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Alfalfa Extracts Which Stimulate Growth of Neurospora sitophila III
Vol. 50, No. 9, September 1961 was taken up in 50 ml. distilled water and centrifuged. The clear supernatant was now washed through a 1.5 X 60 cm. column of 200-400 mesh Dowex 50-X 8 (H+) with 250 ml. distilled water. Normal hydrochloric acid was now introduced, and sixty 25ml. fractions were collected. Hydrochloric acid 2 N , was then passed through the column, and twenty-one 25-ml. fractions were collected. Finally, 200 ml. of 6 N hydrochloric acid was passed through the column and collected as one fraction.
741 Fractions 76-80 were pooled aud treated as one fraction. All other fractions were individually evaporated to dryness, and each was redissolved in 10 ml. distilled water.
REFERENCES
~ ~ ~ s a l ; b , ~ , ~ = G ~ ~ , ~ ~ ~ u zJ.z o , and r (2) Novak, A. F., Jonnard, M. L., and Liuzzo, J. A.. ibid., G, c,,Bowman, G , , and Herranen, A,, J , A,n. Chem. SOL.,27, 1357(1955).
47&1$y2:2;,
Alfalfa Extracts Which Stimulate Growth of Neztropora sitopbilu 111 Effect on Weight Gain of Thyroid-Stressed Albino Rats By JORDAN G. LEE, ARTHUR F. NOVAK, and JOSEPH A. LIUZZO A concentrate prepared from alfalfa leaf meal which retains the property of stimulating growth of Newospora sitophila i n the standard Difco pyridoxine assay failed t o increase weight gain i n the albino rat when added t o a purified diet. W h e n the test animals were subjected to stress b y feeding desiccated thyroid, the soluble concentrate did increase weight gain. It was as effective as a n equivalent quantity of alfalfa leaf meal. H E benificient nutritional qualities of alfalfa T l e a f meal are well known.4 Thus, t h e preparation by Novak, Jonnard, and Liuzzo (1) of a n alfalfa concentrate which was highly effective in stimulating growth of Neurospora sitophila raised t h e question as to whether this preparation would also affect t h e growth of laboratory animals. Liuzzo, et al. ( 2 ) , have reported a growth response of the chick as a result of including t h e intermediate fraction B of the scheme of Novak, et aZ. (I), in a practical ration.
PRELIMINARY STUDIES In testing the effect of the concentrate upon growth of the albino rat, comparisons were made using weanling litter-mates paired for weight and sex. Introduction of the concentrate of Novak, et al. (l), into the basal purified diet a t a level equivalent to 5% alfalfa leaf meal did not alter the thirty-day weight gain of six litter-mate pairs. This was not unexpected, as this purified basal ration has given Received August 25, 1960, from the Department of Agricultural Chemistry and Biochemistry, Louisiana State University, Baton Rouge. Accepted for publication December 6 , 1960.
good growth and reproduction through four generations of our rat colony. Alfalfa and alfalfa leaf meal are among the many crude materials ( 3 , 4) which have been shown t o improve growth and livability of the thyroid-fed albino rat. The basal ration was, therefore, altered by the inclusion of U. S. P. thyroid as O.lyo of the ration and by doubling the content of B vitamins. Animals fed this basal diet had a depressed growth rate but only occasionally displayed any sign of severe stress. The weight gains obtained using this basal supplemented with alfalfa leaf meal or the test concentrate are shown in Table I. It is apparent that both increased weight significantly when compared t o the thyroid-containing basal ratioii. The concentrate was as effective as the meal. A sex difference is also discernible, with males giving the greater response to the concentrate. I t is probable that their total stress, growth plus fed thyroid, was greater than that of the females. These results are not strictly comparable with those in the literature because of the lower level of thyroid in our ration. Still, Overbv and Fredrickson (3) have reported a protective effect with 5% alfalfa leaf meal and of forage juice fed a t the same dry matter level. Ershoff, et al. (4),did not find evidence of protective action with 5% dried alfalfa juice or 5% water-soluble extract of alfalfa; pre-
Journal of Pharmaceutical Sciences
742 I.---EFFEcT O F ALFALFAAND ALFALFA CONCENTRATE ON THIRTY-DAY WEIGHT GAINS O F THYROID-STRESSED WEANLING ALBINORATS
?'ABLE
_-
No. of Pair.;
Sex
Mean Difference, Gm.
+
7
t
Basal us. Basal 50/, ALAIe 11 M +11.5 15.52 2.40* 13 F 6.5 9.62 2.42 Basal us. Basal Coilcentrate" 13 M +11.1 S.9i 4.4.Y 15 F 4.6 7 98 2.20h Basal Concentratea us. Basal so/,ALM 7 M - 3.9 5.70 8.25 O F 2.7 7.98 0.57
+
+
+
+
+
+
Level equivalent t o 5%, ALM (alfalfa leaf meal). 57,. c P < 1%.
a
P<
parative methods were not given. They did find that the inclusion of either 20y0 alfalfa meal or of 15yo of the residue left after water extraction of alfalfa meal iilcreased the survival time o f their extremely hyperthyroid animals. I t is evident from these papers, and others cited by them, that protective action is associated with nitrogen-rich source material and that solubiliration of the protective inaterial has been found difficult. I t is, therefore, of iriterest and importance that we are dealing here with a water-soluble coiicentrate which contains a number of ninhpdrin-reactive substances. The preceding paper in this series (5) describes the preparation, from alfalfa leaf meal of ninhydrinreactive substances which are stimulatory for Neurospora. The relationship of these specific coinpourids t o growth of thyroid-fed rats remains to he investigated. It should he eiriphasized, however, that the alfalfa concentrate of Novak, et al. ( l ) ,has now been found to contain, in soluble form, material promoting growth in the thyroid-stressed rat. I n addition, the fraction B of this preparative scheme has heen found to increase chick growth (2) under practical conditions.
EXPERIMENTAL METHODS Rations Used.-The basal ration contained 20% vitamin-test casein (Nutritional Biochemicals), 5y0 corn oil fortified with fat-soluble vitamins, 3755 dextrose fortified with B vitamins, 2% alphacel, 4yGHubbel, Mendel, and Waketnati salt mixture, and 66% sucrose. The fortified corn oil and fortified dextrose were prepared as described by Lee, Dupuy, and IZolfs (6). This ration was modified t o produce stress by including O.lyoU. S. P. desiccated thyroid and increasing the B complex-fortified dextrose to 6%. The sucrose content was reduced by an equal amount. The alfalfa concentrate was prepared according to hTovak, Jonnard, and Liuzzo (1) so that 1.0 Gm. dry matter was the equivalent of 1.0 Kg. alfalfa leaf meal. I t was introduced iuto the ration at a level equivalent to 5yG alfalfa leaf meal. The alfalfa leaf meal was incorporated a t a 5'17, level in place of sucrose. Test Conditions.-The albino rats used were from our local closed colony maintained a t constant temperature. Three days prior to weaning, the stock ration was replaced by the basal ration. Two days after weaning, one pair matched for sex and weight was selected from a litter and transferred to individual cages. Water and the rations t o be compared were given ad libitum. Body weight and food intake were recorded daily over the thirty-day test period. Thus, all comparisons were betweeu paired litter mates, and each pair of Table I is from a different litter.
REFERENCES (1) Novak, A. F . , Jonnard, M . L., and Liuzzo, J. A., THISJOURNAL, 47, 413(1958) (2) Liuzzo, J. A . , Lee, J . G., Watts, A. B., Fieger, E. and Novak Novak. A. F . . Poultry Scz.. Scz. 39,823(1960). 39,823(1960).
A.,
(3) Overbv. Overby, T,.' K.. K., and Fredrickson. FrLdrickson, R . ' L C3) L..,. J . Nutrition. 71,' 7 1 , iog(ig60j: 129(1960). (4) Rrshoff. R . H., Hernandez, H. J., and Muckenthaler, J. M., J . Nutrition, 67,381(1969). (5) Lee, J. G . , Liuzzo, J. A , , and Novak, A. F . , THIS JOURNAL, 50, 739(1961). (6). Lee, J. G., Dupuy, H. P . , and Rolfs, H . E., J . Nutrition, 58, 433(1956).
741 Fractions 76-80 were pooled aud treated as one fraction. All other fractions were individually evaporated to dryness, and each was redissolved in 10 ml. distilled water.
REFERENCES
~ ~ ~ s a l ; b , ~ , ~ = G ~ ~ , ~ ~ ~ u zJ.z o , and r (2) Novak, A. F., Jonnard, M. L., and Liuzzo, J. A.. ibid., G, c,,Bowman, G , , and Herranen, A,, J , A,n. Chem. SOL.,27, 1357(1955).
47&1$y2:2;,
Alfalfa Extracts Which Stimulate Growth of Neztropora sitopbilu 111 Effect on Weight Gain of Thyroid-Stressed Albino Rats By JORDAN G. LEE, ARTHUR F. NOVAK, and JOSEPH A. LIUZZO A concentrate prepared from alfalfa leaf meal which retains the property of stimulating growth of Newospora sitophila i n the standard Difco pyridoxine assay failed t o increase weight gain i n the albino rat when added t o a purified diet. W h e n the test animals were subjected to stress b y feeding desiccated thyroid, the soluble concentrate did increase weight gain. It was as effective as a n equivalent quantity of alfalfa leaf meal. H E benificient nutritional qualities of alfalfa T l e a f meal are well known.4 Thus, t h e preparation by Novak, Jonnard, and Liuzzo (1) of a n alfalfa concentrate which was highly effective in stimulating growth of Neurospora sitophila raised t h e question as to whether this preparation would also affect t h e growth of laboratory animals. Liuzzo, et al. ( 2 ) , have reported a growth response of the chick as a result of including t h e intermediate fraction B of the scheme of Novak, et aZ. (I), in a practical ration.
PRELIMINARY STUDIES In testing the effect of the concentrate upon growth of the albino rat, comparisons were made using weanling litter-mates paired for weight and sex. Introduction of the concentrate of Novak, et al. (l), into the basal purified diet a t a level equivalent to 5% alfalfa leaf meal did not alter the thirty-day weight gain of six litter-mate pairs. This was not unexpected, as this purified basal ration has given Received August 25, 1960, from the Department of Agricultural Chemistry and Biochemistry, Louisiana State University, Baton Rouge. Accepted for publication December 6 , 1960.
good growth and reproduction through four generations of our rat colony. Alfalfa and alfalfa leaf meal are among the many crude materials ( 3 , 4) which have been shown t o improve growth and livability of the thyroid-fed albino rat. The basal ration was, therefore, altered by the inclusion of U. S. P. thyroid as O.lyo of the ration and by doubling the content of B vitamins. Animals fed this basal diet had a depressed growth rate but only occasionally displayed any sign of severe stress. The weight gains obtained using this basal supplemented with alfalfa leaf meal or the test concentrate are shown in Table I. It is apparent that both increased weight significantly when compared t o the thyroid-containing basal ratioii. The concentrate was as effective as the meal. A sex difference is also discernible, with males giving the greater response to the concentrate. I t is probable that their total stress, growth plus fed thyroid, was greater than that of the females. These results are not strictly comparable with those in the literature because of the lower level of thyroid in our ration. Still, Overbv and Fredrickson (3) have reported a protective effect with 5% alfalfa leaf meal and of forage juice fed a t the same dry matter level. Ershoff, et al. (4),did not find evidence of protective action with 5% dried alfalfa juice or 5% water-soluble extract of alfalfa; pre-
Journal of Pharmaceutical Sciences
742 I.---EFFEcT O F ALFALFAAND ALFALFA CONCENTRATE ON THIRTY-DAY WEIGHT GAINS O F THYROID-STRESSED WEANLING ALBINORATS
?'ABLE
_-
No. of Pair.;
Sex
Mean Difference, Gm.
+
7
t
Basal us. Basal 50/, ALAIe 11 M +11.5 15.52 2.40* 13 F 6.5 9.62 2.42 Basal us. Basal Coilcentrate" 13 M +11.1 S.9i 4.4.Y 15 F 4.6 7 98 2.20h Basal Concentratea us. Basal so/,ALM 7 M - 3.9 5.70 8.25 O F 2.7 7.98 0.57
+
+
+
+
+
+
Level equivalent t o 5%, ALM (alfalfa leaf meal). 57,. c P < 1%.
a
P<
parative methods were not given. They did find that the inclusion of either 20y0 alfalfa meal or of 15yo of the residue left after water extraction of alfalfa meal iilcreased the survival time o f their extremely hyperthyroid animals. I t is evident from these papers, and others cited by them, that protective action is associated with nitrogen-rich source material and that solubiliration of the protective inaterial has been found difficult. I t is, therefore, of iriterest and importance that we are dealing here with a water-soluble coiicentrate which contains a number of ninhpdrin-reactive substances. The preceding paper in this series (5) describes the preparation, from alfalfa leaf meal of ninhydrinreactive substances which are stimulatory for Neurospora. The relationship of these specific coinpourids t o growth of thyroid-fed rats remains to he investigated. It should he eiriphasized, however, that the alfalfa concentrate of Novak, et al. ( l ) ,has now been found to contain, in soluble form, material promoting growth in the thyroid-stressed rat. I n addition, the fraction B of this preparative scheme has heen found to increase chick growth (2) under practical conditions.
EXPERIMENTAL METHODS Rations Used.-The basal ration contained 20% vitamin-test casein (Nutritional Biochemicals), 5y0 corn oil fortified with fat-soluble vitamins, 3755 dextrose fortified with B vitamins, 2% alphacel, 4yGHubbel, Mendel, and Waketnati salt mixture, and 66% sucrose. The fortified corn oil and fortified dextrose were prepared as described by Lee, Dupuy, and IZolfs (6). This ration was modified t o produce stress by including O.lyoU. S. P. desiccated thyroid and increasing the B complex-fortified dextrose to 6%. The sucrose content was reduced by an equal amount. The alfalfa concentrate was prepared according to hTovak, Jonnard, and Liuzzo (1) so that 1.0 Gm. dry matter was the equivalent of 1.0 Kg. alfalfa leaf meal. I t was introduced iuto the ration at a level equivalent to 5yG alfalfa leaf meal. The alfalfa leaf meal was incorporated a t a 5'17, level in place of sucrose. Test Conditions.-The albino rats used were from our local closed colony maintained a t constant temperature. Three days prior to weaning, the stock ration was replaced by the basal ration. Two days after weaning, one pair matched for sex and weight was selected from a litter and transferred to individual cages. Water and the rations t o be compared were given ad libitum. Body weight and food intake were recorded daily over the thirty-day test period. Thus, all comparisons were betweeu paired litter mates, and each pair of Table I is from a different litter.
REFERENCES (1) Novak, A. F . , Jonnard, M . L., and Liuzzo, J. A., THISJOURNAL, 47, 413(1958) (2) Liuzzo, J. A . , Lee, J . G., Watts, A. B., Fieger, E. and Novak Novak. A. F . . Poultry Scz.. Scz. 39,823(1960). 39,823(1960).
A.,
(3) Overbv. Overby, T,.' K.. K., and Fredrickson. FrLdrickson, R . ' L C3) L..,. J . Nutrition. 71,' 7 1 , iog(ig60j: 129(1960). (4) Rrshoff. R . H., Hernandez, H. J., and Muckenthaler, J. M., J . Nutrition, 67,381(1969). (5) Lee, J. G . , Liuzzo, J. A , , and Novak, A. F . , THIS JOURNAL, 50, 739(1961). (6). Lee, J. G., Dupuy, H. P . , and Rolfs, H . E., J . Nutrition, 58, 433(1956).