PLANT PHYSIOLOGY

albus. Proc. Soc. Exptl. Biol. Med. 60: 217-220.1945.

10. MANGENOT, G. and CARPENTIER, S. Action dup-aminophenylsulfamide et I'acide p-aminoben-zoique sur les ve6getaux superieurs. Compt. rend.soc. biol. 135: 1053-1056. 1941.

11. MARTIN, G. J. Biological Antagonism. Pp. 1-576.The Blakiston Co., New York. 1951.

12. NIMMO-SMITH, R. H. and WOODS, D. D. P-amino-benzoic acid and folic-acid derivatives in relationto bacterial growth and sulfonamide action. Jour.Gen. Microbiol. 2: x-xi. 1948.

13. NORTHEY, E. H. The Sulfonamides and Allied Com-pounds. Pp. 1-660. Reinhold Publishing Corp.,New York. 1948.

14. RIBEIRO, F. Influence of sulfanilamide on the germi-nation of seeds. Jour. Biol. Chem. 152: 665-667.1944.

15. SCH6PFER, VON W. H. and AN KER, W. Wirkung vonStulfonamiden und Antisulfonamiden auf dasWachstum von Pisumwurzeln in sterilei Organ-kultur. Experientia 3: 117. 1949.

16. SEVAG, M. G., KoFT, B. W., and STEERS, E. Failurieof folic acid to antagonize sulfanilamide non-com-petitively in the growth of Lactobacillus arabi-ntosus 17-5. Jour. Biol. Chem. 185: 17-25. 1950.

17. SHIVE, W. The utilization of antimetabolites in thestudy of biochemical processes in living organisms.Annals N. Y. Acad. Sci. 52: 1212-1234. 1950.

18. SHIVE, W. Inhibition analysis as a method of vita-min research. Intern. Rev. Vitamin Research 23:392-404. 1952.

19. SNEDECOR, G. W. Statistical Methods. Pp. 1-485.Iowa State Coll. Press, Ames, Iowa. 1948.

20. STOLL, R. Actions du p-aminobenzene sulfonamideet de l'acide p-aminobenzoique sur le developpe-ment d'Allium cepa. Compt. rend. soc. biol. 137:170-171. 19.43.

21. STREET, H. E. and ROBERTS, E. H. Factors con-trolling meristematic activity in excised roots. I.Experiments showing the operation of internalfactors. Physiol. Plantarum 5: 498-509. 1952.

22. WACKER, A., GRISEBACH, H., TREBST, A., and WEY-GAND, F. Der Wirkungsmechanismus der Sulfona-mide 1. Mitteil. iiber Coenzym F. AngewandteChemie. 66: 326-328. 1954.

23. WHITE, P. R. Handbook of Plant Tissue Ctulture.Pp. 1-277. Jaques Cattell Press, Lancaster, Penn-sylvania. 1943.

24. WIEDLING, S. Antagonismus zwischen sulfanilamidenund p-aminobenzoesiiure bei Pisum. Naturwiss.31: 114-115. 1943.

25. WILLIAMS, R. J., EAKIN, R. E., BEERSTECHER, E., andSHIVE, W. The Biochemistry of the B Vitamins.Pp. 1-741. Reinhold Publishing Corp., New York.1950.

26. WOOLEY, D. W. A Study of Antimetabolites. Pp.1-269. John Wiley and Sons, Inc., New York.1952.

THE DISTRIBUTION OF RUTIN AND OTHER FLAVONOIDSUBSTANCES IN BUCKWHEAT SEEDLINGS 1,2

JAMES R. TROYERDEPARTMENT OF BOTANY, COLUMBIA UNIVERSITY, NEW YORK 27, NEW YORK

The widespread occurrence of flavonoid compoundssuggests that their formation may result from funda-mental patterns of plant metabolism. These ubiq-uitous substances have been the objects of numerousand extensive chemical studies and figured in someof the pioneering work in biochemical genetics. Al-most no experimental evidence is available, however,concerning the biogenesis of the flavonoids and thephysiological processes in which they may participate.

As has so often been the case, this situation islargely due on the one hand to the unavailability ofgood chemical methods for small quantities and com-plex mixtures of the substances, and on the otherhand to the paucity of suitable botanical materials.In the first type of difficulty recent developments inanalysis, especially in chromatographic and other mi-crochemical techniques, may afford relief. But evenif the analytical obstacles should be overcome, thesearch for an apt plant object to use in attacking theproblems of origin and function must involve a pre-liminary gathering of much descriptive information.

1 Received November 12, 1954.2 The studies of which this work is a part were sup-

ported by appointments of the auithor to National Sci-ence Foundation fellowships.

For a given candidate the complete flavonoid comple-ment should be characterized; the distribution of eachsubstance within the plant should be known; and thequalitative and quantitative changes in each of theconstituents as the plant unfolds its developmentshould be ascertained. The present work with buck-wheat is part of an attempt at such a definition of asuitable botanical system.

A number of workers have mentioned the occur-rence in the mature buckwheat plant of rutin (3,5,7,-3',4'-pentahydroxyflavone-3-rutinoside). Schunk (24)discovered this substance in fresh leaves, Wtunderlich(28) found it in dried flowers, and Brandl and Schar-tel (5) confirmed both reports. Contemporary work-ers (e.g., 8, 9) have established the occurrence of theglycoside in several species. It has been tentativelysuggested (20) that small amounts of other flavonoidsubstances mav also be present in the mature plants.Concerning the flavonoid constitution of buckwheatseedlings no unequiivocal information has been availa-ble. iNagai (19) reported the occurrence of a smallquantity of unknown flavone pigment in etiolatedseedllings, while Kuilman (16) and Karstens (15)coul(d not demonstrate flavones in such plants.

This report offers evidlence that rutin is present, in

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TROYER-FLAVONOIDS IN BUCKWHEAT

the embryo of the buckwheat seed and that rutin andfour other substances of the flavone type occur inboth green and etiolated seedlings.

EXPERIMENTAL MIETHODSPLAN.-T 'MATERIAL: Plants of Fagopyrum esculeni-

tum 'Moench var. Japanese were employed. Lots ofgreen seedlings were germinated in white sand undergreenhouse conditions and harvested after 7 to 10days growth. Etiolated seedlings were cultured insand in the dark at 27 ± 20 C; samples of these wereanalyzed after 3 to 10 davs. All cultures receivedonlyldaily portions oI distilled water. One lot ofetiolated seedlings, termed "illuminated," was ex-pose(l after 5 days to 2 hours of summer sunlight andthen replaced in the dark chamber. This lot wastaken for analy-sis 2 days after the period of exposure,by which time anthocyanin pigment existed in thehypocotyls (15, 16). At the time of harvest all plantswere dividle(l into their constituent organs. Beforegermination the hulls were removed from one batch ofetiolated seedllings, designed " pericarpless." Ungermi-nate(l seeds were soaked in distilled water for 30minutes and the embryos and endosperms carefullyseparatedl for analysis.

PREPARATION OF EXTRACTS: Some fresh plantswere ext racte(l with several portions of boiling 95 %ethalnol or steeped with this solvent for several days;the cruide extracts were concentrated under reducedpressure an(l used as such. Other fresh plants weregroun(l witlh boiling water in a Waring blendor forseveral minutes; these extracts were concentratedunder re(luce(l pressure and equal volumes of acetonethen adldledl. After removal of the resultant precipi-tates the acetone solutions were saved for examination.Some of the plant material was dried at 700 C andstored in a (lesiccator; this was later extracted (Soxh-let) with acetone or 95 % ethanol. In either case theextralets were concentrated at reduced pressure beforeuse. One ethanol extract of fresh etiolated cotyledonsandl one acetone extract of dried etiolated cotyledonswere subjectecl to hydrolysis with 05Y% sulfuric acidfor 2 hours.

PAPER CHROMATOGRAPHY OF EXTRACTS: Two-di-mensional chromatograms of the crude extracts werepreparedl on sheets of Whatman No. 1 filter paperalpproximately 23 by 57 cm. After spotting of an ex-tract a sheet was rolled into a cylinder the height ofits short (limension and maintained in this posture bystapling w-ith the edges not touching. It was thenplaced for ascending chromatography in a closed jar.Afterwards the cylinder was unrolled, the edgestrimmedl, ancd the paper placed in another chamberfor (lescencling development in the long dimension ofthe sheet. A number of solvent svstems were used invarious combinations for the two developments, es-pecially isopropanol-wN-ater (22-78 or 6-4), aceticalcid-watter (3-17 or 6-4H, or the upper layer of n-butanol-acetic acid-water (41-5). The flavonoidspots N-ere located by spraying the chromatogramswith 1 % aqueous aluminum chloride and examiningthem undler long-ax-ve ultra-iolet light.

SEPARATION OF THE FLAVONOIDS: Attempts to sep-arate the buckwheat flavonoids by traditional solventextraction methods and by ion-exchange (12) and ad-sorption (14) chromatography were unsuccessful.Howex-er, small amounts of the substances-enoughfor chromatographic and spectrophotometric studies-x-ere separated by repeated applications of a pro-cedure similar to that employed by Nordstrom andSwain (21). Extracts were spotted in lines on paperswhich were subjected to extended descending develop-ment in one direction with the upper layer of chloro-form-isobutanol-water (2-4-4) The pigment zoneswere located by brief exposure to ammonia vapor andcut apart. Individual substances were removed byleaching with 95 % ethanol for 24 to 48 hours; thecombined eluates from a large number of strips wereconcentrated under reduced pressure to small volumesand used for further studies.

DETERMINATION OF Rf VALUES: Rf values of thesubstances separated by the above procedure weremeasured in fourteen solvent svstems. For each sol-vent four replicate chromatograms of each substance,of pure rutin, and in some cases of pure quercetinwere prepared. Approximately equal quantities ofmaterial were spotted, and routine assignments ofposition on paper, order of spotting, etc., were madeby reference to a table of random numbers. The pre-pared sheets were placed in the chromatographicchamber in contact with the solvent v-apors for 24hours before the descending development in one di-mension was begun. In each case the total distanceof solv-ent flow was 40 cm. Pigment zones werelocated by means of aluminum chloride reagent.

COLORS WITH REAGENTS: Color reactions of theseparated substances were ascertained with a numberof spray reagents which have been used with flavonoidcompounds. These were 1 % aluminum chloride (11),2 % zirconium chloride (13), 2 % stannous chloride,0.5 % magnesium acetate (25), 1 % sodium carbonate(11), Benedict's solution (11), ammonia vapor (3),ammoniacal silver nitrate (3), diazotized p-nitranilineplus sodium acetate followed by sodium carbonate(27), 2 % vanillin plus hydrochloric acid (2), andzinc foil plus hydrochloric acid (13). The treatedchromatograms were observed under visible and ultra-violet light.

ABSORPTION SPECTRA OF SOLUTIONS: Ultravioletabsorption spectra of the separated substances and ofpure rutin w-ere measured after first chromatograph-ing them on filter paper. Strips containing the pig-ments were cut from the chromatograms and the pig-ments removed by leaching with 95 % ethanol; eacheluate wsas made to a ml with additional ethanol. Ab-sorption spectra against solutions from blank chroma-tograms subjected to the same procedures were readin a Beckmatn DU quartz spectrophotometer. Forspectra in alkaline solution 2.5 ml of an ethanolicsolution, the absorption of wshich had just been deter-mined, were made to 5 ml with 0.1 N sodlium hy-droxide solution (aqueous).

ABSORPTION SPECTRA OF CHRO-MATOGRA'MS: Ultra-

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im\

o. J I *

u 7

On \CI*D\ I\ |-

0-J

I I I I I I, ,

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~SIa.

250 300 350 400 250 300 350WAVELENGTH - MA

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TROYER-FLAVONOIDS IN BUCKWHEAT

violet absorption of substances on filter paper wasmeasured in accordance xx-ith the procedure of Brad-field andl Flood (4). Strips of paper 0.9 cm wide and4 cm long containing the spots to be examined werecut from chromatograms; these, together with a simi-larly treated blank strip, were placed in simple- card-board supports constructed to fit the 1-cm cell holderof the Beckman instrument. At waxelengths smallerthan 300 to 310 m/, the selector switch was set at 0.1,the sensitivity knob turned to its extreme counter-clockwise position, and the instrument balancedagainst the blank strip by regulating the slit xvidthwith the density knob at zero. At wa-elengthsgreater than 300 to 310 m,u the selector switch couldbe set at 1.0. Measurements wvere made in this wayon substance E and pure rutin in the untreated stateand after spraying the chromatograms with sodiumcarbonate, aluminum chloride, and zirconium chloridesolutions.

RESULTS AND DISCuSSIONCHARACTERIZATION OF THE SUBSTANCES: Five sub-

stances of the flavonoid type were found in buckwheatseedlings. Since the only successful method of separa-tion yielded only very small quantities, the usualchemical studies could not be carried out. Examina-tion of Rf x-alues (table I) of the separated substancesconfirmed the hypothesis that the five substances aredistinctly different. Comparison of these data xw-ithpublished Rf xalues of flax-onoid substances (3, 7, 10,11, 13, 22, 23, 27) suggested no obvious possible iden-tities for substances A, B, C, and D. It was at onceapparent that none of them is quercetin. Hydrolysisof cotyledon extracts resulted in the disappearance ofsubstance E, a result xvhich suggests that it may bethe only glycoside present. Chromatograms of thehydrolyzates showved a new spot wvhich resembledquercetin, but this substance wvas not studied further.

The Rf values of substance E correspond closelywvith those of rutin, and a statistical test wvas per-formed to aid decision in this regard. Differences inmean R111 xalues (3), where Rm=log(1/Rf- 1), werecalculated for rutin and substance E in the fourteensolvents, and the statistic t was computed to test theIhypothesis that the mean difference in Rm in all sol-

x-ents is zero. The (lata yielded a value of t equal to-1 .586; this Xvas deelared not significant at the 5 %level (for 13 degrees of freedom 0.20 > P > 0.10) andthe hypothesis of zero mean Rm difference was ac-cepted. It was concludedI that the Rm, and conse-quently the Rf, values of rtutin and substance E arenot different.

Results of the tests vith various spray reagentsresembled those reported by others for flavonoid com-poin(ds. All five substances were vellow xvith alkali

TABLE IRf VALUES IN ONE-DIMENSIONAL PAPER CHROMATOGRAPHYOF SINGLE BUCKWHEAT FLAVONOID SUBSTANCES (A, B, C,D, E) AND AUTHENTIC RUTIN (R). UPPER LAYERS OFTWO-PHASE SYSTEMS WTERE 1.SED, EXCEPT WHERE INDI-CATED. SOLVENT CONSTITUTION Is DESIGNATED BY VOLUME

SOLVENT A B C D E R

EtOAC (H2O-sat) 0.13 0.31 0.16 0.32 0.13 0.12EtOAC-ACOH-H20

(50-2-50) 0.14 0.28 0.19 0.37 0.14 0.14EtOAc-HCOOH-H20

(10-2-3) 0.35 0.54 0.51 0.68 0.52 0.53nBuOH-AcOH-H20

(4-1-5) 0.47 0.63 0.65 0.78 0.63 0.64iBuOH-AcOH-H20

(4-1-5) 0.43 0.63 0.63 0.82 0.62 0.63Xylene-AcOH (1-3)

(H20-sat) 0.50 0.62 0.65 0.75 0.72 0.73Phenol (H20-sat) 0.49 0.72 0.64 0.86 0.59 0.57CHCI, (H20-sat) 0.00 0.00 0.00 0.00 0.00 0.00CHCI3-iBuOH-H20

(2-4-4)Upper layer 0.15 0.21 0.29 0.43 0.50 0.54Lower layer 0.09 0.32 0.25 0.68 0.18 0.20

iPrOH-H20 (22-78) 0.23 0.34 0.45 0.59 0.64 0.63iPrOH-H20 (6-4) 0.55 0.69 0.76 0.85 0.82 0.83AcOH-H20 (3-17) 0.22 0.33 0.39 0.54 0.56 0.57AcOH-H20 (6-4) 0.50 0.62 0.64 0.74 0.71 0.72

under visible light, reacted as phenols with diazotizedp-nitraniline plus sodium carbonate, gave negativevanillin-hydrochloric acid tests, turned red under thezinc-hydrochloric acid reduction, and formed stronglyfluorescing yellow or yellow-orange complexes withaluminum, zirconium, and tin. Within this generalpattern, the five substances fell into two groups: A,C, and E give similar color reactions, as do B and D.Substance E and authentic rutin displayed identicalreactions.

The ultraviolet absorption spectra of the five sub-stances in chromatogram eluates strengthen the con-clusion that all are flavones or flavonols. These aredepicted in figure 1 (A-E) with the logarithm ofabsorbance plotted against wavelength in order to re-move the effect of concentration, which is unknownin all cases. Since measurements were made on thesame eluates in both ethanolic and alkaline solutions,it is possible to compare in a relative way the heightsof the absorption peaks under these conditions (18).A summary of spectral relations is presented in tableII.

The shifts of the absorption maxima to longerxwavelengths under alkaline condlitions indicate thatall five substances possess free phenolic groups (17).In both ethanolic and alkaline solutions substances Band D display spectra very similar to those reportedfor some derivatives of apigenin (18). Substances A

FIG. 1. Ultraviolet absorption spectra of buckwheat flavonoid substances. A-E. Spectra of chromatogram elu-ates: solid lines, in 95 % ethanol; broken lines, in alkaline solution, corrected for volume changes. A-D. SubstancesA, B, C, and D, respectively. E. Substance E and authentic rutin. F-H. Spectra of zones of authentic rutin andsubstance E on filter paper chromatograms. F. Solid lines, untreated; broken lines, with 1 %c sodium carbonate.G. With 2 %o zirconium chloride. H. With 1 %c aluminum chloride.

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and C, on the other hland, show in ethanol the lowpeak or shelf in the region 290 to 310 m,u character-istic of flavonols (1, 26), and the spectra of these twostrongly resemble those reported by Briggs andLocker (6) for some substituted derivatives of quer-cetin. The stability of A, B, C, and D in alkalinesolution suggests that none of these substances possessan ortho-diphenolic configuration.

In ethanol, rutin and substance E display identicalspectra. Because rutin is notoriously unstable inalkaline solution, the data given in table II for thiscompound are not of exact significance; the heightand possibly the location of the Band I maximumwsere observed to change with time. It is importantto note, however, that this behavior was also displayedby substance E: at the times of measurement thelocations of the maxima in alkali were the same asthose of rutin, and the relative heights of the peaksquite similar. The aluminum complexes of rutin andstubstance E also showed identical spectra in solution,with maxima at 400 and 271 ml.

Results of the spectral measurements for rutin andsubstance E on filter paper are shown in figure 1 (F-H), with the logarithm of absorbance again plottedagainst wavelength to eliminate unknown concentra-tion effects. The two substances show identical spec-tra in the tuntreatedc state as well as with the threespray reagents used. Both showed maxima in thefollowing locations: untreated, 362 and 257 m,u; with1 % sodium carbonate, 420 and 273 mju; with 2 %zirconiuim chloride, 407' and 272 m,L; and with 1 %alumintum chloride, 405 andI 277 m,.

From the close correspondence of Rf values in antumber of solvents, the identical color reactions, andthe similaritv in ultraviolet absorption spectra undera variety of conditions, it seems apparent that sub-stance E is rutin. Similarly, on the basis of the colortests and absorption neasurements it is concluded

TABLE IILOCATIONS AND RELATIVE HEIGHTS OF ABSORPTION MAXIMAOF BUCKWHEAT FLAVONOID SUBSTANCES (A, B, C, D, E)AND AUTHENTIC RUTIN (R) IN 95 %/ ETHANOL AND INALKALINE SOLUTION. A. ABSORBANCE; AO. ABSORBANCE ATX MAX OF BAND I IN 95 (/C ETHANOL (X MAX IN MAL).VALUES FOR ALKALINE SOLUTIONS CORRECTED FOR VOLUME

CHANGES

A B C D E R

Bantd IEthanol

X max 351 333 353 337 362 362AO-- 100 100 100 100 100 100 100

AlkaliX max 409 399 415 402 408 408A as oOf A, 109 136 86 148 70 79

B1t (1 IIEthanol

X max 257 270 258 272 257 257A as '/ of A., 97 107 80 95 120 112

AlkaliX max 269 279 270 279 269 269A as ( of AO 147 110 93 107 120 109

TABLE IIIOCCURRENCE OF FLAVONOID SUBSTANCES IN BUCKW-HEATSEEDS AND SEEDLINGS. A, B, C, D. UNKNOw-N FLAVO-

NOIDS. R. RUTIN. P. PRESENT. N. Nor PRESETNT

A B C D R

Ungerminated SeedPericarp.P P P P PEndosperm. N N N N NEmbryo.N N N N P

Green SeedlingsRoot.N N N N PHypocotyl.N N P N PCotyledon.P P P P PPericarp.P P P P P

Etiolated SeedlingsRoot.N N N N PHypoCotyl.N N N N PCotyledonUntreated. P P P P PHydrolyzed. P P P P N

Pericarp.P P P P P"Pericarpless" SeedlingsRoot.N N N N PHypoCotyl.N N N N PCotyledon.P P P P P

"Illuminated" SeedlingsRoot.N N N N PHypocotyl.N N N N PCotyledon.P P P P PPericarp.P P P P P

th.at substances A, B,fllavonols.

OCCURRENCE OF THE

C, and D are flavone-- or

SUBSTANCES IN BUCK-WHEATSEEDLINGS: Results of the preliminary pap)er-chroma-tographic survey of the distribution of the five flav-onoid substances in buckwheat seedlings are presentedin table III. The same qualitative picture was ob-tained with both fresh and dried material, regardlessof the method of extraction used. In the ungermi-nated seed only rutin was present, and this waslocated entirely in the embryo. A fairly rapid elabo-ration of substances A, B, C, and D, andl probablyrutin must therefore have occurred during gerinina-tion since some of the samples were harvested afteronly 3 days growth.

CGreen and etiolated seedlings showedl the sameIpattern with only one exception: substance C, notfound in etiolated hypocotyls, Was present in those ofgreen plants. The " illuminated " seedlings, hypo-cotvls of which were red with anthocyanin pigment,showed no qualitative difference from other etiolatedseedlin(rs.

The cotvledons alwavs contained all five sub-stances, whle A, B, C, and D were never found inthe other organs with the one exception noted. Rutin,on the other hancl, seemed to be present in all partsof the plant, although the amounts in the roots werequite snmall. The pericarps always contained all five;the fact that this structure properly belongs to thepreceding generation suggests that all of the sub-stances may also occur in parts of mature plants.The presence of all five in seedlings from which thepericarps were removed before germination precludes

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TROYER-FLAVONOIDS IN BUCKWHEAT

the possibility, unlikely on other grounds, that theappearance of the substances in the plants proper wasthe result of translocation from the pericarps.

S-UMMARYFive flavonoid substances have been found to occur

in both green and etiolated buckwheat seedlings; theirdistribution in roots, hypocotyls, cotyledons, and peri-carps is described. Only small quantities of the sub-stances have been separated, but these have beensufficient for determination of Rf values in a numberof solvents, performance of color tests, and measure-ment of ultraviolet absorption under various condi-tions. One of the five substances was shown by com-parison with an authentic sample to be rutin; it isthe only one found in the ungerminated seed, as wellas the only one present in all parts of developingseedlings. Properties of the other four substancessuggest that all are flavones or flavonols. Free quer-cetin was not detected in seeds or seedlings.

Grateful appreciation is expressed to ProfessorR. F. Dawson, under whose direction the described re-search was completed. Dr. Joseph Naghski of theUnited States Department of Agriculture very kindlysuipplied authentic samples of rutin and quercetin.

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2. BATE-SMITH, E. C. Colour Ireactions of flowersattributed to (a) flavanols and (b) carotenoidoxides. Jour. Exptl. Bot. 4: 1-9. 1953.

3. BATE-SMITH, E. C. and WXESTALL, R. G. Chromato-graphic behaviour and chemical structure. I. Somenaturally occurring phenolic substances. Biochim.Biophys. Acta 4: 427-440. 1950.

4. BRADFIELD, A. E. and FLOOD, A. E. The direct meas-uiement of the absorption spectra of some plantphenols on paper strip chromatograms. Jour.Chem. Soc. (London) 47404744. 1952.

5. BRANDL, J. and SCH.ARTEL, G. Untersuchung uiberdas Fagopyrum-Rutin. Arch. Pharm. 250: 414-417. 1912.

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17. LEMON, H. W. The effect of alkali on the ultra-violet absorption spectra of hydroxyaldehydes,hydroxyketones, and other phenolic compounds.Jour. Amer. Chem. Soc. 69: 2998-3000. 1947.

18. MANSFIELD, G H., SWAIN, T., and NORDSTR6M, C. G.Identification of flavones by the ultraviolet ab-sorption spectra of their ions. -Nature 172: 23-25.1953.

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