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TEE DEV LDPfizxw c? 3T79333 IH Tflfi SEAN PLAFF, (PHA3EOL39 VthAaIB) By Lou Cornelia Key An Abstract Submitted to the College of Science and Arts of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of YAQTVR 0F SCIYWCE Department of Chemistry Year 1955 Approved 16527<19‘14QE§560/7 I ."" 'tl““"" P fiT iLJv¢i~i3U A study was made of the formation of free and total sterols during different stages of growth of the bean and been plant, Phaseolus vulgaris, variety michilite. The effects of different sample preparations, namely fresh tissue, air-dry tissue and oven dry tissue, were studied. During the last harvest, the roots were gathered and analyzed for free and total sterols. Moisture determinations were made on the fresh tissue and on the sir-dry tissue and all the results were reported on a moisture free basin. The "crude lipid" material was extracted with Skelly- eolve R, and an estimation of lipid content was made. Aliquots of the lipid material were cussed through an adsorption column, containing Hyflo Supeerel and activated Magnesia, for free Iterol analysis and aliquots were saponified for total sterol analysis. Digitonidos of the sterols were precipitated from the clustes and from the unsaponifisble extracts. The digitcnides were washed with 80% ethanol and with.ethy1 ether and then oxidized with sulfuric acid-potassium.dichromate reagent. The excess dichromate was titrated by means of 0.13 ferrous ammonium sulfate solution. The mgm. of sterol present was read from working curves prepared by plotting known amounts of sterols against the ml. of ferrous solution equivalent to the dichromate used for oxidation. The results of the study showed that the bean and been plant contained little or no combined sterols: however, the bean roots or the nodules or both contained combined sterols as well as free sterols. The sterol content of the bean plants increased rapidly up to about the 55th day of growth, then decreased until between the 60th and 65th day of growth, and later increased to about the level obtained about the 55th day. During this decrease in the sterol content of the plant, there was a rapid increase in the sterol content of the been. It would appear that the bean plant, with beans can synthesize sterols at any stage of deveIOpment since the increase in the sterol content of the beans more than accounts for the decrease in the eterol content of the plant alone. The sterol content of the beans increased continuously but the most rapid increase was during the first two weeks of growth of the been. The aterol develonment of the beans paralleled that of the lipid development in that the most rapid formation was during the first two weeks of growth, and both showed a slow but continuous develOpment to reach a maximum in the mature been. The lipid content of the plant tissue increased up to between the 50th and 55th day of growth, after which there was a decrease and later a slight increase. The results for the sterol analysis of the frssh bean plant tissue and that which was previously air dried during all stAges of develOpment were similar, but some destruction of sterols in oven dry tissue was apparent. However, the results for the sterol analysis of fresh bean tissue and that uhich was oven dried were similar, indicating the beans may contain different atsrols from those in the plant. Attempts to air-dry the beans were unsuccessful. “-D'!~""‘“ T‘? "W'P‘D'V'f' w rm"? .77A‘rT U a??? m; .~ ml \J'.‘ «)1: Hal Lb.) In. L‘i 1": N... 1111125 A ' (PWABVQLWS VfiLeAEIs) Ry Lou Cornelia Key A Thesis Submitted to the Collefie of Science and Arts of Fichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the decree of Department of Chemistry Year 1955 AC K301": L?DC- 7'7? TITS The author wishes to express her sincere thanks to Professor 0. D. Ball, whose inspiration, supervision, and unfailing interest made this investigation possible. She is also indebted to Dr. Robert Carlson of the Department of Horticulture for arranging to grow and care for the plants. Io II. III. IV. V. C OTETTJEETS Introduction ........................... Historical ............................. Experimental .o......................... Extraction of Storoll ............... Liberation of Combined Sterols....... Adsorption of Extract for Free StBPOI Analysis 0.000000000000000. Precipitation and Oxidation Of Digitonides sooeeoeeeeeeooeeeeo Preparation of Working Curve......... RBSUltB and Discussion censuses-0000000. Summary and Conclusions ................ Literature Cited assessoeeoeoooooeoee¢io 0‘ UR \fl A) I“ 33 I . I YET??? DU C TI OH It has been known for a long time that the vegetable sterols can be synthesized by both the lower and the higher plants, however, little attention has been given to the sterols of the vegetative portion or green plants. With an increase in the use of certain sterols for the production of pharmaceu- ticals, a study of the development of these sterols in plants seemed profitable. The richilite bean plant was selected for this study and the deveIOpment of free sterols as well as total sterols was followed. An analysis for the presence of combined and free sterols in the roots of the bean plants was also carried out. There have been no comparisons made on the preliminary treatment of samples prior to analysis, so it was thought that a comparison using air dry, oven dry and fresh tissue would be interesting. II. HISTCRICAL Waghorne and Ball (1) traced the deveIOpment of free sterols in corn leaves using air dry tissue. They observed that the sterols are retained in the leaf for a much longer period than the lipids, but finally they too begin to disappear. They also observed that the corn leaf has the ability to syn- thesise sterols up to about the 75th day.of growth but that following this period the synthesis is definitely limited and does not parallel their destruction. Their study indicated that the rate of synthesis of the sterols may not quite keep pace with the rate of growth of the corn leaf since there appeared to be a slight decrease in the percentage value. Blair, Mitchell, and Silker (2) made a study of the factors Ihich influence the sterol content of alfalfa, bromegrass, and wheat plants using oven dry tissue. They observed that the free sterols decreased as the plants approached maturity, while, the combined sterols were detected in alfalfa at all times during growth up to the early bloom stage. ?hey found that the wheat plant contained no combined sterols until heads began to appear, at which time those accounted for 253 of the sterols. Their study indicated that fertilization of bromegrass with sflhnoa caused an increase in both free and combined eterols. III. EXPFRInLHTAL Procedure Preliminary treatment of samples‘prior;to analysis Samples were taken at two o'clock on afternoons of clear days whenever possible. The portion of the bean plants above ground was immediately taken to the laboratory where dead plant leaves and extraneous matter were removed. The plants were then counted, weighed and divided into three parts for the three different preliminary treatments, namely fresh tissue, air-dry tissue, and oven dry tissue. When beans appeared on the plants they were picked off and treated Just as the plants, except that attempts to air dry the beans were unsuccessful. Even when they were spread on a table and the air blown over a hot plate spoilage occured before the beans were dry enough to grind. The part for the fresh tissue treatment was out into small pieces with scissors and mixed. Twentyefive gm. dup- licate samples were then transferred to 600 ml. beakers and enough 95% ethanol.was added to bring the alcoholic content to about 80;. The tissue was then boiled for 15 minutes on a steamzbath. The fresh tissue was then transferred to 33 x 9h mm. paper thimbles by fitting the extraction thimbles into glass filter funnels held in turn in clean suction flasks. The tissue was washed with a jet of hot 95% ethanol and then covered with a piece of filter paper. The portion for air dry treatment was spread on a table and allowed to dry at room temperature until brittle. This usually took from two to three days. The portion for even dry treatment was allowed to dry for 18 hours in an electric oven at 95-100°C. After drying the samples were ground in a semimicro wilsy mill to pass through a hO-mesh sieve and then mixed thoroughly. A portion of the oven dry tissue was transferred to a weighing bottle and dried again for about 16 hours in an electric oven at 95¢lOO°C. The material was then allowed to cool in a desiccator, after which ten gram samples were weighed and transferred to 33 x 9h mm. paper thimbles and then covered with a piece of filter paper. During the last harvest of been plants the roots were gathered after removal of the tops. The roots were very hard to handle and contained a.mass of very fine network and the ones in the same pct were practically impossible to separate without tearing. They were hard to wash and grind. The roots contained a considerable number of nodules, some of which.uere-loet while washing. The roots were washed with distilled water, spread on a table to dry overnight, after nhich they were counted as well as possible and weighed. The roots were then treated in the same manner as the oven dry plant tissue already described. Samples of various portions of the plant were weighed out for moisture determinations at the same time as those for analysis. moisture content of the fresh plant material and of the air~dry plant material was measured by placing duplicate five gran.samples of each in special aluminum pans and drying at 100°C until constant weight in a Brabender Hoisture Tester. Percent moisture was obtained by multiplying the Brabonder scale reading by too since the ,moisture tester was calibrated for ten gram samples. Extraction of Sterols. The paper thimbles were placed in the extraction section of continuous Soxhlet extractors each of which.was previously equipped with a three cm. length of large-bore glass tubing. Three different 18 hour extraction periods were made with Skellysolve B. The fresh material was freed of Skellysolvs B and finely ground before subjecting to the third and last extraction period. One ml. of 0.02% Hydoquinone (dissolved in 95% ethanol) was added to the combined extracts which were evaporated to dryness on a steam bath in an cpen beaker in a swiftly moving current of air. When the solvent apparently was evaporated the beakers were put in a vacuum oven at 30°C and dried for 16 hours.. The dried "crude lipid" material was then treated with Skellyeolve B and filtered into weighed 50 ml..beakers. The solvent was evaporated as before and the beakere were put in a vacuum oven at 30°C and allowed to come to constant weight for an estimation of the lipid content. The dried extracts were transferred with Skellyaolve B to 25 ml. volumetric flasks and made to volume and mixed. Partial purification for precipitation was done by use of tall and Kelly's procedure (3). Liberation o§_gombinedflaterolg. In order to liberate the combined aterola, 10 ml. aliquots of the Skellyaolve extract were refluxed for 30 minutes with 5 ml. of 103 KOH in 99% ethanol. The caponification mixture was transferred to a small ceparatory funnel by using Skellyeolve B and 95% ethanol alternately. The alcohol layer was removed and ex- tracted three times with Skellyeolve B and the washing! were combined with the original Skellyaolve solution. The combined solution was then extracted four or five times with 905 methanol to remove carotenolc, traces of chlorOphyll and alkali, after which it was transferred to 250 m1. beakerc. Adsorption of extract for free eterol analysis. In order to determine the free cterolc 10 ml. aliquots of the Skelly- solve extract were taken at the came time as those to be caponified and run through an adsorption column (15 x 2 cm.). filled with a mixture of three parts of flyflo Super-Cal (Johns-Kanville Corp.) and one part activated Wagnesia so. 26hl (testvaco Chloride Products 00.). The sterols as well as oarotenes were eluted with 50 ml. of 5% acetone in Skellysolve B followed by 50 ml. of 10% acetone in Skelly- solve B. The eluate was caught in 250 ml. backers, placed in a vacuum desiccator and reduced pressure obtained by a water pump. Precipitation and oxidation of digitoniggg. Following the adsorption of the samples for free sterol analysis and the ssponification for total sterol analysis, the precipo itation and subsequent oxidation was done by use of haghorne and Ball's procedure (h). The eluates as sell as the un— saponifiabls extracts were evaporated just to dryness on a well ventilated steam bath. The residues were cooled, taken up with Skellysolve B and filtered into 25 m1. volumetric flasks and were made up to volume and mixed. Five ml. aliquots were transferred to 15 ml. centrifuge tubes and evaporated to dryness over vapors of rapidly boiling 95% ethanol as illustrated by Waghorne and Ball. The sides of the tubes were washed thoroughly by the use of 5 ml. of absolute ethanol, after shich the centrifuge tubes were replaced in the boiling ethanol vapors. After the residue had dissolved, 2 ml. of 1% digitonin in 104 ethanol and 1.25 ml. of distilled water were added. The contents were mixed thoroughly by means of a glass thread which in turn was rinsed with 50f ethanol. The tubes were heated for a minute longer and then placed in pint preserving Jars which contained about one quarter inch of 80? ethanol in the bottom (5). The jars were sealed by means of a screw top and precipitation was allowed to occur over- night. After overnight precipitation, any precipitate sticking to the walls of the tube was loosened by rubbing with the glass thread and rinsing down with 80% ethanol. The tubes were then centrifuged at about 3000 r.p.m. for one-half hour and the supernatant liquid was removed by means of a capillary tube and gentle suction. The precipitate was washed once with 3 ml. of 80$ ethanol, and thoroughly mixed by the aid of the fine class rod and the glass rod rinsed. The tubes were Icentrifuged and the supernatant liquid removed as before. The precipitate was then washed at least twice with ethyl other using the above technique. After the final removal of the ether, the tubes were allowed to stand,overnight in a covered beaker at room temperature. Drying was then completed by placing them in an electric oven at 90° to 95°C for about 1 hour. The tubes were then cooled and exactly h m1. of sulfuric acid-potassium dichromate reasent was added. The tuhes were then suspended in s steam bath to a depth approximately 0.25 inch above the surface of the reagent. »This was accomplished by slipping large corks over the tubes to support them on the steam bath cover. The tubes were left stationery for 3.5 hours, after which they were inspected and any precipitate clinging to the sides or the tube was rinsed down by gentle agitation. Heating was then continued for another 0.5 hour. The contents of the tubes were then quantitatively rinsed into beakers with.h0 to 50 ml. of distilled water. One drOp of o-phenanthroline ferrous complex indicator (6) was added, and the excess dichromate was titrated by means of 0.1N ferrous ammonium sulfate solution, previously run through a lead reductor just before titration (7). Preparation of workingpcurve. Working curves for this analysis were prepared by plotting the mgm. of sterol present against the ml. of ferrous solution equivalent to the di- chromate used for'the oxidation (Figures 1 and 2). The titration values on which Figures 1 and 2 are based are to be found in Tables 1A and 18. The stigmasterol used in this work was prepared from crude soybean sterols by recrystallizing both as the acetate and free sterol a total of fourteen times; M.P. 162-163°C. The sitosterol used for this work came from Nutritional Biochemicals Corporation and was recrystallized from absolute ethanol four times: M.P. 129-l3l°c. 10 "PIXEL? IA OXIDPTIQ‘R O? KT “’0" a”! ATP-Z) PIT OF‘ STIGE '42“; STE,” ROI: Average Ml. gil.Fe** Sol'n. Stigmasterol ELF‘e++ Sol'n. Fe*+ Sol'n. r20 Used Present Mam. Excess Cr207n Excess Cr207- for 23:1dation 0.00 23.50 23.57 .-- 33:2. 23.60 0.25 21.30 21.86 1.71 21. 21.81% 0.50 19.5 19-h1 n.16 19.2 0.75 16.55 16.39 7.18 16.33 16.30 1.00 1h. 3 1h.39 9.18 unis 1.25 10.96 11.20 12.37 11.07 11.55 1050 90h? 9039 1&013 9-30 1075 6.85 6.33 16.7h 6.81 2.00 h.61 h.31 19.26 11 TA?LE IB GXIDATION OF K3033 AHGUfiT 6F SITOSTEROL 6-55 . Average $1.. V1. Fe+*Sol'n. Sitosterol n1. re+* Sol'n. Fo++ Sol'n. 0 50 Used for Present Mam. Excess Cr207= Excess Cr207= deation 0.00 23.0 23.15 --. 23.2 0.25 21.50 21.50 1.65 21.50 0.50 19.21 19.20 3.95 19.19 0.75 17.1 17.16 5.99 17.1 1.00 15.08 15.03 8.07 1.25 13.00 13.11 10.01 13.27 1.50 10.72 10.62 12.53 10.52 1975 8073 5073 1h037 -. 8.78 2.00 6.81 6.70 16.15 1 FIG. I T T I I o h— -J O O N .0 h- .4 b O — d 0 O 04 ID P - N HH 0 l-c 0 +3 - °E _ — O e-vi r40 0') — — 2 O ._. O h— .0 0 I0 0 F _ o O :0 O 10 0 (V H H 901110013410 8m .10 Donn-rue Jc; 199:1 0:131:31] =5='u.10:: 1'11: 9. ++ CURVE FOR STIGMASTEROL WORKTNG 2 FIG. emuouwm am J0 uoneprxo :03 1299000310311 ='U.Ios .75 1,00 1.25 1.50 1.75 2,00 .50 .25 as "[71 ++ Sitostsrol “gm. CURVE FOR STTOSTEROL WORKTNG - T'j'l“‘~"‘l','fi'3 ,5?“ 1\"-"fi*?:7"‘ ‘1 IV. 1......nz31: Lam _'/J..3~..;...-2.)IO.4 The weinht frowth curve (Figure 3), plotted from data in Table II, shows that the plant reaches maximum growth at about the same time the growth of the beans starts reach- ing a maximum. The beans reach maximum drowth much more quickly than the plants, and both fall off rather slowly to reach a minimum at about the same time. It can be noted from Tables III and IV and Figures h and S that the lipid content of the plant increases fairly rapidly until between about 50 and 55 days of sin, after which it decreases, during which time there is a rapid build up of lipid in the fast growing young beans. The lipid con- tent of the air-dry plant tissue and the oven dry plant tissue then starts building up anain, durinc which time there is not as rapid development of lipid in the beans as durinz their first two weeks of growth, to reach a maximum in the mature plant. 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' ‘5'. 3641‘ ‘3’}. 0.1.3" “in "‘5' -: , n. ‘-‘p.- a. ’ -uaAL_ LSQJC .nra -w«»; gfifi or! :1 r o I o 3'». . I .055 21“.” ~4- ‘-J ;l‘ I. .J , (”o-fl]: 3. j 9);“ -.. . v . - . ,'~- . .9!" . ’ ‘ , _- ' . ~w~ wxw wwz ’ L -... .«jb-j“, .\} j“) .v' i; a .6’ a" - r ‘v—w- r." “4‘ r -. rJ¢HUku O o 112:. 7/ c 19 O p; t: ‘F I nvmrxsx \3‘1 \bi‘ ifi z_ "3: c: f r 1" ~. s . 3’33 ~ .u a. a - a q. :- an a an - a no u. I. «- F’N’Q ‘. § 1)) 7"”, ‘I' .39: wJ.J: ..30 ,J;..t 0 21:3. 0 .3 I; '2 .,.r ‘Mfi‘m' .333 09“} W; :2: .upw .J.. S f ’ ‘l r} .13 :I‘VJ-J’ .' O 3 " 2 3 3'” 3. 1" ‘ 0&9: ¢%" -“0‘0-*~u-~-‘¢--~~-0 .323 P3.17 El? {1} avarmge cf at laast E?) fiagnrtefi on maisture I 3 I C 3 fl :0 Q umawunauunnuoouauuuwon i 7‘ 3 .h. .” ) w, \ rftj ILK .L’.:+' 3‘ ‘9'? .0, 4) 3 Q ‘1‘!» fan'- 1""; .- z h—v fr§e bagis -tarol \ '~ '3 l u; .310 01"1? .1\’) Opuklll- .nga f“ ¢=I3 ’ v-J ‘-\ r- -. o a") f .f A ,2 O ' ' 73‘-) J ‘1 O ".1 5 4' ‘ f‘. . ff ‘ v: 5 ,. U ““1. -9 - mumlicfito fiétuPhAL8£LUflfl .313 .337 - - .375 .375 .370 "‘ ‘3’ 0 j 143.! - - .577 o "f"? V, I o '20 . ”rs: - - .{rffi 4%] .L.‘ 414 K.’ o o \ 4.450 «hfi ."V' 5 l \ -..; [U U1 7.’ V” L -K-o l.‘ J. t. u “ u vv— w w— V—v I ,1 I o- 26 TARLE VII Fer; A23 TDTAL STIROL CONTETT up BEAN ROOTS (1) :36 (1) Mgm. Free (1) I’h'gm. Total Lipid Sterol per Sterol per Gm. Tissuoiv Gm. Tissue 2.18 .358 .ZZh 2.22 .3 S . .350 ii .7 «350 .7 (1) Oven dry tissue manm Some: .§& In Io< we; 30mg IE ho aha—modbmn chap 5 ou< oo on or on on ov om on on q # _ 4 q _ _ _ _ 1 3.8.3 hug coho ‘INI 05:3 huoouu< 6-d- 3-33 scorn 1 IL om.o ow. oo.o om.o oo ow. .H 1UBId/‘W3fi —- BIOJaqs seas go qufixem mzmn or 00 chin cu ow< on ov on on 4‘ — ash-«o hug capo can-«u huuuua< 300d 0 £0 .hh q, a _ «— 00.0 00.0 oo.a 0N.H 1UBId/‘W3fi -- atoxaqs eng go zqfiyem n-1Im Hooch». .hhfdm In Iu< new. udbmlhu Amn shun :H ow< oo 00 0v on on 0* A _ _ _ _ — T '1 TI l1 0 ‘4 one»: mun capo [O J onus: 5.on l — P _ b _ _ o .Em 00.0 oo.o oo 'mfifl quvtd 49d auaaq u; BIOJO1S 9913 00 mz<flm z» mflommam AHQ 00 or shun cu om< 00 00 cc A .04 .3» _ sauna» hue capo osoofip nooum o .0wh 0mm 0 N O O 0 v quntd 10a cueog u; 9101019 1310; 00.0 00.0 1. 2. 3. h. S. 31 V. SUEHAFY A‘ED COPEGLUSIOH Neither the plant nor bean tissue contained appreciable combined sterols. Either the bean roots or the nodules on the roots or both contained combined sterols as well as free sterols. The results for the sterol analysis of the fresh bean plant tissue and that which has been previously air dried, during all stages of development, are similar, but some destruction of sterols in oven dry tissue is apparent. The results for the eterol analysis of fresh bean tissue and that which has been oven dried are similar, indicating that the beans may contain different sterols from those in the plant. The sterol content of the bean plants increased rapidly up to about the 55th day of growth, after which it de- creased until between the 60th and 65th day of growth, after which it increased again to around the level obtained about the 55th day. During this decrease in the sterol content of the plant, there is a rapid increase in the sterol content of the bean. 6. 7. 8. 9. 32 The sterol content of the beans increased continuously but the most rapid increase was during the first two weeks of growth of the been. The sterol develOpment of the beans parallels that of the lipid develcpment in that the most rapid fermation is during about the first two weeks of growth, and shows a slow but continuous develonment to reach a maximum in the mature bean. The lipid content of the plant tissue increased up to between the 50th and 55th day of growth, after which there was a decrease and later a slight increase. It would appear that the bean plant containing beans can synthesize sterols at any stage of deve10pment since the increase in the stercl content of the beans more than accounts for the decrease in the stercl content of the plant. (1) (2) (3) (L1) (5) (6) (7) 33 LI'PFZRATUR 1‘3 CITE-ID eaghorne, D., and Ball, C. D., Ph.D. Thesis, Michigan State College (1951) Blair, 2. 3., Mitchell, H. L., and Silker, \. E., Plant Physiolo,‘§é, 337’h2 (1951) Wall, E. E., and Kelley, E. 0., Anal. Chem.,.23, 677 (19k7) Waghome, D., and Ball, C. D., Anal. Chem., 3’1! 560 (1952) Schoenheimer, R., and Sperry, w. m., J. Biol. Chem. 12.62. 7&5 (193m Pierce, W. C., and Haenisch, E. L., Quantitative Analysis, 2nd ed., John Wiley and Sons Inc., New Yerk, p. 219 (19h0) Duke,F. R.,Ind. k Eng. Chem., Anal. Fd.,'£1, S30 (lQhS) LIP? " “" ate Due /V( __ J_L 44m Demco-293 HICHIGQN STQTE UNIV. LIBRRRIES I III II I ll” 1 312930159 2607