the glucoascorbic acid was not reversed or prevented by ascorbic acid. When dehydrated grass(Cerophyl) was fed, however, the development of symptoms was prevented. (72). Another induced deficiency of an unidentified growth factor has been produced by feeding thyroactive materials to rats. It has been shown recently that alfalfa leaf meal is an excellent source of this antithyrotoxic factor (73). In most of the studies mentioned, the unidentified factors have been shown to be organic in nature. That is, the factors are destroyed by ashing the forage material. Work on digestion of cellulose by rumen micro-organismshas shown that the unidentified factor in alfalfa which stimulates cellulose digestion is inorganic in nature. Thus alfalfa ash contains some as yet unidentified mineral or combination of mineral elements which is responsible for the biological activity (74). Summary: It seems likely that some of the different effects reported will be found to be caused by the same metabolite. This is the history of folic acid which has been known during its "unidentified factor" days as vitamin M (for monkeys), as eluate factor (for Lactobacillus casei), as folic acid (for Streptococcus lactis), as Factor U and vitamin B-c (for chicks), and as the Will’s anemia factor (for human beings). From the properties already known, of some of the unidentified vitamins of leaves it seems likely that at least three or four different factors are actually involved. Although it will probably be many years before the chemical nature and physiological importance of these unidentified grass factors are fully known, it is essential that adequate amounts be included in livestock and poultry rations. It is the objective of the producer of dehydrated grasses and legumes to develop methods of conserving as completely as possible the values of the fresh green leaf and to produce standardized, uniform products. This can be accomplished only by means of sustained program of applied research and development. Bibliography 1. Cannon, M.D., and G. A. Emerson, J. Nutrition 18, 155, (1939). "Dietary requirements of the guinea pig with reference to the need for a special factor." 2. Clark, W.G., Proc. Soc. Exptl. Biol. Med. 46, 253, (1941). "Maintenance of adrenalectomized guinea pigs." 3. Elvehjem, C.A., E.B. Hart, H.C. Jackson, and K.G. Weckel, J. Dairy Sci. 17, 763, (1934). "The nutritional value of milks--raw vs. pasteurized and summer vs. winter." 4. Hartman, A. M., and C.A. Cary, J. Dairy Sci. 29, 549, (1946). "A still unidentified nutrient in roughages and in milk." 5. Hartman, A.M., L.P. Dryden, G.D. Likely and C.A. Cary, J. An. Sci. 5, 404, (1946). "An unidentified nutrient in foods and feeds." 6. Johnson, B.C., C.A. Elvehjem, W.H. Peterson and H.J. Fagen, J. Nutrition 18, 527, (1939). "The preservation of the 'grass juice factor in silage." 7. Johnson, B.C., C.A. Elvehjem and W.H. Peterson, J. Dairy Sci. 24, 861, (1941). "The content of grass juice factor in legume silage and in milk produced therefrom." 8. Kohler, G.O., Thesis, Univ. Wis., (1938). "Studies on a new growth factor found in green plant tissue." 9. Kohler, G.O., C.A. Elvehjem and E.B. Hart, Science, 83, 445, (1936). "Growth stimulating properties of grass juice." 10. Kohler, G.O., C.A. Elvehjem and E.B. Hart, J. Nutrition, 14, 131 (1937). "Further studies on the growth promoting factors associated with summer milk." 11. Kohler, G.O., C.A. Elvehjem and E.B. Hart, J. Nutrition 15, 445, (1938). "The relation of the grass juice factor to guinea pig nutrition." 12. Kohler, G.O., S.B. Randle, C.A. Elvehjem and E.B. Hart, Proc. Soc. Exptl. Biol. Med. 40, 154, (1939). Simplified rations for guinea pigs suitable for assay of the grass juice factor." 13. Kohler, G.O., S.B. Randle and J.R. Wagner, J. Biol. Chem. 128, Proc. Am. Soc. Biol. Chem. p. 1w, (1939). "The grass juice factor." 14. Mannering, G. J., Thesis, Univ Wis., (1944). "Newer dietary essentials required by the guinea pig." 15. Mannering, G.J., M.D. Cannon, H.V. Barki, C.A. Elvehjem and E.B. Hart, J. Biol. Chem. 151, 101, (1943). "Further evidence for the existence of specific dietary essentials for the guinea pig." 16. Randle, S.B., Thesis, Univ. Wis., (1939). "Studies on the nutritional requirements of the guinea pig." 17. Randle, S.B., H.A. Sober and G.O. Kohler, J. Nutrition 20, 459, (1940). "The distribution of the 'grass juice factor' in plant and animal materials." 18. Sober, H.A., G.J. Mannering, M.D. Cannon, C.A. Elvehjem and E.B. Hart, J. Nutrition 24, 503, (1942). "Nutrition of the guinea pig." 19. Stirn, F.E., C.A. Elvehjem and E.B. Hart, J. Dairy Sci. 18, 333, (1953). "Preliminary observations on certain seasonal variations in the physical properties and nutritional value of cow's milk serum." 20. Winegar, A.H., and P.B. Pearson, Proc. Am. Soc. An. Prod. 32, 384, (1939). "The requirements of Herbivora for nicotinic acid and grass juice factor." 21. Woolley, D.W., J. Biol. Chem. 143, 679, (1942). "Some new dietary essentials required by guinea pigs." 22. Kohler, G.O., and W.R. Graham, Jr., Poultry Sci. 30, 484, (1951). "A chick growth factor found in leafy green vegetation." 23. Kohler, G.O., and W.R. Graham, Jr., Poultry Sci. 31, 284, (1952), "The seasonal response of chicks to an unidentified growth factor found in forage juice." 24. Scott, H.M., W.A. Glista and E.A. Goffi, Poultry Sci. 30, 930, (1951). "Growth effects of certain supplements added to a corn-soybean oil meal chick ration with and without antibiotic." 25. Scott, M.L., Poultry Sci. 30, 839, (1951). "Studies on the enlarged hock disorder in turkeys. Evidence of the detrimental effect of fish-liver oil and the beneficial effect of dried brewer's yeast and other materials." 26. Scott, M.L., Poultry Sci. 30, 293, (1951). "The grass juice factor in turkey nutrition." 27. Scott, M.L., and L.S. Jensen, Poultry Sci. 31, 986, (1952). "The effect of antibiotics upon the requirement of turkeys for unidentified vitamins." 28. Slinger, S.J., W.F. Pepper and D.C. Hill, Poultry Sci. 31, 187, (1952). "Interaction between penicillin and grass juice concentrate in turkeys." 29. Ackerson, C.W., R.L. Borchers, and F.E. Mussehl, U. Nebraska Res. Bull. 168 (1951). "The effect of additions of dehydrated alfalfa meal to high corn chick rations." 30. Hansen, R. G., H.M. Scott, B.L. Larson, T.S. Nelson, and P. Krichevsky, J. Nutrition 49, 453, (1953). "Growth stimulation and growth inhibition of chicks fed forage and forage juice concentrate." 31. Vavich, M.G., A. Wertz, and A.R. Kemmerer, Poultry Sci. 32, 433, (1953). "Growth stimulating factors in alfalfa for chicks." 32. Cunha, T.J., R.W. Colby, H.W. Hodgskiss, T.C. Huang, and M.E. Enswinger, J. An. Sci., 7, 525, (1948). "Alfalfa and soil as a source of an unidentified factor(s) and the value of other vitamins for the young pig." 33. Krider, J.L, S.W. Terrill, D.E. Becker and W.E. Carroll, J. An. Sci. 6, 187, (1947). "Use of dehydrated alfalfa meal and certain by-products of the fish and distilling industries in dry lot rations of weanling pigs." 34. Wendt, G. von, Z. Volksernahr, Diatkost 10, 116, (1935). Chem. Lentr. I, 3686, (1935). "A recently discovered nutritive factor in milk." 35 Wendt, G. von, Karjatalous 11, 110, (1935). Chem. Lentri. I, 2909, (1935). "Forage for cattle and the public health." 36. Beck, S.D., Physiol. Zool. 23, 353, (1950). "Nutrition of the European corn borer. 2. Some effects of diet on larval growth characteristics." 37. Beck, S.D., J.H. Lilly and J.F. Stauffer, Ann. Entomol. Soc. Am. 42, 483, (1949). "Nutrition of the European corn borer, Pyrausta nubilalis. I. Development of a satisfactory purified diet for larval growth." 38. Beck, S.D., and J.F. Staugger, J. Econ. Entomol. 43, 4, (1950). "An aseptic method for rearing European corn borer larvae." 39. Fraenkel, G., personal communication. 40. Wulzen, R., and A.M. Bahrs, Physiol. Zool. 8, 457, (1935). "A dietary factor which imparts to certain mammalian tissues a quality necessary for the correct nutrition of planarian worms." 41. Wulzen, R., and A.M. Bahrs, Physiol. Zool. 9, 508, (1935). "A common factor in planarian and mammalian nutrition." 42. Colio, L.G., and V. Babb, J. Biol. Chem. 174, 405, (1948). "Study of a new stimulatory growth factor." 43. Gish, C.>, and L.F. Payne, Poultry Sci. 19, 35, (1940). "The importance of herbage in poultry management." 44. Nestler, R.B., T.C. Byerly, N.R. Ellis, H.W. Titus, Poultry Sci. 15, 67, (1936). "A new factor, not vitamin G, necessary for hatchability." 45. Hunt, C.P., and H.O. Stuart, Rhode Island Agr. Exp. Stat., Misc. Publ. 20, (1941). "The effect of different levels of alfalfa leaf meal in enhancing hatchability." 46. Hogan, A.G., and J.W. Hamilton, J. Nutrition 23, 533, (1942). "Adequacy of simplified diets for guinea pigs and rabbits." 47. Hogan, A.G., and S.R. Johnson, Univ. Mo. Ag. Exp. Sta. Bull. 187, 1, (1935). "Good pastures improve the pig crop." 48. Hogan, A.G., and S.R. Johnson, Mo. Agr. Exp. Sta. Research Bull. 332, 35, (1941). "Supplementary value of various feedstuffs in brood-sow rations." 49. Hogan, A.G., and S.R. Johnson, Soc. Exp. Biol. Med. 35, 217, (1936). "Plant extracts in the nutrition of guinea pigs and rabbits." 50. Vestal, C.M., W.M. Beeson, F.N. Andrews, L.M. Hutchings and L.P. Doyle, Purdue Univ. Ag. Exp. Sta. Mimeo. 27, (1947). "Death losses in newborn pigs." 51. Vetal, C.M., W.M. Beeson, F.N. Andrews, L.M. Hutchings and L.P. Doyle, Purdue Univ. Ag. Exp. Sta. Mimeo. 41, (1949). "Death losses in newborn pigs--III." 52. Vestal, C.M., W.M. Beeson, F.N. Andrews, L.M. Hutchings and L.P. Doyle, Purdue Univ. Ag. Exp. Sta. Mimeo. 50, (1950). "Death losses in newborn pigs--IV." 53. Norfeldt, S., Biedermanns Lentralbl. (B) Tierernahrung 14, 87, (1942). "The value of fresh grass in the feeding of breeding rabbits." 54. Shrewsbury, C.L., F.N. Andrews, C.Harper, and M.R. Lelle, J. An. Sci. 2, 209, (1943). "The value of alfalfa and certain of its fractions in the nutrition of breeding ewes." 55. Shrewsbury, C.L., C. Harper and F.N. Andrews, J. An. Sci. 5, 47, (1946). "Nutritive requirements of breeding ewes for maintenance, lactation and growth." 56. Huffman, C.F., S.T. Dexter and C.W. Duncan, J. Dairy Sci. 35, 1001, (1952). "Unidentified dietary factors in dairy cattle nutrition. III. The nutritive value of immature alfalfa and timothy hays for milk production." 57. Borasky, R., and J.T. Bradbury, Amer. J. Physicol. 137, 637, (1942). "Frozen plant juice as the source of a rabbit ovulating factor." 58. Bradbury, J.T., Amer. J. Physiol. 142, 487, (1944). "The rabbit ovulating factor of plant juice." 59. Bradbury, J.T., and R.E. Hodgson, J. Dairy Sci. 25, 704, (1942). "An intravenously active ovulating factor in the juice of corn and oat plants." 60. Friedman, M.H., and S. Friedman, Proc. Soc. Exp. Biol. Med. 31, 842, (1934). "A gonad-stimulating extract from alfalfa meal." 61. Friedman, M.H., and G.S. Friedman, Am. J. Physiol. 125, 486, (1939). "Gonadotropic extracts from green leaves." 62. Friedman, M.H., and J.W. Mitchell, Endocrin. 29, 172, (1941). "Variations in the yield of gonadotropic material from green plants in relation to the season of growth and the pH of the fresh juice." 63. Mitchell, J.W., R. Borasky and J.T. Bradbury, Endocrin. 31, 283, (1942). "Reconsideration of the significance of some plant characteristics in relation to yield of gonadotropic material." 64. Gomez, E.T., A.M. Hartman and L.P. Dryden, J. Dairy Sci. 24, 507, (1941). "Influence of oat juice extract upon the age of sexual maturity in rats." 65. Bennetts, H.W., and E.J. Underwood, Australian J. Exptl. Biol. Med. Sci. 29, 249, (1951). "The estrogenic effects of subterraneaum clover (Trifolium subterraneum). Uterine maintenance in the ovariectomized ewe on clover grazing." 66. Bradbury, R.B., and D.W. White, J. Chem. Soc. 12, 3447, (1951). "Chemistry of subterranean Clover. I. Isolation of formononetin and genistein." 67. Dohan, F.C., E.M. Richardson, R.C. Stribley and P. Gyorgy, J. Amer. Vet. Med. Assoc. 118, 323, (1951). "The estrogenic effects of extracts of spring rye grass and clover." 68. East, J., Australian J. Sci. Res. B5, 472, (1952). "Estrogenic effects of subterranean clover. Studies on the female guinea pig." 69. Gomez, E.T., J. Dairy Sci. 25, 705, (1942). "Further evidence of the existence and specificity of an orally active sex maturity factor(s) in plant juice preparations."> 70. Cheney, G., Stanford Med. Bull. 8, 144, (1950). "The nature of the antipeptic ulcer dietary factor." 71. Cheney, G., J.Am. Dietetic Assoc. 26, 668, (1950). "Antipeptic ulcer dietary factor." 72. Woolley, D.W. and L.O. Krampitz, J. Exp. Med. 78, 333, (1943). "Production of a scurvy-like condition by feeding of a compound structurally related to ascorbic acid." 73. Tappan, D.V., R.E. Boldt, and C.A. Elvehjem, Proc. Soc. Exp. Biol. Med. 83, 135, (1953). "Unidentified factors capable of reducing stress in iodinated protein-fed rats." 74. Burroughs, W., P. Gerlaugh, and R.M. Bethke, J. Am. Sci. 9, 207, (1950). "The influence of alfalfa hay and fractions of alfalfa hay upon the digestion of ground corncobs." |
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