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Alfalfa Extracts Which Stimulate Growth of Neurospora sitophila I
Alfalfa Extracts Which Stimulate Growth of Neurospord sitophila I Preparation and Activation of Crude Extracts By JORDAN G. LEE, JOSEPH A. LIUZZO, and ARTHUR F. NOVAK Extracts of alfalfa leaf meal may increase or decrease the mycelial weight of Neuro-
spora sitofthila i n the standard Difco pyridoxine assay. T h e effect obtained was found t o be dependent upon the solvent employed o r the subsequent treatment of the extract. Water extracts were inactive or inhibitory. Extracts with 0.1 N or stronger hydrochloric acid were active. Increasing the temperature or time of extraction favored activation only slightly. T h e inactive water extracts could be activated by the addition of hydrochloric acid or by the extraction of inhibitory material with diethyl ether or dimethylformamide. mycelium production might alter the developmental pattern to indirectly produce weight increase. The highly active alfalfa extracts always produced very rapid growth without such heavy, pigmented areas. It was, therefore, decided that only fractions producing both increased mycelial weight and the same qualitative growth response as the active extracts would be considered as containing sthulatory material. The problem of extraction efficiency was next studied. Assay values were obtained for extracts prepared with distilled water or dilute hydrochloric acid as solvent. Two extraction temperatures and various heating times were employed with each solvent. The results, recorded in Table I, indicate that temperature and time of heating were minor, though significant, factors in producing active extracts. They also show that the acidity of the solvent was the major factor. Further, the results with preparations 14 through 17 indicate that this H + effect lies PRELIMINARY STUDIES not in extraction from the alfalfa, but is an effect upon the water-solubles themselves. A series of 25 assay flasks containing no added Qualitative differences were observed between culalfalfa fractions were prepared with media from tures containing water extracts and activated exthree commercial lots. These were treated as con- tracts. Where an untreated water extract was prestrol flasks would have been under standard assay ent, growth proceeded a t an accelerating pace from conditions. Least significant differences from the a small number of centers; these might not appear mean, a t a confidence level of 1yo,were calculated for for twenty-four hours or more. The active, acideach series. The greatest LSD was 1.0 mg., which treated extracts, on the contrary, displayed exwas 2.25% of the mean value. A deviation in an tremely rapid initial growth which was initiated assay of more than 5% from the control value was uniformly throughout the flask. These two patterns then arbitrarily considered as being significant, are consistent with the hypothesis that the activaprovided it could be duplicated in a repeated assay. tion process consists of removal of inhibitory A second problem in assay evaluation was the oc- materials. currence of sporulating, heavily pigmented cultures In an earlier paper ( 2 ) , evidence had been prewhen certain alfalfa fractions or purc compounds sented that diethyl ether removed inhibitory matewere present in the assay medium; occasionally, rial from extracts of alfalfa. Accordingly, this prothis also occurred in control flasks. The mycelial cedure was applied to an inactive water extract. weights from such assay flasks were very high. This The ether solubles were found to be inhibitory. pattern of development did not occur with crude The ether-extracted water extract, previously inextracts, although such extracts also produced in- active, was now active. See the results obtained creased mycelial weight. Barton-Wright (3) stated with preparations No. 18, 21, and 22 in Table I. that increased variability was found where sporula- An analogous result was obtained when the inactive tion occurred. He advocated the addition of Zn++ water extract was evaporated to dryness in zlacuo, t o suppress the phenomenon. Under such circum- then extracted with dimethylformamide. The distances, a chelating agent without direct effect upon methylformamide-soluble material possessed inhibitory powers. The residue had become active. Received August 25, 1960, from the Department of Agricultural Chemistry and Biochemistry, Louisiana State It should be emphasized that both of these proUniversity, Baton Rouge. cedures produced insoluble solids which were reAccepted for publication December 6, 1960.
shown (1) that 0.1 N hydrochloric acid extracts of alfalfa leaf meal increase t h e mycelial weight of Neurospora sitophila when added t o a medium previously considered complete for growth of t h e mold and still widely used in a biological assay for pyridoxine. A concentrate retaining this property has been prepared by Novak, Jonnard, and Liuzzo (2). T h e evaluation of the desirability of procedures to b e used in isolating the factor, or factors, responsible for this increase required the definition of assay deviations, qualitative and quantitative, which were to be considered significant. A means of assessing t h e efficiency of separation and concentration achieved by these procedures was also considered desirable,
I
T HAS BEEN
737
738
Jownal
TABLE I.-ACTIVITY
of
Pharmaceutical Sciences
OF EXTRACTS UNDER VARIED ylformamide solubles, suggest that the mechanism CONDITIONS OF EXTRACTION AND TREATMENT of activation is primarily the removal of inhibitory _____ material. Whatever the cause, extreme care must Assay Prepn. be exercised in applying this pyridoxine assay to Extraction Procedures Value= No. varied types of extracts, for the mycelium producWater Extn. at tion may be governed by treatment of the extract 25" for 18 hr. 92 1 and not solely by pyridoxine content. 121' for 10 min. 102 2 121" for 30 min. 100 3 98 121' for 60 min. 4 EXPERIMENTAL METHODS 0.1 NHClExtn. 25" for 18 hr. 135 5 Microbiological Assay.-The assay method was 121' for 10 min. 140 6 as described by Novak, Jonnard, and Liuzzo (2). 121" for 30 min. 144 7 All assays were conducted with that amount of 121' for 60 min 147 x preparation equivalent to 1.0 Grn. of alfalfa leaf Extn. at 121' meal. for 30 min. Assay values are relative values with the control Watw .. . .._. 93 9 assigned a valuc of 100. 0.01 N H C l 103 10 0.1 NHCl 137 11 Extraction Procedures.-Commercial alfalfa leaf 12 0.4 N HC1 140 meal was extracted with solvent in a 5 mi. per Gm. 1.0 N H C l 144 13 proportion. Extracts prepared at room temperature were stirred thoroughly to prevent caking, then Treatment of Extracts allowed t o sit for eighteen hours before filtering. 82 Water ext. prepd. a t 25" 14 Heated extracts were autoclaved a t 121' for the 80 No. 14 heated 15 min. at 121O 15 No. 14 made 0 . 1 N HoCl, then 16 indicated times, then allowed to sit overnight a t heated 15min. a t 121 133 room temperature before filtering. Initial solids No. 14 made 1.0 N HCl, then 17 removal consisted of filtration through heavy cotton heated 15 min. a t 121" 164 cloth sacks followed by manual expression of fluid. Water ext. prepd. at 121" for 15 18 I n preparing extracts 1 through 13 the p H was min. 93 adjusted by addition of sodium hydroxide or hydroDMF solubles of No. 18 76 19 chloric acid, the solutions were centrifuged until 141 DMF insolubles of No. 18 20 clear, and the volume brought t o a 4 ml. per Gm. 21 Material from No. 18sol. in 50% ethanol and in diethyl ether 55 alfalfa proportion. 22 Material from No. 18 sol. in 50% Where subsequent treatment followed, as in prepaethanol and insol. in diethyl rations 14 through 22, the crude filtrate was centri148 ether fuged until clear, then divided into the requisite number of equal-volume aliquots before proceeding. Control = 100. In each case, the preparation was centrifuged after final pH adjustment and brought to a 4 ml. per Gm. moved and did not appear in the final soluble frac- alfalfa proport ion. Preparations No. 19 and 20 were prepared by tions. It was evident that the apparent activity of al- evaporating an aliquot of No. 18 to dryness in vncuo falfa extracts was governed by the presence of both below 40' in a rotating evaporator and extracting inhibitory and stiniulatory material. Further, the by shaking the solids with dimethylformamide until balance, presence, or availability of these was the extracts were colorless. The residue was disgoverned by the nature of the treatment of the solved in water and again taken to dryness a t 40' in vacuo. These solids were then redissolved in extract. The objective of formulating a unit of water for p H adjustment a s previously described. activity to be used in assessing the efficiency of extraction or separation procedures was, therefore, The dimethylformamide extract was evaporated abandoned. No attempt has been made to estimate to dryness in zmcuo, then treated just as the residue the recovery of, or contributions of the various was treated. Preparations No. 21 and 22 were prepared from fractions to, the original activity. No. 18 as described by Novak, Jonnard, and The activation process may be one of making available, or of producing, stirnulatory substances. Liuzzo (2). I t may also be a question of removal of inhibitory REFERENCES material. A definite mechanism cannot be offered until the compounds involved have been isolated. ( 1 ) Novak, A. F.. Garna, A. R.. Liuzzo, J. A , , and Rubloff, However, the inhibited pattern of growth displayed E. B., THISJOURNAL 42, 581(1953). Novak, A. F., jonnard, M. L., and Liuzzo, J. A,, ( b i d . , when the crude water extract was present, plus the 47,(2)413(1958). inhibitory property of the dicthyl ether and dimeth( 3 ) Barton-Wright, B. C., Analysl, 70, 283(1945). ~~
0
spora sitofthila i n the standard Difco pyridoxine assay. T h e effect obtained was found t o be dependent upon the solvent employed o r the subsequent treatment of the extract. Water extracts were inactive or inhibitory. Extracts with 0.1 N or stronger hydrochloric acid were active. Increasing the temperature or time of extraction favored activation only slightly. T h e inactive water extracts could be activated by the addition of hydrochloric acid or by the extraction of inhibitory material with diethyl ether or dimethylformamide. mycelium production might alter the developmental pattern to indirectly produce weight increase. The highly active alfalfa extracts always produced very rapid growth without such heavy, pigmented areas. It was, therefore, decided that only fractions producing both increased mycelial weight and the same qualitative growth response as the active extracts would be considered as containing sthulatory material. The problem of extraction efficiency was next studied. Assay values were obtained for extracts prepared with distilled water or dilute hydrochloric acid as solvent. Two extraction temperatures and various heating times were employed with each solvent. The results, recorded in Table I, indicate that temperature and time of heating were minor, though significant, factors in producing active extracts. They also show that the acidity of the solvent was the major factor. Further, the results with preparations 14 through 17 indicate that this H + effect lies PRELIMINARY STUDIES not in extraction from the alfalfa, but is an effect upon the water-solubles themselves. A series of 25 assay flasks containing no added Qualitative differences were observed between culalfalfa fractions were prepared with media from tures containing water extracts and activated exthree commercial lots. These were treated as con- tracts. Where an untreated water extract was prestrol flasks would have been under standard assay ent, growth proceeded a t an accelerating pace from conditions. Least significant differences from the a small number of centers; these might not appear mean, a t a confidence level of 1yo,were calculated for for twenty-four hours or more. The active, acideach series. The greatest LSD was 1.0 mg., which treated extracts, on the contrary, displayed exwas 2.25% of the mean value. A deviation in an tremely rapid initial growth which was initiated assay of more than 5% from the control value was uniformly throughout the flask. These two patterns then arbitrarily considered as being significant, are consistent with the hypothesis that the activaprovided it could be duplicated in a repeated assay. tion process consists of removal of inhibitory A second problem in assay evaluation was the oc- materials. currence of sporulating, heavily pigmented cultures In an earlier paper ( 2 ) , evidence had been prewhen certain alfalfa fractions or purc compounds sented that diethyl ether removed inhibitory matewere present in the assay medium; occasionally, rial from extracts of alfalfa. Accordingly, this prothis also occurred in control flasks. The mycelial cedure was applied to an inactive water extract. weights from such assay flasks were very high. This The ether solubles were found to be inhibitory. pattern of development did not occur with crude The ether-extracted water extract, previously inextracts, although such extracts also produced in- active, was now active. See the results obtained creased mycelial weight. Barton-Wright (3) stated with preparations No. 18, 21, and 22 in Table I. that increased variability was found where sporula- An analogous result was obtained when the inactive tion occurred. He advocated the addition of Zn++ water extract was evaporated to dryness in zlacuo, t o suppress the phenomenon. Under such circum- then extracted with dimethylformamide. The distances, a chelating agent without direct effect upon methylformamide-soluble material possessed inhibitory powers. The residue had become active. Received August 25, 1960, from the Department of Agricultural Chemistry and Biochemistry, Louisiana State It should be emphasized that both of these proUniversity, Baton Rouge. cedures produced insoluble solids which were reAccepted for publication December 6, 1960.
shown (1) that 0.1 N hydrochloric acid extracts of alfalfa leaf meal increase t h e mycelial weight of Neurospora sitophila when added t o a medium previously considered complete for growth of t h e mold and still widely used in a biological assay for pyridoxine. A concentrate retaining this property has been prepared by Novak, Jonnard, and Liuzzo (2). T h e evaluation of the desirability of procedures to b e used in isolating the factor, or factors, responsible for this increase required the definition of assay deviations, qualitative and quantitative, which were to be considered significant. A means of assessing t h e efficiency of separation and concentration achieved by these procedures was also considered desirable,
I
T HAS BEEN
737
738
Jownal
TABLE I.-ACTIVITY
of
Pharmaceutical Sciences
OF EXTRACTS UNDER VARIED ylformamide solubles, suggest that the mechanism CONDITIONS OF EXTRACTION AND TREATMENT of activation is primarily the removal of inhibitory _____ material. Whatever the cause, extreme care must Assay Prepn. be exercised in applying this pyridoxine assay to Extraction Procedures Value= No. varied types of extracts, for the mycelium producWater Extn. at tion may be governed by treatment of the extract 25" for 18 hr. 92 1 and not solely by pyridoxine content. 121' for 10 min. 102 2 121" for 30 min. 100 3 98 121' for 60 min. 4 EXPERIMENTAL METHODS 0.1 NHClExtn. 25" for 18 hr. 135 5 Microbiological Assay.-The assay method was 121' for 10 min. 140 6 as described by Novak, Jonnard, and Liuzzo (2). 121" for 30 min. 144 7 All assays were conducted with that amount of 121' for 60 min 147 x preparation equivalent to 1.0 Grn. of alfalfa leaf Extn. at 121' meal. for 30 min. Assay values are relative values with the control Watw .. . .._. 93 9 assigned a valuc of 100. 0.01 N H C l 103 10 0.1 NHCl 137 11 Extraction Procedures.-Commercial alfalfa leaf 12 0.4 N HC1 140 meal was extracted with solvent in a 5 mi. per Gm. 1.0 N H C l 144 13 proportion. Extracts prepared at room temperature were stirred thoroughly to prevent caking, then Treatment of Extracts allowed t o sit for eighteen hours before filtering. 82 Water ext. prepd. a t 25" 14 Heated extracts were autoclaved a t 121' for the 80 No. 14 heated 15 min. at 121O 15 No. 14 made 0 . 1 N HoCl, then 16 indicated times, then allowed to sit overnight a t heated 15min. a t 121 133 room temperature before filtering. Initial solids No. 14 made 1.0 N HCl, then 17 removal consisted of filtration through heavy cotton heated 15 min. a t 121" 164 cloth sacks followed by manual expression of fluid. Water ext. prepd. at 121" for 15 18 I n preparing extracts 1 through 13 the p H was min. 93 adjusted by addition of sodium hydroxide or hydroDMF solubles of No. 18 76 19 chloric acid, the solutions were centrifuged until 141 DMF insolubles of No. 18 20 clear, and the volume brought t o a 4 ml. per Gm. 21 Material from No. 18sol. in 50% ethanol and in diethyl ether 55 alfalfa proportion. 22 Material from No. 18 sol. in 50% Where subsequent treatment followed, as in prepaethanol and insol. in diethyl rations 14 through 22, the crude filtrate was centri148 ether fuged until clear, then divided into the requisite number of equal-volume aliquots before proceeding. Control = 100. In each case, the preparation was centrifuged after final pH adjustment and brought to a 4 ml. per Gm. moved and did not appear in the final soluble frac- alfalfa proport ion. Preparations No. 19 and 20 were prepared by tions. It was evident that the apparent activity of al- evaporating an aliquot of No. 18 to dryness in vncuo falfa extracts was governed by the presence of both below 40' in a rotating evaporator and extracting inhibitory and stiniulatory material. Further, the by shaking the solids with dimethylformamide until balance, presence, or availability of these was the extracts were colorless. The residue was disgoverned by the nature of the treatment of the solved in water and again taken to dryness a t 40' in vacuo. These solids were then redissolved in extract. The objective of formulating a unit of water for p H adjustment a s previously described. activity to be used in assessing the efficiency of extraction or separation procedures was, therefore, The dimethylformamide extract was evaporated abandoned. No attempt has been made to estimate to dryness in zmcuo, then treated just as the residue the recovery of, or contributions of the various was treated. Preparations No. 21 and 22 were prepared from fractions to, the original activity. No. 18 as described by Novak, Jonnard, and The activation process may be one of making available, or of producing, stirnulatory substances. Liuzzo (2). I t may also be a question of removal of inhibitory REFERENCES material. A definite mechanism cannot be offered until the compounds involved have been isolated. ( 1 ) Novak, A. F.. Garna, A. R.. Liuzzo, J. A , , and Rubloff, However, the inhibited pattern of growth displayed E. B., THISJOURNAL 42, 581(1953). Novak, A. F., jonnard, M. L., and Liuzzo, J. A,, ( b i d . , when the crude water extract was present, plus the 47,(2)413(1958). inhibitory property of the dicthyl ether and dimeth( 3 ) Barton-Wright, B. C., Analysl, 70, 283(1945). ~~
0