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('52) £23.73. at: of 2.3.52“ ...: 122.22.232.01 on 1:22.. 1.20.2'2‘2.*'2232‘.22..:.33 0.2. C 0
1723; 13-1391 in :33 [42.37.53.117 pea m :23 (-.'rnzhu 31‘. EC) 0 o 9 g :1}

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1727.2. 2:37 2.77 {7.7:}: is .7 -27.7 1.02.31 [5.239.131.0113 2...:2 ...73

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6.74:... '7;32:27 in :7 1137:2127 937273;”. 272.227 212:." 7:; 5:02.217 of
{napkam COOOOOCOOOOOOOOIOOOOO

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12",: 7.701722. ta 1.271..th 77271 12:2 (.2737: 22.227 7.72337...ng
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171727 solvent 2732322717 u-butaml: (27.222.722.271: 7.22272277'

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1322:2272 72:27:27 £21.27 a: “..‘.-x 2 (721.2:27701 extract 2:17:22. 7.27:;
77272717.:12277 of C"-hua2312271 2777.3. 02:: 17:27: «72237777977273
1:7 71177770176772: system whats-77:71: grist-75.2727:

121212C11'(v33‘3 cocooooooooooocoooo.

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the '1 77:12:17.7.” "272:7: 177 7:173 2:271. $27772: swam:

p‘rfiiy“ .233 £17ij 72127271701: $817.02.“ (7" (’6) o o u a o o o o

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solvnz‘m brad: 1:1 tiling}: w 1. L160? :01: water

(7:?36)cocoa-000.000.000.000.

(a) C11:- 2.211.952.3133 of cm balsam! pitytic nci1£
untum pt: :13 1.1 cm: 3012:0212: 6:," tea: biz-ate. zolsm
"Cavutl‘LrJQ-szzi‘) o. to I o o .o‘ o o o a Q a

(31) 3'45? “”3773 mums segmratiem of p“:-*2’.;i a932,:
:-...-.1:.r.ax i '03 1:2 as: puss in 1:22;: ash-2:31" .. U}".:£"u

n-fifr‘aruxzmlc swat-la: v: w: (58" :1) o o a .' r . .' 0'

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nutmfi «2'23 3.31 [:1 :6 no). .2131. 3:22:12: kahuna-vie:
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(.1) C": .aa'nmtu of 1:! 'vfiic acid of pom in the solvent
swam t-bgtausl: formic «~25: 22:32:93 (.;,;:“"3:‘ . . ,

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- {macs in ti». “.....- 3;»:ch 1.301229 gmwlg annual:
“423*:(731‘5‘) oocoooooooonooonng

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£2322:me entrant chains}: 2" mm 9339 f: .1 0"“ dzmizol

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335.: 2:1“: mama of 1.323121 at vs-ri "a tint; ai’tc: izzjcciiam
of Cmnim: ital 11:20 L..tvar:,.13 pea pale: (Exp-original: VIII)

waozrpcmtion as? C“'~1m..ii:ol into "12:: barium phytutc
(-1.! 12622133 :2. ' my.» 1" fuc ions: at Wham tire 111211 ...-1.21::
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rhytic acid is a {53332913055}!ch derivative of ap-imnitol, one of
ab water column vitamins. Mtic acid is probably tho miy biologically-
mdmd mlecule containing; ain- phoaptnto enter groups, and certainly
an .ly knmm compoum raving nix such groups on aéjacont carbon atom.
fl ch basin of the rather liczitmi evidence availmlc, the structure
M below in the one we: generally accepted for the compmnju

0'?°3H2. 0'P03H1,
0-P03H2.

“1059-0

0-903“;
Phxtic acid
Phytic eel-3 ix: universally present: in the plant Ling-ion in the form
of a aha-l calcimuomggmsiw cal: called "phytin". martin was first

describei by Panama (1) am} late: ma sham to ba an inns to! «:29an

by Eiintemmin (f‘) after its extraction from seeds of Inlian custard

 

by’GCECic acid and precipitation.by 0130313.

Pbycic acid has assumed comilemble importmca in nutritimml
atwfiies, ecpccially because of its effect on the: availn‘bllity of metals
such as canine-.2, umgueaizm, iaron cm zinc, 3111 also in mantras, 172x220
it in use-3 for am mammal of iron Ema: vines: (33) an! cogsyar from
branlim (It).

fizytic acid hm been sham to lint-ac: an ixfifibitory effect on the
peptic ingestion of ovalbmiin and aimed: (5); this effect is believatl
to be relate-r1 to its ability to form immune emulsimztziom uit.~ pm-
mint: baiow Emir: irimzlc-cttric points Le. :22; a {:21 which cosfamztymfia to

the optima for the action off (again.

5:22:22}. 16:23, 21:17,:ch ...:in ca .4121. :2 2222;”. Name: £2222, are bean-.1 very
acrmgly by pIz; “III: 2:22:11. T1223 high cmwsmtwtiou 03 {21231122 22:11 in
cereals ire-22.12222 calcium umz-vailabia 1122.1 is 1212...)! one :22." '32.: 2:52.225. .33
of flaunts in infants. T313 122 cc. randy 02 *2:-222: concern 22:21 12:; mrtama
122 parts of 122% 2'14 2:122;er mrcfimd cereal: fan: a cousilemble par-
tion of fin: daily diet. p.21... y 023.} R2rri2mu (2’. ) 522‘- he Edi-2.22222- that
ciao mama-3322112: action of 22.3222222123 camels canning {3:232:13 222.121 1::
norm-.113: 212:3 to the ixfliibition of calcimu abaam 731. 31021 52-022.: the: 22112; ~2zta. ;

21221 and not- 1223 to the unamiI. ability of pIz 2322214: acid pin-322913212222 32:2!
flurtIaar that 2:;-.23 making 2:: ...-1c 2322:1232: of cereal: can be «vamp-2.2.: by
3.3211233 22:22:22 calcium to III-.3 2:12:42. 23.32.;in 22:..121. (*3) £02223} tI..2: 2:312:32 phy-
tata replaced pot-I222: 2122:: {£203 2.312... '2 :c 123 a 9:321 mchitogculc or Blightiy
tacI-Iitog'mrucfi .222,- mm .Imclama typical 2:..c.2..t1c a {“32301‘223; 3222;223:2322
phosphorus £311.23 cog-21v: protec'. .1921 even 22.32:: 12:52. 2323 21232213131 the
(22:32:12: of 23.12322...) P. The 2:; 2.922222: mo of 2:222: .2212: 3312;220:223 mum b2:
maglaiml on tin b22312: of the fact: tI'Iz-t the 2:22:22}. 1022:: 2222ch 2222 (3221221222:
8213 ngmsim 922223.322]: in the diet 222:3 mu strong. Jy by 2:22.. 22 1:322:12:
acid 2110122222210, 2222121233 film 1.22.22.22.21... 1‘12: appemance of tickets 21.2.29
to cmcium academy tut-.43 been. c-oui’im'marl 12y Ikllanby 2223.1 1211:2130“ (S).
Fu'i'fixzr, Emmi phytw 36.3 123 not 1:22.:Z'I'y 11;..1'23‘3261 to .mtvay 12103322112:
ptnqzhata 2221.1 L222: rcsultim 9.33.1'1 1.2.. ...--c22-..cy 2:02.11 be an 21.1.3251
factor canariauting to 1:222: awcmme 02‘. 1:12:22»: £222.

MIC-23 and 1’13 21231:: (3) haw: 222120-13 ..I...: I. piaytic M211 is re 2.23927 52721231512!
for 2222 U23132-221222: Ileficieacy 13 2122-2222. 2:222: 22:3 142‘; 60.21.2223 3323;232:220 in
their diam. In rats fed 802.131.2223 pk 2:222:22 in Cheri siietu b33323: avvmmly
£11 ans-1 (Lilition of calcium or ixzcxziial I32) the diets b::o2.a;I’2I: about m)
1.232....» , Itczsma £211.. 2:;". mix: 2..-..1... 02: 1210221201 2.3222312122222232 :23 res-25mm

for the illmnzs. 23.31021 0332.... 323...... to the 5212:2222 2.222221%.) 12: 12:22:02.2:222222:

3
in tin cmflition 05 tI'm rats. 112222.223 922231.223 start-:33 the Immortame of
the 1312.31.13 of 22:22:21 102123 by phytic acid in 521123211 mtrition.

32.2221 (12-) his state: that phytin was tea-.2113: soluble in 522:: 2:12:
juice and. was 2212220212ch 221212022: lrflrolysia, but Isa—3. to be: split 1:: 02:21.22.-
t‘Imt the bout-.3 calcium b0 release-:1 and utiiiacd in 130222: for-..aaciou.

23332231 02113221 (11) éc::c2:.b«221 the anticslcidcatian effect a? rice.
11222229. 322253193 of anticnlcifiicacion eff-22c: mam carried mt 2:221:23 rats
which were fed brown or polisbel rice. 'l'I‘Ia anticalcificauon 2:. .; 3:21:26
to 1222201239 the precipitation of calcium by 9223:2212: act-.1. 17.223 amnion
of 0.6-1 52’: calcium carbonate to the brown rice. 21:19.2: produced better
calcification tI-um 23.1.1 than fxfliuhad rice .1161: , but on £3.25: other 12:52.1
the brown rice 21122: {gm-e pom-c2: calcification in the aabacxm 01’ cm
mzoziicncoua source of calcium carbomtc. This clearly 322272-23 aim: the
mxticzalcifiwtion effect; of pIsytzrte can, be counteracted by suits-I212:
2.3613102} of calcium, to mmom the calcima deficiency clue to precipita'
tirm by 9:237:11: 22cm.

Little is Imam about the role oi? pIzztic acid in plants, .2222. it
123 found 121.1er «Iistfflmted and in 0.121512222225122 wsmentratiozw. 621221210112:
and 1.21223 (1.“) conchnmd £21m their 22:22:12.: £2222: £1223 phytatea 12: pmnta
act: 2.23 0 atom of pimsngms Watch my be 222211122321 by the plants (1222-1223
amtim. 1213 cpiuion was further supported by the fine: that: the
comettfiration of free tmsitol increases in several Giffcrcmt type of
ace-la 2.22:2. a; main: aim.

III-113210121 (15) has eugzcntal tint: pizytate-cz serve as; corners for
trace metals nae-3.321 to 121232.22: normal plant grmsth.

Tm 111051.22231222313 of p‘Iytic 22.311 2.222531 remain: magiomd 2222.1 121 fact

no direct: approaches; have bean mic to the. elucisjation of this. 92-02222...

1:

2-103: of the 2202'}: 1:: 1:110 322122-221 00220 has been 00225310221 01221.22: to the
’001'51410100 of 2:00:21 p... [2:10 0002' 23202: or to 22030222201100 01:222.? 03 00 :30
32123110 0013 22201001010. 2! 2:22:10: 01's 02:21.1. 2:020 22:3.- -0022, 002:0 00200-021
2.11.922 eh: 10001001 12.20.9002. 00. 11.01: 100100100 22.0.3 cimrczctcriwri

tn varma plum: amok-.0 has 1:000 0::texw1veiy 1:002:00 0222:.

10.1012: 0:: (11.51") 0:11 .30.an (1.1." ) can-...! out 2.00.20.11.10": 01‘
pinaphcric 02:11 000020 01’ 10001001 in 251021: bran and potato 12:02:00
teapectively. Further 2302;231:229 00 9123:0000 9320002202000 2100020210 110213 10

V0010225 9102:: 9004322000 92200 00 co .1, 00:2: 0221 0002:0131 cache: 0... 2213 mm:
90221000221 by 30.20022221101' 00.! 1:012:13 (1?), 1:09:00:- (13) 00.! 123222202: 0213
11111102220 (13-).

21:0 6001100 on 0203201022 of 93:31-12: 2:0 [22:02: 222021.29 10 tit: 1223022100220
plants, 022000101137 11:20 0220, 121.10.: 1:: 01’ " 011022102: 00222000 122 213.20 7.200300:
2202.2, were 2100:: by 1201: ( 23), 1’00? 0: (:‘1) 021-1 Sammit“”ya (5 i“).

T120 910221010 of D100 03.220013 of 322.2010 001.1 10 an 10012120103 000 £0:
0 02:11:01" 0- mamas: 91002121200 02.20223 21:- 00 1:: £220 2212123220 2302311102: 02?
00:72:10 00121 :20 the m0: 1:131:32)? 00309102710001 00.000001 1:20:30 12: 2:012:20.
It 10 90.3010 :0 0002:0100 of 0:00:01 0100300320010 90:32.03'0 Em: 502170201021
01 1:32:22 phyt‘ic 001.3 0:221 1:320 0010': 100221001 polygfimcpimtm.

file 000:: 001102.13 001202.20 2:02:11 10:01.00 3 02:0 21-100 0102;12:1«3132'100 100 01‘
nay-1213:2100}. 121 2:120 press 0000 01’ 00010:: 02‘! 122001101 0.21 p 0"0‘10'“ 2:02-
10091001 221022003. 0m 2700122 120-21 0:110:00 €330 12021.0», d1-0'2101fi212220910t00
of 10001201 to £2: £221.00 00 1010:: 72.1.0120 1:1 122:: 12.003.021.22. 1123 of phytic
M121.

In 00100 01' 0 222222202: of 02:10:20 -. '31:: 21.12? £2 00221 I 000222001223 :20 one
22:10 0000 03.210 to £10.! any Irma-9.220211% "izmsital 121.1200 0011.... r. 1.0.

01:01 + 2‘11? ......) 100:1001-0000171200-911'310 + ADP.

5

Hafrfrmzm-Oatc-31‘mfi’ «:3: :1, (5’3) have mammal fiimling some evil-gimme
fior than enzymatic piwzsgflmrfiufiva off fa; o-iswfltczl using a cmflza ixnmlimsa
133%;er aim: i‘mm was t: .

tic-22min: m3 Muvtlm'm (11.1»,95) 1‘3: ortcd that cmla rat. liver czztmc :3
mm wwbie of caszulytziug cm fur-mam: of ismzitol :mwznisofzpiasao flown
LI? (casein? inc Higgins piazza) ..'.-ml izwsitol. Timm are) this: only re: furnaces:
in tin war-L able literature Lanna-3.1.1.3 the; direct easygatzc swamaia of
1.519;.in nazrwkzmfize ta.

Since um: in min: Labnmtoriaaa Ins Ellen-d to corroborate eitlwr or.”
tmaa £111.21ng , ii:- is quite pom-3:310 that izmitcl nmmmmmmm 13 no:
Eon-med by a kimam macaw: but 13 in actuality an artifact: arisim Em
film £03201;le 0:3 cifi'fiwr bloatitol gin-:apimtiulea m." Oaths}? finnzgztufigrlatcd
flfliitOlfi .

B should be mntimd here the: 121031.301 magma-spate, 1:19:13 ulth
varima 133351301 polyvimsspimtm, t3 clam famed by the ear-zymzztic hfimlwm
of pinsytiu by the "phytzuma" pix-went in 3&3} plants, but this: is a (1:3er-
tiva 93:13:35.3; t8 5:229}: £12.43: a blwwttmtifi mthmy. magmas on the {virolyfiic
121:0.ch ca 0:? 922mm anti-e: have been cm‘rmzi cm by ink-mm (:33) , “wry
mar} Courrtoia (5‘?) , I-kCoamicZa and Carat); (2‘52) a; 3 flowchart my} Flagrant ( (‘3;

fimfim {1:912:13 biosyuttmtic 3'th kWild be: am in u’azgicin izamital
mamapmgtnte 1:: Eon-a1 in tm system by £132 hyiraifiic lavage 5f 911.3391».
umu’mzlizém and this: mars-1:01 tmmgfi&2plmt¢ £19 imammmtel 1:239 1113:; ital
wlmflmszpmztma. 'E'Eds 1.21.1114 amxear to be a goal possibility, :31me free
twice! can be tmozrpammi amzzrfintically into imagine]. ptmagtmtiic and
further this wow-41:3 help anglain the fiommtim 0:3 1mg: :91 taxmpmwhma

in than aabaamze 9'3 transits! {nix-mm.

1:3

A mafia-er of £2rmetigetora have been actively engage-:2 in the isolation,
characterization (ml bieeyntineia o1? ptwsemimmitfies.

1‘ nay-been p; 10.121-3ei'moitim has be an premrej 021:1 partially charac-
ter‘lsel by 31001113: ( 3-31), F0101: (31), Carter 0?." :11. 3:7), fleetbtme 61111
6310233131 (3;) am 80210121011 003 2100000 (3313.).

mmspbeimeitiiee have b.1001. 301020.! from cat ..en 0e 01 by 0100:: (3:: ,
Linea 30.11 by 2121:0100-6131:102,: 001 17.21.1220 (517.) , 1:32: liver by Z'kchberzsm 0:11
Lucas (37), her-3.0 an}. 110;; liver b J .11.... 1.1.1.; ( '33-). A 1.1131100312131110310£3¢
firm brain can 103021201 by -eleh (j; ;;1)’,- 011... a tr1:::1109;11101000111.10 free:
b02313 brain try 111.111.1300 and 201100 ("1) 0.1.1 Eroci 212321031. ’00.! 131221100 (311).

{fegcnfxmebt 02:01.: (315’ ) 01.11.01.001 0 platpixaixurslti-‘ie E100 1320911111001
£170.“- :00 90110 012.3 re'mri: 0.1 1:001: tee 0012;100:111 £3 a merry? ~-~~>..0..m..-..z..e and
probably the only {11035110100311.1310 present in the peas.

flu: same-1mm 01. mm: 1.0:: 11.10:: 1.“- ~10 110:3 been “per“?! by 1.. Um.
1:410th 01: (-1 ). To: 13.0300 001 Be 1100 (110) 012.1 2100.10.1011‘2 012.!

3611100 (311) also carried out strut: rural. studies an the [11030210103031.1111
00:31.10}. 012' bee-ii brain. Tigris- atuflee have confined the presence of!
p'nspummwrmdttie in beef brain 010:0; : 1.1111 13.1- 00.1 t molars-22111011030101.1120,
and their pr pose-.1 structure for the wz‘wxaincehoime11:11.10 3:000 111:1. 01.0
cm Wren-d out by 113.3001mchtrt.-1, (111;).

1011112010123 0012 Belleu (31-; ’3) am Brocmrho. 2 0013 11.2.1100 (511) have 01110

0200:0002! to 01:201. 101.0 the structure of! dio, 00.1 triptnzgireizmifimea of

110 beef brain, 1:01.111? 01’ :01: Lecw’edoe, 01.00:: deem/lat 1021 10:11:: ca 91201--
(3 01:0 1.211011 er“ 1.. more manila to scgcrzztien an} Gamer-0:01 0113}. 23-13 time
are the inks-cl; 13.311133. The 1-01.11: 11-20120 aleeeylameJ v3.21: 113230 3:311:11 1100 an}
the resuming mixture 015 deecyhte-i 01100;.1Iwisx30131100 wee eegxzratel by

ion exchange ciu'mto;re;1tr

7

1* ~‘- - .“t' - .~1 1 I, 'C- - , 3‘, -y~. ‘ _2. w.» x r‘ u 1' v -_ . . -
11:13:11: mug-1-31.133 £36.;a25 1.1.12.1,113-53 3:321: W:.'u'.§3.:36§ o: L. 1.: 321.3116}. 11:313-

1;.

' O24- '- . - . .;n I» ’4. .q'.’-.‘. “Q .5-...x.
111033231 gown-hots, 32;.11111211o3. mtg-3:10.311»! 1.1.3.: 3.1: 1:112, 6.11 1.1111 3:3 L191: -..

O

ova-3.11.331; $91 $1":- 12 1'3“: 13351;. F12: cam-.113 Malina 112.1111 L‘iooto c3233 his:
firm coma-12.3.3 11‘ so 4.....- 11-...13‘113 1.111: .1114 a 12:1}! 11:1 {to 1. .21: a 1 .1111:

93:41:91.;15111ylozzion of! no“. 1 ...-1:173:41; ;1Ewinaz':.3o 1:23 1211.11.11 3'11. 111:: in 0:1: 111* £111}-

it}

25.3-33. “inn... 1 12. T16) 323:1.‘3..:1:1?.:i.11.’c «1. 123.1111 31:11.11: {13.61; :.w.w.3‘.-3o3 31:13

13:11: "1; 33:11:11.:

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“1039-0 0
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' H—o- coR 1
QEPIIOSD3‘Q” 11031321311 (l-pMSplmtidy‘i-L- ”1110911101 h—pEwSphate)

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“4.11 10.. wan-11:11:: (l-phois . Lattiyl-L-myo-imsi 1h , 54111111333331.3219)

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...-4.3 on; 5.5.3.1111: .. "31:13.11: - 3' 31:31:11,191 mew-1:311:11 1.1.1.1 1.11:1 Luau ......4a-

1.

   

. 3 §
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("| 0’31“) ‘EQLJ 3p 0.9%63-1‘“; M'- Di u

exciton ty .-r'*2;;1:11.no1;13 1‘3... 1"“,13 (I: }, ‘1‘? 1:11.: 1.-.: :11:
.. 4, 1. I. 2"
fifié‘EkIf-ay (HS ’36". 3 .
.~ .. 3.! f". . ...... «.1». . a...
m on..." 13:31: mount: 3.23 1.1.3:: imot‘no. 3:12.111: Co. 1.1%: 3.39.1.9} 1.12.1.3

1119-1111133. no to; 13.111.11.113 £12 ”1.1. 12.1 :31 213.212..- ...1.x.m o; :3 111123.: 03’

1: ...3'!~_. ...! .. ,r ,. 9. 1. .. - .2 1.: .
wot»: 3 mm 31332315": ‘11?" 2; “H“JQM'CVT’ le‘;‘;flfi.) «filwihflnytwta airw- fi;:i:.~om

..‘Eipimmimre cholizm Wk: :11: a" -‘::?:~:tc'rx=1:; 1::; ”firm-9353" ca‘: r1(‘
bonfit'rnj 811.3 c33‘22'2...£: E {y "Angry-p 22:37.1 9:” (3 E (:1?L;3U
(1,3,2: ) , '23:) ctgp‘; £3.13 2‘21»; (2:53:43? 0?? CEWP-winfina: in 11:93:25..
by 5:11:11:ch (53?. (3 35‘s fuiiuz: 3-»

I.:--"("';';'.1 37(51‘57‘01‘3201 ‘91.”? {:2 4- 7‘ Fifi-C‘w E3353} —-—-) p! n - *2,

91:34-12:35; 3.115.»? $95.3 «l- Cf-ZEP ...
U ' -. .~ >'- -
C)?» :1. I "(VEi-it, 4- 1.x: .1.

Egg; -,;:;!y 2391.3..32 :23! .1.» .y (’3?)
52¢.er ‘15: a? inc-titre? mo 2:32: :22 * $2.115. 3::
through in": 50:? 62 5'15; 2"»: v.21. 2 0:: ..'1‘3“

L mg}.:1c<:2.’apiwr93:52:10 4» " FCC:
pizoaptutl iic tail + (IT?

C‘?~-§£;f 1‘ yani 3:: 1‘» 1.1.0.35.

11:93:5- OLTZCTJREIEOZXS Em}: 421:: ti to

{thaw “332:0;

1mm , '2'. 1:32:21: t pi'i m:

ul'mk

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thra flirts-z: cmzicztzaatiml 05’ n to. ,f};);fi;§.§

pinata to yiml a {3.9.x 33,-:"h:

. q ‘33:): 13¢? a 6:;?* ‘1.- 2 if,"

9m"

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or a 12"““z‘w

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of. 2'2: 1 c knits??? 1.2:: a.
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in ‘11:: '21”: 2 tr: -2335!

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12.29 Gnu! L; 4.23.13} “3 $3-:li} 5.9:.

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$433 ;

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1:30:33.ch dams: 2m: 333393.33 its {23 mid-3-};

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$11.9 035 (’13~EC§.1\13..:~. 6. 3.3.01}. £33: {MAJ do? 2.3231 ‘3‘ \‘vu'i'm;::.-31{: 1. x. 33

'13 33033:: in m 1~n do. :33 9:331:12;

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34.03311333‘1 :33 “33.46543.“ i;‘m?~.ii,u] 1:3 in £13333, £332.34. pan? .-
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13

Dctcrmimt‘m of the Llfltin 3?}??‘333w‘2fi‘fi3- These ficterninatiom were alga

 

carried out on mature see-3.3, 3, 1?, 15, {‘53, £94» an} :33 Jay 01;! plants.

The ethanol- preserved samples wrc driel in the forced-61:- oven
at 55°C for 1.? hours. Tm grams of the sample was takcn and extracted
with 83-75 etlmuol in a Sable: extractor to rem free sugars. The
czzgar-fmc (sample was than extracted by shaking for tlmcc hours with
1.2;? hyarochloric acid coctniniug. 13",:- anhylrouc coal-cm culplutc. The
suspension was then filtered cm! the residue «linear-.193. b. :35 mi. 93:71ch
of. the chow filtrate can diluted with 9:3 ml. of distilled wear and to
this solution vac exiled 15 ml. of a 0.2“?" ferric chloriie solution in
0.6;"; hytlrochloric acid with 510%} stirring. Tm solution mm alluded to
stand overnight in or-lcr to allow the catmletc precigsitction of merit:
phytatc. The precipitate was filtered off using a Gooch cmciblc, mama
34‘ (2113338 with 0.6,”:- hyirocl'zloric ccid-aaiiw sulphate solution, to rmfm'e
traces of non-phytiu phosplwrua cmagmnla. The precipitate in the coach
crucible was miatcncd with h 612'093 of 53% mggnccium nitmtc solution
and dried in the oven at 133°C for half an hour, after which it was ignited
overnight in thc electric fut-name at 933-306. ...-*ftcr cooling, the precipi-
tctc was dissolved in 10 ml. of concentrated hydtochloric acid, dilute},
filtered into a ‘73:) ml. volumetric flask and made up to voluma with dis-
tillcd water.

Far the colorimetric determinatiana of phytic acid phocphoms, 1:) m1.
aliquots of the above solution were taken and tho proceaum follows-:3 was
the name as describcd for the: tonal placp‘émrw: dc terminations.

The results of these exp-estimate are also 93mm in Table I cal Figure.

I.

Mg. bound inositol

(per plant)

1600

lhOO
1200

1000
800
600
hoo

200

1h

 

. -_ ..- l.._4 _-:._

l_____._T__.._T___.T..__. ...1- .. 7 ' ,_ . .

O h 8 12 16 20 2h

Days

Figure 1 (a):- Changes in bound inositol
during growth of peas.

Phytin phOSphorus (Mg. per cent)

15

50 ‘1 \/ \ Total + 300

 

 

 

L—__T"‘_T“'“T”_‘*T““' r‘ ‘1 ~ i“""
O h 8 12 16 20 2h 28

Figure 1 ‘h;:- Changes in phytin and total
phOSphorus during growth of pea plants.

AF Ph tin
25 —j fi//J{\\ T 150

Total phosphorus (Mg. per cent)

Percent phytin

phOSphorus

16

 

1
30 +
Phytin phosphorus as
per cent of total
phosphorus.
2O 4 \\
\
\
\x\
10 r
X
I x v 1 1 T’ r *"“

 

01+ 8 1216 20 21+ 28

Days

Figure 1 Lcfi- Changes in phytin phosphorus

during growth of pea plants.

27

(1 “’55-2: _u‘gn. igcu”.§.¢na -V.H...‘?‘.¢og.
L I ‘. ‘3! rl-l'.'~ . \n‘ - L" ‘. I.«‘- :‘l. - -' pr.‘ .-

(A) g~fmrzm obeys-limomoist-$94:- The proporation of of m
inositol-U-C“ was achimol by fooling C3813? to poo pods or plants for
varying lamtlm of! time, star which thsy Lore sacrifice-.1 our; the CM-
inositol isolate-l and purified by pop-or clu‘mzotogrsphy. In those: studios
both the: motoring pas pols and yoga; poo plants more used for tine prepara-
tion 0! 63-1305 :01. Typical prooo‘icros follows-3 are {lascribod bolas:-
a) This method involved the fooling; of 6143;; to poo plants which more
approaching maturity. In a typical csoc ttm pea plants clout EsJJ-chty

old wore rowel from tho flats in tho growth chow-hon via-arc (233;; more
gracing. Each of thee plants was flmoring and each 121.} one ii cm lam;
pol attached to it, in which tho coo-.33 onto just: bozizming to form. The
plants were rot-moi from the flats slow with sufficiott romicolito to
protect the roots. Tho vermiculite was held in plscc by two layers of!
choose cloth. Tho plants mrc kept in c stopperod jar which was conmctod
at one: and to a filter flash in which a total oi’ 5.3.5 "summaries of"! 61433
was gamrstoé, and at tho ether on} to a tuho containing soiiun hylroxiflo
for trsppim ctrcoss Cmop. ‘l’fo plants more kept: orator light only :.i:urin3
the day on! were supplied with C3463: as follmzsr-

-- tho plants wcro for} 81409 for five hows during tho day so}

 
 

 

tinned for 1.5? hours after which it z-zss atopgma} again.

tan[21:1 :Esv- the plants were supplied 0’" 83 only for six hours. fit this
point the plants were sacrifices and CM-imsitol isolated.
1:) 2111s casa ixwolws the foodisg of? 614%» to camisol pols from hfé-Asoy old

plants in a leifimrg tube. In one stool); two pea goats, crash 5} cm long so!

153

containing 611: lam-nature peas, were used. The pols were reamed from the
plants: in Such a way that: I abort portion of the atom can still attache-J
to them. Those pols were plcccl in a numbers tube with the stem portion
dipping in water to the bottom of the tube. The Timbers tube was then
evacuated col connected to another Timnbcrg tube containing 531! sulfucic
acid an! 0.1 Milieu-tic of; 2335:1403 to the side arm. '33: 0340;. was {generated
by mixing the contents of this tube, and thc pod-containing. tube was 019ch
w the oyotem allwcd to attain equilibrium. flue pods vex-o fed 01409 for
70 hours, after which tiny were sacrifice-d cm CIA-imsitol can-acted.
c) 11118 experiment is I modifitcacton of tho one described above. km
.13 pct: pain (from hOorJzzy old plants) were placed in two different maniacs-3
tubes, with tho atom portion of the pods lumrocd in water at tho bottom
of those tubes. both tubes were evacuated and than connectci to a chit-J
Mary, who by means of c T~jc£nt. 1111:: third WMscL-g tube contained
0.5 calllcuricc of Faapcmog; and had excess 53 sulfiuric acid in the 61.18
cm. The once was generated by pouriog the act) in the site am into
the cube, and the ‘rhcnbcrg tubes containing the pols were opcned and the
C“O;_. allowed in. fiftct I feeding period of 5 days on: of the Tlumbcrg
cubes um discomsactcd and the 90.23 were acct-{flea} and. coated up for thc
isolation or: cl‘ocmuol.

Tim 90:13 in the cocoa! tube were ice-:3 Cl‘GQ for eight days, afitcr
which time they were also oacrlficcd on} cM-lnocltol isolate-:1.
d) In chm procclure fist-clay 014 pm: plants and pads were fed 63'4cpfcr
91.3111: days in a vacuum desaicator. Ilcrc this apparatus cool for generating
Cu’op m9 awoke: diff‘c‘ccc from :20 one alrca-Iy dcccrlEm-‘i; also, lactic
acid ml 3331403 acre: user! for scum-cums; 0140;; luaccci of gel furic acid

and 2333431403. A acparctory tum! was commence—.1 to the evacuated dcccicccor

19
containing the plants and the 90:33. T1313 separator}! fume! cantaimd
5 3:1. of lactic acid. In a swan banker in the dasaicator was placed a
ausmmicn of 82181403.- :‘310 lactic acid from this separatory Emma]. was
allowed to drip into the beaker containing; M01403 and evolve 63"403, which
was taken up by the plants. The pads and the plants were eacrifice-i after:
eight days and used tar the isolation of 614-moaitel.
a) In this case, young pea poda were use-:1 for that preparation of CIA-tmsitol.
In one expert-mu: than inflame pea pods (about has cm. long from hQ‘dfly
old plants) with a small section of stem chill attaches}, were 91:11:56.6! 121 3
numbers tube in such a way that the 813:33 of tire stem were untamed in
water at the bottom of cm tube. The tube rm than evacuated and connected
to anotim muuberg tube in which we placed 1:5 microcuriea of nacl‘os,
with lactic acid in the side am. The c1609, was rammed by mixing chm
contents of this: tuba, ma evacuated pol-containing tube was ttmn apemd
an! the sys- tem pen-11 tteJ to come to measure equil ibrium. The pod-con-
taining tube was than clams, 0011*.er air was pemtttea to ante-2r the
generator tube, and the two tubes were reconnected and spewed to draw more
6140;. into the pod tube. This whole macaw was repeated five times,
coaveying mat of the available 61402;. in?» tin-a pod tuba. fiftar three days
the pals were tweed from the tube, and. worked up for than isolation of
cuotmicot.
f) In this: er-zmfiwut yea plants 13-15 flag/a 011 were used for the prepara-
tion of labelled mammal. In a Egghical mgmrimnt 1:) plants were 1'er
from the flats in tin: growth czmwer, vim-m they were wring. The vet-
miculike from the room v.23 resmrwl am! the meta time-.3 1:12;}: {unfilled

water. The roots of each 16m: were then laced. in a 1273 m1. crummy“:
P P

to
£1.23}: cm: ”131113 51110112: 50 1.11. of a mtrivsnt aviation 3*. Tim flasks mm
place-.3 in 8 11113512111131: and the cieafiicsztor cvscua‘ca‘i. The evacuated
1113111119: was emanated to a nwuLc-rg tube, in which 1111.! Men 91:11:13.1
1.0 millicurio of 31101403, with lactic acid in the Mk: am. The cl‘og
was gomratua by 1111111113 1::-.1 Caz-11:12:11.1! of u... 113112115013 tube 311.}. the. evacuated
dcsaicacor containix 1:11.- p 1:133 11:13 05:1ch 1111.! the nvailgfila 6140.... 921111;):
on: of 1'11: 121111111113 as inflamed in (a) abmo. The. 1111131111311th 1111:: then
kept unfier constant fluomsccnt 11111 1112111111 scent light; for 1.} days , aEter

which the plums were 03:11.1..ch and Cléoimsitol 1:113 1111; 22:1. sin-.3.

 

71:01:- 1'131 plants or the 90.23 £12.! 0 40:. '..12'1 '13 cut
into 3111111 pieces am! 11011031111111 in {31:21:33 etnaml in a W. 21:19 Wmmniucr.
Thu 1101303121111 .:e um c1: treated 11111.33; 12111121101 by refluxing in a Smxhlec‘
extractor for {‘96 110mm. Tm 1.0,". (111111-1101 extract. was eve: «ante. ton-11:1:
dryness 1:11.312: vaczmm, rcfiissol'md in vat-111', decoloriaed 1: 11h 1:11:11:ch and
filtered. Tim filtrate was: 11111011112211 11th fix-barlita 111 11“!) (I!) and
EU. M30 (1’3- :1) :1: Mi ”'. T1393 aolutim 1.119 than evaporated to 1 31111111 L’fllun‘fl
in a stream of .111 water at 1390311321: and ctu'mntogmpi r111. ‘1‘111: cuuimattol
was putt-2111.1 by rape-.1:- 11.111111113313913.

Five 1152:1111: solwn‘; ayatc-ms \ere 1.112111, the éetaila of which apparat-
in the motion on paper chroamtography.

7111: pzzrlxica: ion of! the Ola-inositol 831139108 was fella-:33 by running
:31. 1011111203 '1'1113 .

Widen). 3:11.“.- .utogram were run by sgaz'muug c.1011: 5-533 microliter of

W The stamlurd nut-:1 a: 3011:: ..an con-11:31.11: 1 gm. C11(.1’.)m)---1.30, 0.1" ’5 3n.
K3333, 09:) C33. augfgi‘3430 ‘ver. ) 3'3}. 221‘, {33-13-10 121 am 11:33 Of $111233; {$113.5
solution V1113 dilute 11 1:5 in this c3111...

5‘1
labelled samplca 1n the fer-m of a bani on thutmxm 3 as paper am]
using pyridine (I): aha/1 alcohol (7): water (C :) solvent syn: tam. {-fccr
Ch hours the papers new air dried and then exposed to iii-ray films for 3-5
days, affine: which the film were dam-elem}. The pure twitol samples
swear as one clear bmfi, win-urea: the presence of labelled lmptmities
1n the Cmohwoitol Bflitpiefl ma 1:34-th by t' we appearance of more than

cm band on the K-rny film.

(D) 1113.112; (2:329:17533 '7‘ ..th I; 3-1’ 119*“: “If-CM:- A muster of: etzpcrmzents
were perform-.1 tn which ra-iioacti'va imam). was fed to develoging pea
p013. T43 éiffictcnt types OE feeling aperimnta xxx-a carriod and

1) realm;{Bu-@3313)! tit/2'01. in 2729 etc-.253 of acid pots:- In this case
the pole mm 1'0;ch 1'2. 0:: the pleats with about one inch of the stem
atlll attacr *an to $133.11. These pods were placed, seem dorm, in heaters
cwtaining a Cmoiwitol (solution.

In a typicalm mmle, thrc-a pols of about Ms cm length more ram.)
from h m1 Hy-oll plants an! place-i in a arr-.0.“ beau»: in such a manner that:
tin atom um amt-.30.! 1n 5 m1. of water containing the ratiioac'cive (manual.
The beaker with the pain was then placed in a large dcssicator coatainm
a tray of water to prevent excessive ovammtion from the poi-containing;
beaker. The 1mm: contaimd an mama: of angina). equal to 6 a: 1553 comma
pert. 31mm. After three Jaye a!» pole more Emmi infected with some kind
of nmlal or fungus smith. Sow of chi: less uzficctc-J portions were heap-v
3911th with 1"}:- erochlox-ic mi}, platal on n plane-hot an] countmi, but
showed maligibla incorporation of la‘ml.

"line was cz-zperimnt was re:- rent-cl 1n the Open 111., Mm water had to
be misled from time to the to mgflaca that whit}: evaporate.) out. of the

inositol solutiam. The poi-constaizfing beaker had 111383.301 czrgual to

9.? x 1'34 comm pea: 1113:3333. .IEtc': a 1.333%113 pals were 1:313ch and
hoax-15333333 with 5‘5 tzyirochlorlc act—.3. To the. lmmogemte 333 3333-1 90
ml. of 17." 5 nichloroacettc 331.3 and the mt: turn was 31133311 to 31:31:13 for
about: an hour to cm'zgolctc 1:113 precipitation of 1:113 1333:3133. The. pro:-
mint: were 1511:3333 off 31113 the 2111:3333 1:33 3:3th m a 13.3: plate, than
create-:3 with 1": “5-3 11.130115 an min solution until the 3311131011 1: .13 332:. 31:23::
to 1133113.. :‘n 013-11103 of this solution, 301133 131.113 313333.37 03313113.: phos-
phate 33331.33, 33 31.1.1333“ by paper chromatography, 1.33.3 plate! 311.! wanted
in 01.31.33 to 33 3313321113 the extent of incur-poms :103 of 0-1333: :01. The
total imorpcmticm 1:33 £03.33 to be 1333 that-1 113-31312 of cm: pet can: of
the 3.1.13.1 counts 3111 because of 1:23:33 very 103 incorporatiom: this type

cf £331.13; czqmrimnt was 3 3333:1123 ..

. 4 . ,4- ‘.o-. JHP' a“! . 5 . ...11— R an, 1.,
1:11.: M133. In 3.1-2:. t “.142 31.13. 3’31. g0 $.13

$3.333 1:31 ~11

 

   

14 . ..
33:13.73. T113 1131:3103 com:£::~t~::-3 3.1-1 11113333113 (3 43033331 3011:3103 taco

 

than interior space of the: pods, by wan-3 of a 5-3 A 1311;231:311 311333.33'1'1333.
7:213 9033 1:333 135: 32123333 to 1:113 [113112.23 in or ”13': 1:131 tI .3'9' 1:133: 3331:1333
to 1131:3139 naturally. Variatimm of. £113 fallm'img 1213:3033 were 33911-03311:-
3) P33 91:21: 3233331 3 to four 1I111r..;3*1L--f3-e33y-011 plmnt 131:3. 111333333 1:“311
the follmzing 190131.323 09 a. CM-ri‘fioaitol 3031:1213]:
Pod I o 5' 315.3303.
Pad 11 «- 1'73 1.13::30333”

P03 III '- 7 micro 35.13
1’03 II" 0 .15 1313.013.

”13:";
’0
0113
Cit-.3

1:3

The 111533333 (Sm-inasitol 1133 9-5 a: Iii-3‘ con-111:9 1:121: 33:33.33 p.33: 13.5
u'icrolltrcm 311.1 the total. 1311311133 132133131111 33313 to the four {2333 3311331333
305 a 113‘ twat-.1 pm" minute. The p313 331:3 3.333: 33:23:13.3. to 1:123 plants:
3313 $1101.33 to 3.3.3133 in. 3313 13’- mmtory 1.11.1.3- 3333:2331: fhomw 113 331:1
13331133333113 light. Sitar 3. 1:333: the: {.1033 11:33 11.13231 33:01.1 1:313 1.1.1 3.3123,

F "'3"!

cut: 5.31:3 313311 {113333 311.: I'm=1.=;;3ni:,:cl in 1"} 3.1. 0;; ;, ,1, 3......101 in a

‘.

£3

Virtia 1103033313313 fin 311.5303 of 3:113 hosvgmaate was [1132123 313.! 3.03:1th
to determine the tots-1). recovery 33 13331 (T3313 11), the 333931131311 33.3
centrifmcd and the summ'amnt 3131:33'133. TI“: precipitate was 31: 11313.1
to remove £33333 of 33313301, 3113 then stirred for 1:311:33 hours with 13} 13.3.
of 135 tricIfioz-oacctic 332.1 to extract: acid-3311:1313 phosphates. 1'53
:-:tract was 31333 basic with 0.53:3 33:13:: 1133303133, chine-«3 for 8313:1331
hours and centrifuge-=3. The: precipitate was 13133011333 in 1'75 hyirochl 31:13

acid an! aliquots used for cI-u'matography 3:11 903333.213. (T3313 12).

T1133 II

14
Incorporation of C 1530:: 111001.331 11:53::th
13:0 maturing, p33 5-3513.

EOE-’31 3. 32135413

P3! to 3 pols 35,991)
Total recovered

in mmgcmzte {9,333
333311.133 after

:1: extraction MOD
Super-331.3 from

33:11.33 precipitation 53:12:)
53:13:11 precipitate €13

 

33.311 figmres are for count rates corrected
only for 11332333333, no correction has)
been 31333 for counter 3301113313“; 311 were
1.11151211wa thin plates.

fh
b) This experiannx was run to Setcrmine the extent of incorporation
3 . .
of C"oiaositol into phytic acid W1Ch increasing titm 9031049. Seven
different c:;;x:ri::nnts mm {act-formed in which the poi; mm 1393
,, a M - «I: u
varying amounts ; C ~1nosttol and teen caczir;ced at dixzercn:
um inter-.1213 , an! the Rectum-221m of the label into various fram-
tiana atuiiai.
Tin feilmm‘g sciwm sham the premiere follmrzz-l in isolatig
the 92233:, lipid on! barium ptzymtc: fractions; from the p033 after injec-
- M .‘A.
tion of C oinoaaholz
Pois tnjectel with CL‘-1nositol
’ 1) hmmgenim with 8-"??? ethanol
:1) extract with 32.71:) ml. of 3":
ethanol in a Sociflec zztractor
for 15 haurs.
Q 9

fiasflw 8' 021133391 fractio:
(containing comely auburn)

 

 

(Extract: 1:12.: 3113’.) m1.
of ethylene iichlorije

in a ‘30:.‘2'2103 extractor
for 13 hours.

 

“......

€ V

negate 0:13;},ch dic‘iulorue Extuztion
or

1191! Eye 3103

era-tract: with 11353
trichloroncetic aciJ
Ea: three Imsa

Jr ' W

Resting 135 tricleoroesctic ecu extract

 

 

Lei-:3. £3.52! barium hj/irozi-fie

V ‘ w
barium FEW-33° barium wavy-mate:

 

2'55

In exparimnt I, :20 pan {.0313 from *2:-lay old 91mm mm injected
with clé-inacii-ai equal to 9.5 x 1733 mums par urinate. fftct a me}:
tin pain mm wswmi 53:93:: the plants, cu: into small pic-2cm and ban-x;-
ganimd in 33:1 4-2223;me in a Virus hmaogenimr. The hangezmm was

{:er tie-:1 wi 2:21 33“

:4 athezaol to get the: sugar fructi . T132 20:31th 53m:
this tmarmnt ma 3:.-123 and azztmctcd again with ethyiczm “ichiarizia to
get a 1191-}. ”auction, MS was further amazed for the cmmstion oz?
phytic midi. 1511:11th 31' tit-am fractimw were pinata}. and counted.
(Ta-{21.2 III).

In Ez-zpermeu: 11, three. pain were 1:110.ch with CM-inaositol 3'2;th
to 8.1; a 13:3 counts: per ciamize. ffter can flaps time mm were sacrificed,
bxwgenimi wit! 391% ethanol and the labelled avgars, 119135 cad phytic
aci-zl fraction: isolated 86 state}! earlier and (cm extent of labelling
determimi. Results upma'r in Table III.

Eupérixmw III to VII amt-ta} with the injection of if?) microlitem
of cu-inos so! (erg-U21 to hot? 3: 1133 counts par mimte) into each of: ten
pole. me. miss new sacrificai in paim at 15 mizwtm, 5‘3; 1mm, 11,53,
an] 13 days an! were cut into 5:321}. pin-can and hammer-aimed with Ca; etimml
in a Virtia twangeniscr. 515m lamagamta um gramme-l as iniicazei earlier
far the isolatim of sugars, lipirls cm! phytic acid fractiam an! the
mat-23:11:40.8 labelling fiatcrrimd in these fractions by plating ali-‘guac and
amazing. assaults are 32mm it: Tatie I".

c) This experiment was devises-1 in each a mam: that tha marpomtim of
cn~imaitol could be améicd mule-r equalizimm of centimmaa light and also
units: cmtitiom W'fiml‘e light was éiscmttimwfi at night for aural: sin: to

seven hours .

£33. 5.: £333 82. :- 2:8
gsuauggéawugu?

 

 

 

9.49218.“

_

 

 

 

 

A A
. 1 A . . :1 Ag 3
085a 5.1a Ham 1 85 1
. A A H“
1% Lfl 1:? - Ll
:
: _ M
“when S
23 inch “8.8 83 38 a
w , u

 

 

 

 

 

 

It... .

Til 33.11? 831+

 

 

 

 

.1100.» no «use an. an
luau!“ ugfiukwa

 

. 1 ‘ r a r _
— *3 g x, @333 I]
..- E In «03.35
3d! a cab

 

 

:8. Sfiiigiagfiégflu
alo- aggiuoiflozuifiaog

5 EH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0383 “w

 

 

 

 

.31 non nausea.- g .93qu you: 3...: 33.39
8831388 no 1.35 3283 go g3

 

 

 

 

IN?!
0 U

T‘BLE V (:3)

Effect of dark parity] on the incorporation of CM-
iaoaitol in the maturing pea poia. (Erperimnnt VIII)

 

 

 

 

 

 

 

8% P17th Barium
Tim Ethanol Lipid acizi apex-mm Resume
2 days 5922 9:93 856 571 567
’3 «ya Mm 675 11m 81:} 637
8 days 3331 361 £651. 152 655
12 days 2801 $6 9&6 51:2 997
16 days m5 use: 2587 532 1:310

 

 

 

 

 

 

off for 6-7 hours each m1

:11
h

t.

t was matted

 

Effect of dark patio: on the inflammation of c“-

1'9

2:31.12 v (b)

Waite! in the maturing pea poie. (Exporimnt IX).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E—ifiji- Fay: ic marina W
Ethanol Lipid acid superman Residue
20,956 935% o m: 233
‘99565 9503 O
17.1% 10,193 0
12120 5,990 m
19,115 10,335 563
10,395 M 695
10.575 5750 1375 “9‘3 675
11.150 3050 1535 515 795

 

 

 

In this «apartment the light supply was continuous.

33

:rperimnt VIII was performed unier conditions where light was
discontinued at night for 012: to seven hours. Ton 9039 from hon-larch!
pocplanta wore used and in each case the pads were injected u ch incaitol
equal to 2.1 x 13‘ counts per minute (specific activity 2.5.0 3 1:36 counts
per nixmte per udllileolo). 1119.30 90:13 more sacrifice-J in grmpo of two
after 9, h, 8, 1.1." ml 16 days. The pods were cut into 8125311 pieces,
‘bouogcnizcd in 83,1 ethanol in a Virtia homogenizcr and the hocogcncto
proceooed for the isolation of various labelled fractions, for each of
which the extent of labelling was determine-.1. (Table V).

In winch-cut 13, the Cu-imsitoI-injectod pods were kept under
constant light. Born 13 pm: 90:13 were injected with cuoimsitol cqucl
to 9.1 a 10‘ counts pct minute (specific activity 2.0 a: 13° counts per
minute per milliz'aolc). The pods were sacrifices two at a tim at intervals
of 15 minutes, one hour, 61:: bout-o, 9h hours, 9, h, B, 1?. cm! 16 days. The
9913 once again were cut into small pieces, hocngenimd in 8351 ethanol in
I Virtis homgcnizer an! the Sugar, 1191-1 and phytic acid fractions iso-
latod and counted. Results are about: in Table V.
5) £13023-‘ttion 312:1 pat'tial (barrenri‘tstion of 121-311?! 5:.;;:._:31 11315.3

and fimepbosgglatc! jig-Mitch fWgnsa 33!? f9“! (in-inooitol.

J

. 4! , . - fi-fi , .
a) relation :3“! i-Ecfitizicsrlm cg cmfismese of := office—:21 f:r.;3ct.£:m:~

The 83:1 etiwnul bmogczwtc of the: pale fol (in-imsitol was filtered,
and tho res-1.2m extracted with about 373‘.) ml. of 83:1 ethanol for 13 hours.
The filtrate an.) the extract were then cowbimd. The 873:1 ct‘iwnol fraction
thus obtained contains mostly the sugars and imoitol. Tao rccilue was
furtmr treats-l for the ox: action 0! 11.91.33.

The 313-1 ctmml extract was o‘cgamrato-J to a small volume color vacuum,

traction} with 1301-120 an} Eiltcral. The: filtrate woo vicionisod with IR 1-"0

31

(B) and 111?: 1.9) (CI!) resins. Tim aoionizaod uolution was evaporate-i to a
small value union: 3 spot light in presence of a continuous stream of. air.
An aliquot of this extract was plated m3 counted to determine tin extent
of total labelling in tho. 833'. ethanol extract. The moulto of Ezaporixmuto
I to 12‘; are about in Tables III to V.

The 535$ cthaml extract can chronotographcd in the pyrilioc (7):
41:;qu alcohol (7): vote-t (6) solvent system and 5120.ch tho presence of six
cat-Wits” inocitol, sucrose, gluoooc, xylosc, glycerol ml to organic
phosphate eater.

In or..1or to find out the extent of Li‘mlling in those oix corzrooocuto,
a portion of tho 323:: ethanol cacti-cot was run in the form of a strip on
Ethotmon 35 no pooch Reference spoto were run along with amniotic owl
the position of the various components located by spraying the reference
portion of! the paper with mmtmiacol silver nitrate. The atripa were out
on! the at: com-momma eluted from the strips with motor. Tho wotor ex-
tra-ct was cvcoorato—J to a small volume, plated and counted.

Those éotomaimtiom were married out only in experiments I, VIII
and 123. Thu results are slam in Table VI.

It has not been possible to itootify the organic piwapmtc cater
present in tho 83;? cthoool :ztroct, deopito a number of attempts. This
cazwpounl appears as a blue spot on pope: chromatogram sprayed vi th the
.“ccolrod col molurszzy (55'?) moiification of llama and LEhCEUDOd mlyhtloto
can. . limos-or, on hydrolysis in 3:! hyfirochloric acid for 5 hours at
130°C, this organic phosphate ester though not completely hydrolycod,
showed the presence. of inocitol, glucooa co.) inorganic phasohato. f-notior
spot trailing the glucose spot cogent-oi on the paper chi-om: grows; this

to probably duo to tho prcocmc of lryfitochloric coil in the hy‘itolyooto.

:2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

28 8a 8.. 5 8m SE 8:. u

8% 38‘ m5 Ru 3.. can BBS 9

$3 2.8. 33 Eu «8 ,8.» in: a

$3 “a. 8e 89 88 88 98‘ a

8R n8 an 8... 2m ’3: 8n: n ,5

$2. om» E. 8: an. R: 35 a

:8 «no 8.. RS ‘ Rm _ Raw 8.8— a

:8 5.. . a: an. , 8.. as , 38“. <

m2. 3 mm a. 3 _ ca. wfiu u

33 R «2 no a. .3... 88 a .

89 8. n3 8. 8 .3: an» 9 flag
. .3 r 8 8m . 8. a. __ 68 as. n _ .

83 5 3a m: «9 RD 8% < . .

$9 «nu a? 3.. R. m5 :8» u
llama"

a} . . Pi

 

“Bu-...?!» .3 a}. 8.. :26 353% 8.58. 185. 3a la no Inn-38!:

3n» a!“

  ‘, p"

 

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

933030 a *-

 

 

 

 

 

2. up gm.

$8.3 *8.” m: Rod ‘8.“ 3.: _—

Eda “vi *3... Em Km... 31...... o
«QR $08 “8.3 $8.: nod 3mg.“ .—
Ewan 8.3 “8.“ good” End $0.5 n
awn?“ $96 mafia Sin *8; 3.5 a
“2...: *8.“ man... mead :84 E46 0
$33 $3 Sod ta.“ End :3.“ a
sand: 56 *2.“ man; «3% $3.? <
813 *2.“ *8... $86 “03.. than a
$8.5 “no.“ :86 Sm.“ $3.“ $3.8 a
«3.3 «86. m8... ERA Roam than 0

3.: “8... Rod Sm... ER $3.8 -
“Ram Ed “86 $6.2 33“ 33% ¢
RM.“ m EA $2.: :36— Rafi t9: _

A . £« ,... 1;}; “4.. . ...< ‘ I. ‘
I. I8 unluc- iuo moo In no Sal-unan-

 

 

fix: res-212:1: of the chromtwgm'gshy of the hyirolyzate cm penetrated in
15)

extracttm was dried and er: mated with 3.???) ml. of ethylene dichlorila

C?

3013:1011 6" the 11:21.! f7: ctiun

    

- 'me teak-Jug left nfiter (36915 ethanol

     

for 13 hours in a Sozahlet extractor. The ethylene dichloride extract.
thainixrg we 119L351 ms evaporate-:1 don-m ta 3 sax-all volume. The roamue
from :m 1191;! extraction ms stored far this extraction of waysic acid.
In aliquot of the 1191:! extract was plated nul‘countod; results on the
incorporation of label in the. lipid fraction of Experiments I to I}:

appear In “tables III to V.

 

it: cf :33: 13.3.4: fractianu ma 1191-1 fraction was hydrolysed an}
some of the away-pomnts in the hyirolygate were detemzined by paper charmin-
tography. 3329 method fella-".3043 um esmutizzlly the one described by
wagcnluxzcht £423.. (345?). Tim lipid fraction oE Expert-manta III to VII
were pooled cagocmr, cmccntrach to a Gflfflll vol-um and hydrolyéesl.

1.1 W” «Emilrn-g-im- 2.1.! an“ byximlysis was carried out in a scale-.1

   

tube In an :‘blcrhdicn pistol. In the aealed tube was placed 1 ml. of
consummated lip-1.2;! extract an} 0.3 m1. of 0.53 sulfuric acid. The lawn-0157310
was can-19.1 but for fh hours using water (as a reflux-:13; solvent. The
hydrolysam was azjusmd to pt! '4» by stirring with 30-2. «32-4? (3230.) main.
Tim resin was filtered off and aha filtrate was taken to at: .33 by
lyorghiltmtion. The thrice! regime was aimn up in distilled water and
chroma tag; ’51 pin-3 .

Spay. new imircfijsgw .fnot‘her 1 12:1. of the lipid extract: an} 0.5 m1.
at 62'! bylrochlouric acid was placed in a male-'3 tube. in ma r’blerimlaficn
pistol. The smapio was hylrolysegfi for em hour using abnetlaylcellosolve
as the refluxing aolvent. T133 hydrolyante mm 2.1 justed to pl! 1.5 wish

Down-2 (1130.) resin. The resin was Eaten-a! off and the filtrate mam:

55

 

 

 

 

r" /"\

3 w s +--- mum—4m -
'U

m a) a)

u m u vo up.
0.2 >aucu H0 H0
w-l Fun-14': com mu
Gad-a coon-a -oo 'oou-I
2.23 32.33 :3 as
use: hunch up: us:
00.0 4300.0 moo m-H

Figure II (a):- A typical paper chromatogram of the organic phOSphate
ester hydrolysate in Methanol: formic acid: water
(80:15:5) solvent system. The paper was sprayed with
sodium periodate-benzidine spray.

 

-..... H_ rufl--‘_--

 

...--..- -..‘ “H

. . --...- ...-

 

36

 

 

 

if)

/

 

 

(“P
i
{
$9
I

'U

o m a)

a“ £03 Ba 23
«4.2 pew-In cum mu
no.“ coon-o 'UO '0?!
moo Mama) do am
0004-! 'UODOU on «to
$4.130) {hi-1.130) “Fl 4-4::
and) some» moo m—H

Figure IIgfib)

A representative paper chromatogram.of the organic
phOSphate ester hydrolysate in one solvent system
methanol: (80);formic acid (15); water (5).
Chromatogram Sprayed with ammoniacal silver nitrate.

 

_‘ --‘....k-A- ‘

If?
to (1mm by 1:70;: 1.1.llization. The 11113.1 residue; 1:113 313301ch in distilled
water and chromite-3211:1113}.

31.239.11.232 1: .1‘wi‘30 5.111311% htimlyaia was can 1.4.1.1 out in m finnwlic

   

potassium 11321903113. 0m :21. of the lipid extract 1...:3 «1153011121 in moist
banger» an! to this was 3:. 1.1121 one ml. 013 1.113.}! mtimnouc potasslmn [191 ro-
xLio. ma bfiirolysis 1:113 carried out at room tenant-acute for 9’1» 1x31: rs
with occasional 321111113. The reaction mixture mo filtered and the res-1.1m:
mo washed with 31:: 5 ml. portions of benzene-11211131101 (3:1). T113 rcsiiuo
me than 1113.. olvcd in distilled 13.1mm: the p11 311.justod to 3 with acetic
acid, and E1 to solution than ontractca 1:12:11 petroleum 33‘ act. The other
layer was discaricd and the aqueous layer filtered through Cclite 5‘5.
T113 filtrate: was or. ctrwcml three than: t—i‘ 1th ethyl other to fQIDOv’E acetic
acid and was then lyo 11:11:39.1 2115.3 hyirolysato was 1110331331 in water
and chmmtogmphod.

Tm chro 31:03? .aghic details of lipid hyirolymteo 32:12:33: in tho acction

on paper chozm togra pray.

c) LtvacJM of." 3129114: M11:- f variety of mthois were tried $301: the
extraction of phytic acid.

I . 14.

Dorian the early stages .of £1113 scaly when C aims: to). 1....3 prepare}

1‘.£A}Oi31°' :01 U33 :9.) to thing

by £00.11ng 13361603 to tho pols and 3.13 0 that C
maturing pals, £111: procoojcro for the isolation 01! 911371.13 3121.! began by
homogenizing tho pols 1:1: 11. "‘ ’ 13120121110313 acid.

In a typical case, two pols from lé-flaaixzy-old plants, which new fed
E38140; in a vacuum 11c- 1:11:3th for ten 1.3331 .1, mm 113.123.21.131 with Cf-
hy-Jrochlm 1c ccié. To the 1111113.:roc12131'1c act-.1 c2: tract 1:33 3.1...ch 15 ml.

of 1’2"?” .- t3: ichloroacotlc act} with con: 1111.01.13 stirring. 1‘2th one hour the

precipitate! protcim were 1221mm} off and the alarms was 1133:1321 on l

'5
{J

‘0!

hot plate, first: treetewi with 1:32;": aqueous «awnia solution 11:11:11 the
solution 1:113 slightly 1311;311:113 to 11:21.13. The solution was 115131321 for
a short 11:2, 1111th to cool 11:11 1:}: 1.111 emcee; 31:31 11:1 or we ......1. On
eve: rattan 1.135.151: curate-13 sweater}. These cryfistale mre fiiaaolved la
'10 1:11. 0? 1.3}: acetic act.) and 11:13:11: for a 31101:: tin-:11. fifzer cooling
an excess of 31131113 3111 (3:311 ethanol was 3.1.1321 11:11 the solution was
chilled acruight. A white 31131111113131: appeared and the sciatica 1:33
clwmu‘atograpEw-i.

23.1.1 111311-1111: solvent system were 1:21:11 for p13, tic acid cineenao-n
grapzry, he details of which are mentioned 1.11 the paper cEwomteg:mphy
section.

In the later etxniies variatiam on the mo main methods of extrac-
tion mm trial. The first mums-1 111133113 the use 0.. 13:5 acetic acid
(55) and the amend 111110.: comiete1 in a trichlereacetic acid extraction
£011an by precipitation of barium 1::-1.11:3 at pH 8.2? (515-).

fives 3 1113111113 were first c.4113}: 0.1 for the extraction of phytic 31:11
from nature we seeds in an arts-1:9: to device an Meg-3mm mum-1 for
furtimr e: tractions.

Pats; 11-21131; ("2 3.23.3.7: fer (2:2: 3:. eat

 

:- T1111 {31:31:13 of the pea see-.13 were
30:31:31 in water, broken into small pieces an! time etirrel 1:1 '11 13“ 1.1.
of ether fer three 111113. “the ether 11:23 éccmted off, and the man were
oven-11:11:31 until the 1:13: trace-3 of ether bani been rearward. 1113 might
of. the cam-.1 3:.-4:12.13 was: 3.1? {11:11.13 311.! these ace-313 were nae-1 for phyuc ecu
ezzcrecum. .

2.! yenmmamu. 1.6 effing 95 c221. 13-12-1132] arse-3.3 were stirred 1;th

 

5 1.11. of acetic 3311 £01: three hours. :‘fter filtration the extract was

concentrate} and cbxomtogrephei.

39

_5égilpr0309tic acii‘gxtrartinnzo 1.6 grams of the ether-fiwieé aaeis were
truaced 31th.?" :21. of 1:315 tricfizloroacctlc acid an} maimaically stifle}
for three ham-:3. The summatm: 11.2.3313 was. collected and. 0.1713 barium
hy-lrozzua solutian :‘aa a. 19.1 to a p31 of 3.0 to 8.5. It was then set «2.9.3.3:
for an hour an} the precipitate, Lidch wually fora-3:.! 1mm:- 913132135 was
«110.13.! to settle. Thu p‘mcipitatc m3 1:23.33 spun Liam in e ccntrimge
an! vaginal fire. 3: with distilled 33.2.. tar, than with $33,“ 9.1mm]. and finally
with distilled mtar again. 1130 precipsitate was dried in n vacm dessica-
to: o~:em1;;.~.t in tin ccatrtfiuga tube. B3111! of the precipitate was aus-
pcmled 1n 0.:“21 Irflrochlaric ac“ 3:31 the other 13.212 in 153?..- acetic «:11.
fit: precipitate was 6.11:1sz to 8:33.! for about an hour, the tan-.Eisaolved
portion was :53ch by cent-213.333.321.011, 43:31 1:133 windows were used for
paper c'zzromtoz;m 33;.y.

Far the ontruction.of’phytic acifi Eran pea pods injected with C1‘-
130511101, the "barium ezztrnctizm” 02:21:04 .333 fella-Jed. The 904 regime
left after 11.91.] extraction vac extracted with l=‘),‘5 trichloroacctic acid
for dime hams an] the procedure described above was fella-3&1. The barium
precipf. ..ata was taken up 1.112,. 133* .rocadodc acid and ace-tic sci-J for paper
aka-0233301333321“ (.lctails in the soccian on paper cmmn Lost-.3 phy). :‘n
aliquot of the sample we plmta-i an} countczl to find out the labelling
1n pirytic nail. Results am shown in rabies II to V.

The supernatant: solution left afitcr 331337th sci-1 precipitation by
bans-1.1m van concentrate-:1 to a small volts. 3-1: unlicr 3 am: 11323:. I‘ portion

of this solution L :39 plate ..3 42:11 cauntad.

m: 2111...}. resime 1.32:: was 9&3»...ch in water, plated an} counted.

333

16) Qhrsm 20"?83‘311‘. : :3 33.73.: t 13:: .

fl) Column cfirOfttsgfrggg;

9:911:31: clwnwztcgrnglty of the b'triz-zwoéehtle Ernest-£5333:- Ettampts were
male to identify the cagmncnta of the supermtant fr..;.:tian left after
the precipitation of the barium phytata.

filo methods were use-J for aegmrating tln bar-ism azuzx-rmte cowmmata.
The first £133:le entails the use of varying concuntratiom of lithium
chlorile an tbs elutiag solvent (331), while the second mthol utilims
clam-dun Eonmtc-Eormic acid as the eluting solvent (5'5).

(8) l.

evaporated dam to a trail volume, at which point a white precipitate

3231232.“: chlarile 55"fif’tt'f‘l'lfi‘fl!‘ The aupcrnntzmt fraction was

   

started to appear. the suSpenaioa was then treated with In}. 112:) (H)
resin and tin precipitate went back into aolutirm. The resin was fil-
tered off 33.! the filtrate was made strongly basic with cyclolxazquaas-inc
and evaporated to dryness unfiar vacuum.

The resilue was dissolved in hot absolute ethanol and the insoluble
was (which appeared to contain an organic phozeplwtc) was filtered off.
The filtrate was taken to dryness, the reaifiue was dissolved in 15 ml. of
distilled water, and this solution was aubjectcl to calm-.31 chronatogrepw
and pap-m.- chrormtography.

m2 ion “puma resin use-.1 was Dower-l chlorile (17:53-27:73) mesh);
column size 36 cm by 1.5 cm, rate of (lot: was adjusted to 1 ml. per 3.5
minutes.

fitter packing, the column was lowed with 1’) ml. of the sample and
the calm-3n trauma-.1 with 53-63 ml. of ..'.-istillol water. A mailed gradient
elution tcclmique Lana nasal, £12.32: keeping {‘59 r21. :31 water in the mixing

chamber and 0.133! lithium smart-la in the reservoir. titer collecting 9'3

hl

fractions of 15 ml. each, the concentration.c£ the oluting solvent in
the reservoir was clangcsl to 0.1%»! lithium cl-xlorilo witlmt charming In
contents of the mixing chamber and another 75 fractions were collected.

The total pheaohatc determinations of those fractions were carried
out according. to the method of Barley and Dcturk (3557)).

The column fractions were then evaporated! down to a small values
and subjected to paper chromatography.

I (b) Formotc ocgorotionzn Tho mupcrnotant fraction after treating
with 1m 1??!) (E!) mail: was extracted tln‘ce time t: 221 hi) ml. of other.
lhe water layer was chuc d in volume and put on tho column.

The ion ouclmngo rosin user-3 rm! Swami-E? (lira-TOO cosh, farmste);
column 31:6 1.5 cm‘by 13 cu; rota .: flow 1 ml. pct minute.

The rosin.wos first washed with fin hyflrochloric acid and than.uith
SH ammonium for-mm until free from chloride. Tic excess of filIDi‘liLml
foramto was: rest-loved by momma with water.

fftcr loaiias, the column was wnohcl'with 393 ml. of distilled
water and then eluted with the following solvents:-

a) #30 ml. of a solution'madc 0.313 in formic acid and 0.Q3 molar in
ammonium furnace; 55 m1. fractions were collectoJ.

b) MI) ml. of 0.01:! famic acid . 0.63.1}! mmnima fox-mote; 35 cal.
fractiouo were collected.-

Thcac fractions were concentrated to a small volume anl aubjcctod
to paper chromatography.
921mm cftrcswtogar‘xfl o! t‘32 $7“: othonol outrocfififwrr go 157“” fi'-..!_g:1:

.nitolso The procc'lure followed was the saw as motiomd for the chro-v

mtograplzy of barium supernatant fraction (fa).

as?
The cluting solvent system was formic acu - armonium formte. m:
83% “trawl extract was cmentrated to a small volume and put on the
column. The column was washei with abaut 130 ml. of attuned water .
tn mm the sugars, and after that the rest of the procedure is the

same as mutiomd valet (B) «have. The water wash and the oflwr fruc-

tions were taken to a small volum and ctzrozmtogmpmd.

 

a.m;.or-*ra'fih'~ :— 1! variety
6! solvent BWteum veto caed for the 1330138101: and fictification of Cm-
imaitol from the 347:; ethanol extract.

In most cases asccniiug; chrauuatogrmns were tun 1n clu'amtegmpfiic
tanks for :24; hours on uwathed Whatmnfi" 1 93:33:. Each jar was covered
with t polystyrm cover or a cardboard box in oar-.32: to protect it from
air currents an} condefiantioa.

the following solvent system mm trial in order to find a solvent
system which would give the beat separation of the compomnta of? the 33’
ethanol matract.

1) Wm: ( :2). ethanol (1). water (:7)

2‘) n-butanol (h): pyriaiim (1): water (I)

5) pyridine (7): may! alcohol (7): water (2'3)

h) n-propanol ((2): sum-Janka byword-dc (5): water (1)

5) lac-butane! (S): acetic acid (I): water (.5‘)
Four Of them; solvent ayatem (wt-hers 1, 5?, 3 an} 2‘») gave a reassembly
good neparzztion. '13;me reanlts of these separations are 911mm in
Figure m.

Fm: the vimmliaation of spots, the papers were first air clued

and than sprayed with the mmziacal silver nitrate away of Ballou uni

h}

 

 

 

 

I
__~ _‘I .__———-———_..—— .-.—...- ...

no
N\
L, 1

 

--.. ..- - ‘

1

glucose
sucrose
inosit 1

14
Figpre III (a):- A typical chromatogram showing separation of C -

inositol from other components of 80% ethanol extract
in the solvent system n-butanol: ethanol: water
(10:1:2).

 

up” ~——

 

 

 

~—-...—. -_ v

- _ m“--~ ~.---”—. --—

 

 

- .— ..- ~-'—....—'--' ...“ m—-.~.~--AA- 4V o

 

\
<

A \ -
/ _{L-
\
L...
db-
fl”
.1»
‘!
i
l
‘.
l
1
1
0
\

A
v-I H
O O
u c:
H m
m .C: H
O u 0 O (D
2.2:” 2; :3 8
IQR H m o
:1: go 0 O :3
H (I) :3 I: H
003v U) ‘H 00

14
Figure III (b):- A.paper chromatogram showing resolution of C -inositol
and other components of the 80% ethanol extract in the
solvent system n-butanol: pyridine: water (8:2:2).

 

#5

 

 

 

 

   

 

1r _— ' h
.L
4}-

 

 

..fi I. ____+__.__ _l -_._,
_I
A
094
HO
a m '3 8‘5
m m u or.
O O ou-l mu
3 3 8 o.”
H :3 {-1 H131
so a) q-l DO
(I)
V

Figure III (Clt' A typical chromatographic separation of labelled
inositol from the rest of the labelled sugar com-
ponents of the 80% ethanol extract in pyridine
(7): amyl alcohol (7): water (6) solvent system.

Ag-._lv .— ..

 

h6

 

 

A “-..Au-o.

 

I___+_. -._~____+ ._ r _ -c_+l__ - , -_+__.-- _l I _. _ _

A

H

H O

a) CD o t:
a) a) u a CB
0 o ‘H H u
u H m an:
3 3 8 S ‘”
on m H 013%
O

00

v

Figpre III (dl:- Separation by paper chromatography of C14-inositol

from other components of the 80% ethanol extract in

prOpanol (6): ammonia (3): water (1) solvent system.

4-‘4-A

L7
Index-sou (55) or the silver nitrates-30.1mm Izmir-curiae dip mogcnt of {not
and Reynolds (5L), uni heated in on own at 1‘; F06 for two to throw: minutes.

Further chmxtomophm smiles ohm-and :3!- nc pyri! inc: :2:-3;! alcohol:
water solvent system mm the boat resolution of the compensate: of the
”V3 ethanol extract: in {‘34 hmrs.

For wriflcation purposes the 82313 at wml extract was run in the Eon;
of a ban: on Huntsman 5 13-1 paper. Raf-creme spots were. aloo run along with
the stnnlardo ml the 0' 413031301, gloom o, maroon and other lo tell a!
cmqmonts were loco ted by Spraying the reference portion of the gas-par
with amoniacal silver nitrate. me corrosponlim: strips were cut from
the paper an.) the (ZN-inositol eluted with water. The water o:.:trsct v35
evaporo ted to a stall volum in a stream ofi air in the presence of a spot
light an! c‘m‘ozmtogroplwd again in the ammo solvent. This praise-lure was

repeated until a chromtographicslly pure samplo of Cj"-1moitol was

obtained.
A D n -' ... c‘ r!
C“?O"""’3"*""1”" 2, rmi.".c*“:n of corfiocfntr: or; r‘-.‘ ethos“, c: trot; no“:
M “‘ _1 JC: 7 III-Ill.-

 

 

wit: fol Cl oinmitolx- For tho L’entificntion of cannpomnto of 1:0
80,? etlmnol extract, asconfiing chrmtogmm were run in the solvent
system mntiomd earlier for the purification of BEA-inositol.

Once again, most of the clmomtorrno were run on :3 2e pyrtelinoa
any]. alcohol : voter solvent system for .’L hours (moon-.1 23.21:) and the spots
develoycd with mzarnniacal silver nitrate solution. The results :3! on!
the presence of six different. conpmwx‘zts namely:- inooitol, glucose,
sucrose, :tyloso, glycerol and orgzmic pin-39mm ester.

In order to find out the Latent 0" lo boll any; in tho-so co"; moors,
the 33% ctirxml oxtract was run in tho farm of a strip on Hrztmn 3 Ill

paper in a doseonling tank for 33 hours in tin some oolvent system.

1.3
Reference spots wore run along with tho scomlords and tho position of
the cmwnonts was located by spraying the papers with silver nitrate
spray. Ila strips more cut and the air. componmts elutod with water and
tho extent of labelling in then determined. Bonita of the chrnmtographio
separation on presented in Figure 1?.
o': f “.314 h Miro}. ..

cltrorrntooro fll" The lipid hydrolysotea wore

       

cmtmophod in four difforent solvent system:-
1) isopropaml (3)8 acetic acid (1): water (1)
C?) arbumrhol (1s): acetic eel-J (I): water (5)
5) n-propcmol (35): acetic acid (1): vote: (1)
h) pyrifiim (7): moyl alcohol (7): motor (5)
Out of those four solvent oystom only number 1 on: four abovo new found
to 311:. a cloer resolution.
The spot-o mm devolomd once again by using the silver nitrate!

spray. Results are presented in Figaro V.

  
 

.. ml inositol polsqalmonlmtoozo For tho
separation of. plrjtic acid and inositol polyphoophotes, various solvent
system were tested for both doocan-lm an! oncoming; popor thromzogrophy.
The nine solvent ayatoom tested were of than following, ammo-anion:-

l) t-butonol ((3): formic acid (1?): water (’7')

f?) t-butonol (8-): 13% h groan: water (:5)

3‘.) t-lmtaml (8): plcric acid 5.5 grams: water ('37))

1:) ootlnnol (80):fomic acid (15): onto: (5)

5) mtlmnol (C): main (1): voter (5)

6) ioobutyric not} (13): 0.1??? nos-30121:: (C)

7) n-propanol (53): ammonia (34-3): water (1:?)

1&9

 

flu ‘— _‘

W’M-M“ E's-m J. 53 Pic mm shaft: {the s-mzw'UmI of ca*5‘:15*..t5‘3 of 533553 52-5

cum-15.: 1 Can't-1532‘: of ' If; instant :12! cm 21.55191? CE;?.”0§.E€.O;1TC52-EJ
a": in £23.50 {51:31.20 (7): 9....‘13’1 {llamas}. ('2’): Lilia-5." (.)
$015011: 3:713:53. .

H. (3), (5’2). (‘5) 55:51 (5: ) 5:52.522: 5: 55555255555 of! 5.22555 13-53

city-mm} e.::f:'.-::::c£: 01’ p.333 (watt) 3", L, 53, 1'"- .52-1-.’-. 1.”; 53:13-53

(reapcctivcly) af er ingcm $133 of 014 ~inoaitsl.

( 1") . stamina-1 15:331-201., ((5) 5- Sta-n 3155:.1‘3 gimme, (31) -

stealer-.1 L510 0, (I) ~ 552:. 25.124! 32: - 355:), (J) - Maniarl

inn: 1 :01 tammyta: phi; to .

 

u i.l|‘llllut' ‘!

51

 

at Houwokaw
m
. 5. Heuwmoaa
x/llllx _U ovum

25/; m mwmhfiouvhn
mama

\\y :5 can.“
\\

\IIIIIII/J mwmhaouvhn
. v«uu.uaou

gangs

 

mfimhaouvzn

;; .5 can» cage
" wfimfin

 

 

solvent system isopropanol: acetic acid: water

Fi ure V a :- A typical chromatogram of lipid hydrolysdteéain the
(3:1:1)-

52

 

 

\i/-

I 'i'Ii-‘r I! ‘1'": ‘55]! 'I- , 4| nl.. yuxk nll'l"|ll»l. .

 

'-’I itll“)--"‘ 1’ I'~l.‘l. 5 I'll" '0‘: n

 

 

mwmhaouvhn
omen
sessfi

mwmzsouwhs

Uwom
.ucoo flange

mamhaouvhn
vwoa

wade

eemsfl

Heufimocw

Honoomfiw

pyridine (7): amyl alcohol (7): water (6) solvent

Figure V (b):- A paper chromatogram of lipid hydrolysates in
system.

 

5h

 

il'!‘ 'I'IIIV

 

 

 

 

oomuuxo kum
caucunu
-ouosn em

UUQHUNG
taco owuooo
uofi

 

5I\.\..II»IIIJxU UH O“
.r

fiflHwifi-\L.:i!r1 owuoom KOH

_ ououumauoum

aaauon

 

.
_

vwoo afiuofino
-ouosn em as
oumuwmuooum

_ Bowman

fir|\lll\lltilli!li ;- vwoo
4 - .,;. .wv owuhnm

oumsmmonm
ofiaomuoaa

 

phytic acid from mature peas in t-butanol: TCA: water

Figure VI (a):- A typical chromatographic separation of the isolated
(80:h gm: 20) solvent system.

 

55
6:3) isoprozaanol (3:23): picrio acid 1 first water (3‘43)
9) isomopaml (7:5): org-moist (12:): motor (2'52?)
0! these solvents six of! timm (mowers l, 5”, h, (5, 7 out} '3 above) were
{mi to be sotiofactory. am of the typical results arc presented
in Figure VI.

that of tho ctnmntogmphic ooporotions manned how we re carried
out either in isobutrric acid: oars-mic or isoipropmnol: ermim water
solvent system. Tho inositol :2! Splinter: caved very almvly in tho-55o
solvents am! the spots were much more coupon and clearer than EFL-woo
obtainei in any of! the otmr‘solvonto motionei above. The solvent also
travelled very slmzly on the paper £2713 therefore throe to four days. tmro
accessory for the dovolopoont of the chromatogrem to get a good rcsolutim
of the compomnto.

For the detection of Spots, the papers new sir dried and sprsyed
with tho Amlrod anal B Mural-Ly (5.7") mlificotion at time llama om! 1:5:er-
mod wlybdato spray for the detection of organic and inorgsmic pfiwephotco.
The papers mm then heated in the oven for 5-5 minutes at 15.55306 end

z-tpoaed to ultraviolet light.

5’4

 

Iii 'Irl ..'..

 

O

‘-t'l..| II it . . I‘llllnf [IA II 1 I II . ¥

 

 

uoouuxo owoo
ofiuoaso
-ouusn mm

 

.\12.4. L. . uoouuxo
PI IllllllleHHv owoo owuooo

moH

\ju 33
.r

AAHIH.:-‘ ilk! ofiuooo fioH

. ououflmwooum

Esquon

owoo owuoano
[Illir nouomn fim ow
ououwmwooum
_ Bowman

 

1

owoo
owuznm

 

ouunomono
owaowuoofi

 

Figure VI (a):- A typical chromatographic separation of the isolated

phytic acid from mature peas in t-butanol: TCA: water

(80:h gm: 20) solvent system.

 

55

 

 

my... ._

-.‘-

4 ,,_.._fi...

0

o

O
O

\

/'“\

-- “'--""-'*'—~

 

r '. .... A..-

b
a; C3
\4

1 4 Wu"--- ------ -
Q1 U) ‘
. o
I'D 'r-{
'o mow-c o-U
'3 ‘3“: 33 on»
on “a uni ~44.»
«44:0 w-l :2
u go. w-l gonna m
'14 «432:: HO. coo.
u HUNG HOH'U no)
2‘ fifiata‘ 33:23 8.8
a. pan-Io nan-«'6 0H0-

Figgre VI (b):- A representative chromatogram showing separation of
phytic acid isolated from the peas in the solvent

system n-propanol (5): concentrated ammonium hydro-
xide (’4): water (1).

 

E

 

56

 

 

 

 

 

 

- u...... ’

    

 

W'”‘~ " '
4r-
fl‘” M; -.._. ‘ - “a" .-
.L
I
+
P
I
P
I
—+—
1
3
p
l
l
I
t
l
9

o

I'U 0:!
'U mO-H mu
«4 uuo um
0 «wow ‘30 00
cu uh um an
0 '335-3 galiuz 5.3
H gw-nszu sou-Io con.
a saw: sacs 33
.5: “34:14: “Ht-'10 £143
9- u-DO-I‘I-IO ronl‘F'w HO-

Figura_VI_(c):- A paper chromatogram showing separation of phytic
acid of mature peas in the isobutyric (10): concentrated
ammonium hydroxide solvent system.

57

 

~-o-__qp.. -- .— “—.v— -.. ~1r

- .mwflwwm.a~-..‘w_q ,. _

 

ll.||. ' +.!‘|11'4'

Adv

 

\Lj

 

w

 

r

l

 

 

uoonuxo
vwoo owuoHno
ass am

uoouuxo Uwoo
nausea moH

owoo

«dunno fioa ow
ououamfiooao
Bowman

owoo ownedno
-ouvsa am as
moonwaflooam
aowuon

vfioo owuhna

muonmmonm
owdowuoow

in the solvent system t-butanol: formic acid: water

(6:2:2).

 

Figure VI (d):- A typical paper chromatogram of phytic acid of peas

58

 

‘1'».‘11 ‘1 -..:I£f\| «(Tot I) rF.‘i : ... . y . I.IJIII"L‘ :l , urn: ( l . ‘ l.

\\\\:;IIII/ .t oumsnmonm

ochwuoca

\ I i '

Kill-l

/ -... Q 38 033a
. , . ‘ .‘$.|\Ivrl.ll\.!. ..D n/[. _. ¢\.

.. :-a 5. -xy nmxxxnanlvaz1ctnx. :11; «,swu
\\ , 3/ 33%
\\\\\I‘ ,o l:

w
.T \ .538 .Bm
+
m
_

:a/x .\ .: r ,- “tr vaoo owuofi
\ // .. n 2M: \../u£oou?3 mom a.“
xx5;:‘ 1x ; 1 . oumuhnm Baguwn

d v x.. ...- ..tquv vwoa owuoow
// 1 P \\a![U mod 3 33.39

aswuam

 

 

'1 'vii All .15.!!!“ II'.‘ I"Ill:l||. .Ar ..I".lv-"u‘l. Irfi

Figgre VI (e):- A typical separation of phytic acid of peas in the

solvent system isoprOpanol: concentrated ammonium

hydroxide: water (7:1:2).

 

runs“ :2:) 11111011331131:

In the press at 11933111113111: study, prior to 1310:1113 any tre cc: $012313;
e-.11sri.nent.., crazies were earths} out on 1:31:11 Earzmtioa OE 911311.111 in t’m
growing pea plants, with the intention of (firtflifliflg some information 1.121-
gar-‘mg the 1:11:11: at which phytic acial ayatl'tcsis cgzyr'osczws :1 11:11.3»:

ate. Previous investigators ('71), :71,’,L- ) 1:301 6 1:21:11: ty of 1219.11.10.19
to determine the phytin 111'- 911021115 ccmccntrs 1111011 in 1111:2112: , 1112131113 from
the diminuition in color of a ferric t Mocyamte solution (517) ( :tich by
a suitable choice of concentration of sci-.31 and of the reactants can be:
male almost proportimuzl to the phytatc concentration over a limited
range), or aspara‘ing phytin 3:11 ortlzgfiwspz ate chemistogrspl‘dcal1y on
paper by use of animus solvent syrto 1.9 and than es mung the spot
area or spot lengths after developmnt of the c3:r<n:ato:;ra::1s (3+3), ans/or
the stamimd colorimatric determinations of: £a.w,1aaxc phosphate beware

and after heron-sis of 1:21;: ptzytin 3111111111211: (33-).

l) Ghana-as 1: 11:11:11: 13‘1“; 2:11:13 in misting to Marni: crt‘1a"" .1:3':.-:s.

A's

  

These determinations 11ers carried out accoraziing to the 21.19.1101 of
Earle}! and Bcturk (’49), and tla wlorimtric detemfimtiam of plies-pints
were done according to the procedure of Mahatma and Ersy (5:1). This
particular xmthod of Barley car} strut-3.: has a'111'ants;;cs over the methods
devise-:1 by 11011: ($11)) and Powder (313). In the method of Bolt, where
phytin concentration 1 as mmsurei by the :11:.:1m:it on in color of a ferric
thiocyanace solution, certain anioas (110* ably orthop‘msrh. mtc) are 5012111 to
interfere and tha ortimphmhsm commutation string 1.111111111111113 of staf' y
here is quite high. S-mllarly, the 119131013 of Fm'ler has the disadvantage:
that the estimation of phytin phosphorus an! orthopbospbate by massrisg

the spot area or spot lea-3.1211 and or wamring the. zicnsity of 1:111: spots on

("1

O
pans: chronmtog rams is more a quaiica at}. .11 pros-c lure than quanti cattva
and will give lower cmwentmzion vshms.

F11. 033.321.1013 1.11.3 3:01:03 (51) have shmn in r. mates of' 0.1;. 1.1.11.1 plants
tint "1141033” inositol (lacrosse...) sharply «35:121.- gsmimtisa, zcmins cars
or 1039 con.- was for a while so}. than slowly incl-12:22:11.3 as the giant
approcckza 13.:‘1; criiy «2.13:1 5.1.1.1 mammoth. 53 11.21141: 3:11.3sz Esau-m 0'31 171::
cimngcs in 1:03:31 1.210311310111113 00.3 p}. .ytia 3.11.0139 bogus 0033:0111: of the p.13
:11: .013 in: 3.0003 that 91.1th com-1.0:: 1.2.1.011 1:11.13 sh»: :1... 1.1-. " :13 scram.-
£1013 and mmiwa at an cascathzliy c0"'t:.mt 1w 1:11.01 1.1112121 catnrity.
Typical results are sham 13: 11.1.1.1: I 111133 Fig. arc I. It 13 spimrca: from
figure I 1:103: the results of 21011111133011 and 1:31:04 (51) and this study
are in agreement 1.9.. the dccrcssc ta "banal" inositol drains; and just
sits: gsmfimtioa 13009 in fact rcprcscn: 1.11:.st brcaifiam a! phytin
phosphorus. It: is wrthn. ...-a to motion bars that stuc'iics of 111.113.211.191:
and Malawi wars on warned with the cats! 11.111113 11m' :01, 1::-{wrong in
the 0:2th case only the 931313311 91:01:91.0... was domains." 3, but the results
arc still in cg; ..cuxnt.

These results 1113111310 0 arc-.1: difference in phytin concentration 111
the! flouting pas p1. mm an} the mturcl ma pa. .1 , 1'13: 13:31.3 commutation
1:1 relative? .37 higher, 33. 111;; 11131.1.-.1113 of its sync! 1.21.2319 11311113 scci
fat-cation. There. fore, it was dcci Isl to carry 01.21: tracer fooling expert-
mots with the Lam-1mm pea pols, 1:11am the seals am just beaming; to
form 1::. Beam 0.. ccsc in 1.111111 .113, c 11me vs"1cty"l.1:alc zml" was

selected for tracer studios.

C) r0350. ”0- ’bgfi , C'ZLW-li 3:31-1:13.
( ‘1) , ,fiAhA-k‘. 6 I“""‘"“"‘j" .: p. 1 .1§'(‘ I.“
EC~LM_¢. L.-'I ... ”33¢ a '1. : 1.4, J ...-'11 p. l: b: ..Q 7‘ '3, I

For the re: sons :3 snaiomJ in the 11120111121100, '1th ..s1c......1 to 1 so

l‘ I
first weevmesitolvc as a eoeeible ;:-re ..urser of 312.2111: ecu. m 22*
cemm1 13 an: attainable: mercielly, end several mtheaie of {archer-aura

had to be tense: befere e setteble 3.9!: 2.2.2:: was e-u. 2122.213.

Th: eta-.1125 of. Elisha-deem eel melee-1 (31) inflame: 1.1e111.., 2*- of
tam-11:01 both 1:: twisty 011 pee plants an! 12: e: 5:15:21 metering pee W15
when meme-.1 ea a CEO... etzeephere. .1 mesh-e: of! rte eme- were fellweed
1:1 earlier to 1::. Le: a make! again: fer the leeletien of CM-kmeital
4:211 1121...}: 2.2.21: .31": '90.! y 31-33.

1:11:13! are. 193, where the flare eea plants were erected to a
(1)402 atmx: phere 1a a 3-2:, as we var. peer results due to a very 11m.
time aware of plants: to c “0;; ermegsher 3:21 also to twee than eaieeuate
creamre cf pleats to light re- cities. tn meter depleden free the plant-:3
and comieneetien of this meter on the wells of the jet. 11:12.: 61 so recent-21
1n the e211H 2:;; of plants. Overall meal: of this: experiment: we a poor
moreoretten of c1 0.3 into the meet .01 freedom, which was «11.0 in part:
to the feefiiza rim period and to the fee: that humming pea plants are
slow to incorporate 6140.; into 12109311291. The small amount: of 61463 in-
corpotnted 1mm them plants went: largely into the gluceee m1 sucrose
fractiens.

in early smaiiee where. the 6140:.» was fonreJ m1 fed to 9018 within a

eagle Tureen tube, the («2: ...m: of lab: 11m" over a per 10.1 of 32-5 Jaye
nae mostly mixed to the glucose and sucrose fractions, and these fractions
were alea pearly 1 themed. , vimreee the Label ling 1n the 1110:4101 fraction
can arsenal! 1:...

In later cases, where young pea p013 mm expose-.1 ta an etmeehere 0?.
Cue... in a 9212:1111}; evacuatei mmberg tube for three «.1... ,3 ea! t: mm lactic

act-1 we use-:1 for gemratim'; (2140f.l ammhet better reeul its new outeimd,

1:22;"?
but no: 50921 999931 to be 9:91 99 a 129991.99 c199 2.9.J £9: 1.29 1301912199 9..
cu-Lnositol. Fee-11:23 {29:19.19 of 999 to three days 99129 twat-factory,
since this 29919: 1999119.! wmncnts com-991wm'291 to 99922999 9.2.21.1 31999229,
with 19991991 I 199992.- 99... >9992at. F1. 9 99:99 3:22 .29 1291:9912 3.2.1.2111 of 219993.991.
Frau one five clay 91.232912119121211: in 2.111911 1.?) 91111992319 91'? (3140;; 2.2.2229 999.1, a
total of 1.0 9219220991219 of [292.225.33.921 19991.99}. 9932.119192] 1.299- (1121.93.99... at: the
0999 time the combined glucose p.193 9299:9129 1322999199 1.29.2; 91.39 919992.! 9.291
gm 7.} microcurim.

In 191:9]: 2.222921212122299 the. 9929991999 1. 9:: 2.2.2912. 2.3923. :1 to 01:9..19 12...:11221.
amount 0:! 19129111213 5.2: £1229 122991.991 53129991922 2912.139: 211.2212 19 the 9199999
and 91292939 2129991999. 122 this 99:11:19.1 9120991129229 1." to 1.. 93.93;.911 plants
were 1:99.13 they were enzyme-.1 to 929 99:92:59129 of c “On in a vacuum 292.221.-
9912012 for a pet-1.921 of 1:) «1932.22. 129 a tygical 9999 1.0 9111;191:2219 of CMGQ
was 2.9921 to $299.1 1.) plants 92.11 52191.19. 5-5.0 39912199 of! 99919191
inositol. not only 521093 £1219 12933129399: 9 32.299: 1.292292229999919 21.2911,
but also (:19 9999 of! purification 03! L219 19991991 999 3- .catly 1999999 2921.
In all 1:19 9990.9 1.112.999 the. you», 99:: 999.111.1193 were used, 2:922 .91 incur:-
poratian 91' c1403 121:9 9:19: 99392- 5129991999 1.99 1993 than 9922- 9112191 91’
the incorporation 1.2120 1909:1901.

From tho 923912952211291192;92:1’293 9992-. 9 it 9222299129.! 1:99.12. 9:119 12.991: {22.20-
991212.220 for the isolation of C“- 229391991 con-9 .9991 in 599.1193 of 61409
to 1.1? to IC-day—cm planta £91: 9 992.2191 953 10 c1932.: :19 a 9992.. .9 (19.291.991.922.
this b99919 the 91:91-21:19] {22.991.291.129 991 1:99 9.22 .2291 1’92: the: pmmmtion of

6144999122: 1 9129992292 22.993911.

wave

The CIA-19991991 was 199199921 9921 992.21.31.91 £1299 2:119 ...-2,.- 91:1 9.991
extract by paper 9.920199912129913; 932.1 11:9 99121321999199 L2: .9 139119291 12;: 9.99922

01 29.111999991391229. .1. 99:12:92 01’ 8012999: 93.299919 2.292.229 999.! £992 93.42.991.13, 9

av

L5
zoo-.1 separation of imoitol from the otimt sugar ws'mmnfzs present: in tho
8335 ethanol extract. Out of! tho £11m elifi’o'mnt 301mm: {:3rsc.;-::a twio-l, two
of them, namely n-propamlz concentrates: 823mm 22333021112: water (5:5:1)
and martian: any! alcohol: moor (726:5), seem to aooomto the components

wary renaiily, but the latter solvent was mad moot offton in film pmgmmtim
summations. It 33w a better roaolutiou of the: whole 33% (221mm). extract
and the spots new more cow-11c: and clearer than worse obtaimi in any of
then other solvent: nae-J.

(B) Fooling e::gx<:1:i-:m1-‘;3 wiffiz :‘s‘mm-izm-nii'nl-va‘cm:-

Varm attempts mm {El-.10 to 5:33.}: 1::-m incorporation of Ola-inositol
in the developing poo polo. 111293 goalies gave a clear 1321.23.55.01; that.
the best: way to Eco-1 (EM-inositol to tho whoring pen pain on} study its
incorporation consisted in injectim tho CM-tnocitol solution into the
interior space of tho pods by mom of a Licrozzyrime.

Variation of mo min tremolo was test“ for effoctivmwso. In the
first case, when muting; poo pair: veto removed from the plants cod
placed in beaten containing tho inositol solution, very poor tncorpora»
tion rooultezl. This type of foo-31%, boat: in mammal desiccator an}
in the open air, resulted in fungus 3:.-man on the 903.3 ia loos Hum three
days not! maligiblc incorporation of! lobe). into the polio.

In one: came tho polo were a} {and to grow for at}: «133m in bonkers
containing; a Curtxzogitol solution. .‘f‘aor 9.1:: days the pods were awaiting
infection by mid and were morii’icol. Inauguration in each pol one
fours! to be 1353 than mac-11:3” of «mo per cent. o! the ant-3&1 commas. (5).? 2
13‘ counts pot minute of inositol).

This type of feeding was swam-m (zoo to 10:: incorgomtioa and

repeated contamination of the pairs by moi-do or mucus grmmhz.

£54

In the second typo oi’ mourns; cngrkonm, Gag-imam}. woo injectofi
into tho mtorior space of tho polo am! the pols more 1::-ft attachod to the
plants in orig: that they might continue to develop naturally. Hold or
fungus growth was not observed in oozmution with this: type of footing;
and this enabled the study of imatpotcation of label at various time 2.3-
torvola at least up to sixteen days, tritium: major cngl icatimw.

In a typical case whore four immturo pods of! about ":3 car: lam-{:31
mm injected with a total of 5.5 K 113‘ owns; per minute, a town day
incorporation study ohmaoci a 33;? izmorgmation of the tujcctc-ii inositol
in some m-alcotwl-cxtractafilo form in the pom. Moo, own: 31 of the
tncorpomtoa emu-inositol could be located in the barium phytnto fracticm
as sham to T2223; 11. Twso results were encouraging no.1 it was antici-
pated the: aviation: lacormatim for precursor otudioo c0221.] bo obmiaca-zl
by fooling higher lovcla of CM-inors'ltai.

Later stunts as? tacormmtion of CM f‘i'flfl inositol 13:0 phytic acid
fractions with hematite; time imitated that about 2:;-5"},‘3 of tho incor-
porated 121061201 could actually be locate-.3 in tho horizon pixytato fraction
after a. mzixmu fooling patio-I of? 15 days. 313:), 1322:2119; car-hon to»
cavcrioo tn the: phytio acid fraction were very lot: bemoan too to four

3:3 am! in fact there was: no itinerant-Mirna at all into plrgtic not] within
the first ttmnty-‘ZOur hours after foaaiiug CM-izmoitol. Tit-.2534: percentage
incorporation valmo veto calmlmoa on tho basin of the total count

0

rocavotefi from cm. poi tw-occzwztoa on} not no tho .t—sio of the total counts
fed as Cm-Lwaitol.

3336:: on those obsowattom, fax-ama- attempts were mlo to locate
tho Intro! to cotzzaomnta own: than the 23:21am p‘izytate fraction. For this

Fatima, cm pod hot-.mgomtos vote outmotol’ wit" 33;; cthmol to got a

55
sugar fraction. (32‘ 330130 VII) The 13203200 was fut-1.1313 ezrt 00:01 with

ctbylcm 2110111013110 which 9,000 a 0-2. '21:: 12.91.! fraction.

8-3133 01.112.33.01 attract (“133.210 VII) W hen 090::th on cirromto 3:8.."23
indicated the presence of 01;: 211E50tont cmzpmtcuta, manly: 1.0.1.1:01,
312. c.0000, 0001-030, 3223/1030, glyca 1'01 and on 0050010 9110.33 2:11:80 critter. 2‘11
of these 51:2. cccrpmxonta 11:1 100089000th lab-:1, with 0011313610 13.011-
121,, in tho or;- 2:010 911039‘080 ester 02.11 t‘ 20 tnxitol. Tim 1013-0113203 of
the rest 01 £1.20 0123080 on} the. 33110001 1200 10:2 0 aigmii’icsut and tin-
variations 22080 very 011.3211: f0: 00 100:; 0.22 the 020.2100 2. 2:0 00:810.! out.
£29100! results of? 02.11.. written: VI II 8120 3121;; the 22138811308100 of activity
in these 00090001180 are magenta-.1 in F133 0113 VIII.

Ibo result 0 1.211100% 0 high 2.01.0090 00210210 0£ 1.12101 In the 083212110
913039111100 actor. 1110 100:" ty of t"~ 0 00:13.10 pim9113t0 cater 10 08111
not 12002211, 21009180 v0.2: 1000 attempts. {1023033022, 002.30 of the products of
by} 0013301 3 03:22.10 cmfzpmufl have: 120021 1.102181271021 by 9:290: cbrouatwfiapby.
The hydrolysis was carried out In 6N 12.321002211102122 001.1 for 5 1101229 at
Two»: 0111 tho. by—lrolyaato stunned. the {18000000 glucose, 1.2» 01:01 0:13 10-
0830010 91103211121120.

$2.0 0:: 3,113 0210 21101 d0t110 :ztmct 002280101225; the 1191210 0:302:03 rapid
and 2280001340 incorporation of (314.11107312291, '2: 1110!: 1232 probably 212.30 to the
incorporation of C u’iWitOl int 0 (:00 9120391131021201210dt120, 2:123:00 t '21:)
10 the only 1110018150 130901-2202: 1.0 90:22: (32?). This is clearly 0120030 111
quro VII. :20 1.21008 mtion of 13. but-.1 1.1180 the 1.913 fraction (slum
1n F1300 VII) 1'0 3 found to be W11! r229 32.11 11-1232 3.1.3.0013: after 1215:. 001011 of
13001301, with 0 1:25:02 lowering; in 101.3301 and commiwnt £5.30 in the

1020290022100 05 1012.01 into the 9. 3:32:10 0013 2323008100.

6

Counts per minute

66

 

2h,OOO 4
22,000 -
,1
QO’OOO -\f -—-—- 80% ethanol extract
--.”. Lipid extract
18,000 ..
HExperiment III to VII
16 000 o—~-o Experiment VIII
’ CI“

x——’-x Experiment IX

 

 

 

3§ 112,000
a.
o
5 12,000
S
tH
a 10,000
o
u
8000
6000
#000
2000
—r j l ‘
6hr 1 2 h 8 12 16 Days
hour
15 m.nutes Time

14
Figure VII:- Incorporation of C -inositol into the lipid

and the 80% ethanol fractions in the maturing

pea pods.

fl

f-n ecil hy-lL-o 13min of the 113.211 fraction m 2: carried out in an
attempt to Lietemim the labelled cmzpemet 1.3 the lace tile.

The 112,213 hylrol gate on clmmmtegmpey eta-rel the menace of
1:101: ital an} gLytcrol, but only £2.26 £22m ice! of the ignite} yeete agzywred
to be 1." .belle 1. 21:13 wee deternincd £36019 the 1""t11. chained an?“ amt-1;;
a paper ctzremtog rem through a aimciel page par-25:21.9 on mice.

Later feeding armaments were run on a cemmmtive tceie in melee

1‘
to atwly the c cixuoeitel insorgmL-etimm with imam-ear. :2:; tine.

In Ezgerimcnta I and! II, the res-nits of which are [:2:-enema 111 Table
III, the fee-11.21:; pea-£01 eerie}. from a week t9 ten days.

The meats sum: tmt 2mm: £1115: fee 3...:23 {Jeane-.1 in E: pen... .3th I gene
a tensile-rel. 1a increase of lei-ml 12: the 11521.! Erection, Lime es the
incorporation of. label into the ep‘izytie {Test-Jen 1:: mite 121.2.

"in-3 tnerea: :e m. t .29: feeiiqz time to 12‘) $2323 in Razperimnt 11, gave
Maize: yieua of tumorguoreted 12.1201 5.3 pE-Lytic eeti, wlmreee the labelling
in the 11:25.: Erection declined. There 1.3 further an memeec of label
in the €372.3c tame! e2: treat, Lil-Len email 903.42; 157 be Line to the tree-5:3 .9121
of the phosphoizmiti-Io presume in be 11.23;: ext. act or of some other
tam-31:01 derivatr-m.

In i2::;.:eriment :5 III to VII, ween 90.3 were Eel a tot: .1 oi Inf} 2: 133
counts per mimzte and sacrificed at 13 minutes, 23+ beers, h, 33, an} 1:?
day intervals, a steady increase 5.2-1 the fixer-mmfiea of label into t‘m
phytle eels! em observed, star: ting w' .21: 20:20 incorporetm -i'ter IS
0312th and immanent; to a mmiemn of 11.3,: of the total tmee‘peretion
after 1:? Lieye. “the reeulte are presence} in Tet-1e IV.

The new cry at he total late ---:1 in tee hmsqzeeetee is also decreased

with hmreeeim the, which my be a remit of tee tremloe: :tzlee oi" the

Counts per minute
(total per two pods)

68

 

2800 n
2600 - T
2h00 - ,\
2200 - ; l
l
l
2000 ~ 1
r R
1800 - l ‘ 1- Organic phOSphate ester
; I 2- Inositol
; 1 I 3- Xylose
1600 - j , h- Glucose
‘ , K 5- Sucrose
. g . 6- Glycerol
lhOO ~ f
n i /F\--
1200 ; X
g i/
.I I/
1000 - , \
/ ‘~
I \~
800 - g 4—1
\ f
a x N 2
600 ~- 1’
e/
i
too a
*2
f“"‘*
\
200 _ \*\h ”ritéx\\
-x m"-\ _‘M“
é\*“"”’“§*‘~<:¥if‘
I I I T T“ ‘ k ‘
0 h 8 12 16
Days

Figure VIII:- Incorporation of label into components
of the 80% ethanol extract obtained from

pods fed 014-inositol (Experiment VIII).

u.)
53-21 C “-11112321201 to 031122): ports of tho-'1 plant, or may to, .132“ :12: 103232322
cine: to mtobolio 12.5.1. 1.2.0213 to CG: 1221.1 not“;

no results of Emporiimnts VIII not! 1:: am sham: in 22!.an V. fins-so
mo ---rimnta 2mm mrformd to 11:12:33: the choc: o! «stori. pestm M3 on the
iroxorporotim pout-.211. In both 22:22:22: the posts: were fol the ooazzo 2127;022:2221
(3.1 .1: 13412012212213 Wt minute) of CM“ ..nooi .201 of goo-:12” .1: activity . ”.0 a:
13° counts per minot per 1211211210121.

In Etzrmrimot Iii tin ponio 1mm 5:21:21: 1:11.212: 12222121121: 11.11.21: can...tzmn
thereon in 3:.-:gmriiznn’c VH1. he 15.33212: (supply 1.2223 fiiocontiunol at night {or
about G"! hours. 113:: resnlsia coco ago in iniicoto a ates-.131 ion. ass-so of
incorporation of 15.31.1131 into pliytio ooid Emotiono in 1221.: E1 (Ho oztrpot'imnto,
81 though the total rooovorios am 12.11.1131: in 22-22321-1212212 VIII than in ISL-sport
mat Iii.

mom is on initial rapid moiwotioo oi 8141212203201 ioi. o the
1191.3. function; enter («’2 More ti.o 122‘ no}. 12121233} to 111223212222- rspilty 1:212:11
t‘m 13:12:81: (1.21;, of tar 1:1 .1, tin: neuron-.212 of! 12212121 to 12-12122. mrti‘mr it is
also 1:10.22: from 11:11: V that the incorporation of Cmoinositol into 1211;. *tic
acid in firtporisfint VIII 19 greater for 1:21... 2 total potions? than in Estooriimnt
I? in town of tots! counts per mimics.

filo result of incorporation of. (in-inositol into 2.2210123 22. notions
of Earperimnt VIII are shown in Fig-.220 IT’S.

The mammal imorporotion of Clo-hositol into phyizic 211211122121:
cowli tions of continuous 11;; ”of: email be 22.231122 121121 on tho voodoo that the
pea polo are eymtbooirsing 1.11022 to! 2111112213 5121211213 of ions ital 17111 be
contmrotinoty moi) arcs-22221.2: in tin: prcoonoo oi contitmmm light. This 12012121
moon that tin injected Cmvimoitoi, 12mm in... {scenic} 1:. tin 215.213.11.225

nail, is in 00222122121022 1. :h t" .12: 11.03 2201 for. 21:21 in tho poi :2, which accounts

7O

2
12000 4 +/\. 1-
.\ 5-
3600 . 1.-
: 5-

' 11

3200 _ ~

 

 

 

80% ethanol extract
Barium phytate
Residue

Barium supernate
Lipid extract

,. 2800 4 \
m .
3 '8 \ 1
a a" 2200 ‘\
22; 2 \4.
H //
2.2. X “/2
m a. 2000 _ , _/*
4-! H \ ./
c: m 3, ’
:3 u .
881m0- /*
v \ /’/
1200 - ik’
. I, - 3 3‘
\ //‘t-‘-—Jfi
800 .
. “N. u ..
uoo - \\\y sv/i
-______~* ’ 5
._ I I """T""‘"~"T“ ‘
0 2 z. 8 12 16
Days

Figure IX:- Distribution of labe at various times

 

after injection of C

4-inositol into

maturing pea pods. (Experiment VIII).

71
for the lessor incorporation of Cid-inositol unjcr contimxoua lififzt con-
ditions.

c3190, tho. low total rccovcrioa of label in Emoticon: VIII my be doc
to the: foot that during the cork potiofi, when photocynthosiovliniwd amt-3y
production comes, the. plant say-stoma are metabolizing cn‘logcnwo untcrialo
and one such available catagmouni is the injectml CM-inmitol. Since it
is Imam that inositol is converted to glucose, £3,716.16 out! oucrooe etc.,
this could provide a route for its evcntucl commotion. Translocotion
into other ports 3 the font may also be momma! curing dork pea-103:3.
73:13 pmzcibility was not investigated acting this} work.

A. portion of; the carbon of the ted inositol is; also dcpooitod in the
cell walls, which is inflected by a constant increase of label in the

131.21 rooiluos with increasimg (iii-i! petiole.

{long with the constant incrcooo of CM-inocitoi incorporation into
phytic acid, the ctzpcrmtont solution loft remaining after barium pmcipi-
cation of phytic com also shozxzd increase in labelling-.3 up to a period of
four Joya and then started to decrease. It is poaoiblc that this who!
lost from the commutes: fraction betwcn four cad eight cioyc opgncoro
in tho barium phytota at eight days, since the: rcoulta imitate a 3:.-cut
increase in the incorporated label in the barium phytctc in going from
four to eight days. Results agapcor in Figure 1:. Emcmr, the decrease
of labelling in the lipid fraction can @130 account for such an increase
in the barium phytoto fraction cm! that-3 might mu be true in Experimnt
Ea", when the drop in tho ia‘ocllim of lipid froctiam is cmiicmblo
in going from four to eight cmys. In tizio coco, inmnver, all. of the. da-
crcaso cannot ho accounted for in the horizon p’xrytate fraction. 1!. sitziliat

conclusion cannot be drawn in taperimnt VIII, atom loss of label from

Counts per minute
a (total for two pods)

2h00

2200

2000

1800 -

1600

1h00

1200

1000

800

 

  
  

_ -—- Barium phytate
--- Barium supernate

4 0—. Experiment III to VII

g ,/ CP-—<> Experiment VIII

. /

T / A / X——-X Experiment IX

‘ ' /

l 6 x 3/

.1 / PA

i “

J /

‘r

i I t)""-C

‘ /

1/

9% x

a

l

 

I

£2 £6 Daya

H-
m-
#3
G)

6hr

hour Time

15 minutes

Figure X:- The incorporation of Cl4-inositol into the barium

phytate and barium supernate fractions at various
time intervals in the maturing pea pods.

73

113111 13942133 in going; from four to 31;;‘33 13: #33 13 eaucutiamy :33 1131121
coca-33:31 1::) mm 103:: 110-3 :33: 13131323 333333.332“: fraction 33.1 313 0 cm mm
ta (:31! itmruflam 1.2: 1:113 Elvis-u 332131316. It 143 933313313 tin-3%: 1123 coi‘igmmnta
of both 2112-3 1131-1 fractioa 3232 the 123111.“ 33:31'33'33211: 1333....» 303:1" 3'33
to £133 barium 3133mm 1333111: :3.

From 123'. 3053323? ifitlfii‘tfl the 550116;:1215 WikIUQIQL‘LJ can in 33:32:31:-
!) Tim: ‘53) 133331231 614-1332312101 agagmrs in :33 33312.33 phytata fraction
03117 3:23:33: 3:33 Mum an] 13mm: that £1113 121w; 93.31.33 133333333 with
times.
2) Tana 131231112313 31 113: 111.213 133(21103 33.331333 3 2233:1133 3: 5:223 333 of
the first ” Mara and than 3:33: to 13333333, with 3033121333313 1333 of
labelling being; 312332335 133-32321 3m 23 1332‘ 313323.
5) Elm: nix-1 constant incorpm 311311 a” “33- 123331131 121 the M3113: super-
natant fractian u; to 1031' 33,33, 53113333 by .3. 331.3321 1033 31' 132231 from
four to eight: days 33...? film 3 92133131 3333-3323.

4.1

1'1) 11:3 2330333731 12.21231 1:: time» S; 2 311232131 1’: 33:; 1021 333333.333 1322 1.12232.

715
1123 mm: 2:5 at? 2:12:33 amines we. whamtenz with a. & mi: 03 $112

4

E4213. rim pmmflne pattzmys {or that bimmimsm of phytic emf-5.53

glimme: ,
angry-3 (@1103 :1 _, )
0 {:0 . 1:13:30 pm ta

7 Gawain and X
9-) ; !\ Luz-aim!
' . ..J " gamma: \ ,
‘1:.‘a0::if.‘£'.01————> puc-zzpuoc 1:203 col ...};t-gytic

fat‘mczi‘E‘lde' - - , - polygflmfgfimm ' anti-.1

\w ._, J 4 1,. /
3:313“ sang-fi-

1:10;: i LL16

3123-13039 ,
sugars (:cylmze, )

e to: . ‘ - ...:ws: ital .

a
/ pimps: 91".; ta \
b) 11.193130]. -——-9 pbozs 3:11.11 m ‘

imrziml _9 9213': ic

'cmrmrn! K ' - palmo-thzzfimm acid
. mugging: K / -
p110? 79in?- pox ypiws {sh-.2: {to
l ‘4;‘&‘ 1'5
pixmgtm- xylem, gly’ccml
ligiia etc.

c) inositol <——‘ Shame ‘9’...) 31y cmllclrfla 2:31;“;1201 {91731.13
9,3*fliz:iwnpigate tetra... mi .1
phosy‘imfia
9330121333 fiugmge
COIvamml .3.

73

1377:1151: 3.72:3 Frat the pate'mv it L- see: yet paeeffe 1e to 17.77.174.43 even
171 13:13:19.; 6. 71.1.4“: laziemy (c),1sa7.'eue:, enema lees-3- 1211713137 than 05. the?

(e) 0:: (1:). 3171-7, inewguw‘mn 62 CM 1.21.111 1.72:4) verifies august? 1.73
7:111:13: 71...; 51:34:11 .auum ta 0:11.27. CGEWWS, eepee 7711;; 1.37.1 v1 .27.: 01'." the
steefiy immeee 1n inmvparetzan ...nta aim "tie acid. Secondly, niece 1.2:
my be expectui c1117.: glee-pee 7.3111 133' 1111777411. .euaer tepmiy into the cell-
vall acacia ,1; {5121.19 a: ”7.2.1.; en aetiee Luteznaed‘We £7.71 £1127;th acid
formation, 1: sheet:- prefixes: a relatieeiy regial inbei‘liee 17.: rise resume

material. all.“ ctzartmctian. Jetway, this euterfiel Enema lalleietl only

13
i

very elarly (@1111ch III, I? .~.:.;.3 17').

1.153.117 one 0:: the 931777: of the pet-1713 seam, or team cmiaimtion of
time, errpxeeeezr 71:11.: 66131.9 e3: eergmzwe of 1712;161:1033 in 5597.:- gmtiea a. pie-tic
(11:13 1.7111 bears to emu 2.1172113 c..e..1.a...1.z.ua of the e777 t .2737: e axe-i relee

a i

of tree "ptaerpmm empomd .1 , ...-71;: ...»)...awmxufi” .133, are. tee various

"Q

7 - - a 7 -~ th‘ 7: s-I. (1 7 '
6&1.“-%R10;& 1.1:.- £11213 7 13 he: ear ‘1’13’762‘. ”~qu .7
5":

5.
C7.
7.

11.
11?.

13.
13:.
15.

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5'53.
51.

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....-.

P31173571“: 17.0: 3' 31°10) 31:12“) (17-7111 0
Ermteretcm, 2., Der. Dtecb. Chem. 6433., 11, £5.37) (1373-7).

Capt, 3., (313mm. Felereue aseaie amlyms 1::-21.7., Leeeazme, Suits)
11 £122. Gebiete Lebemn. U. 17193., ’51, 31331 (135i).

Coriomzief, 11., mm. Imt. 111:1: . Lecherche carom, 5171.7. 11., i, 17;)

(193:): 1.7.1:: .331 113.1. (1.521713) I1, 11'! ( 7,135).
313ml, 11., Inn. 18mm. Era-7.1m, £1, 16!? (12155)
IILAILJ, £0, at}: I; rte-3011, 1) .CQ, neat-.1101}. 30,12, 1:53 (1):?»

tozty, 1:03., :wllé‘ro, “.12.: {3.21.} mi”: EILO’ Proc. -..ZILI'. 3‘06. 3-010
Chbtfio, J. BiOlo fining, ‘02»: )3 (1)-";

3961.420, “.11., and ihnliu, 301.310, 33.73.31.313 1hr, 8?:5 (1):!3).
Came, 11.11., Nature” (Loni-en)” ”, 379 (1’): 3‘.)

3.2.7.7121 ..‘..1'" 111.13: OEJLI 112::21301 .57 19337: -7, P. 175 (11:77.: You: 11116.13. 11:23..)
CWQSQ

Robe”. M31711." 133.370 to Shame-yo ‘1 87.: (17,77,773).

c2113:- ‘91.“; VCR.' and Lira, 39L], PEOC.13060 E ytlo L101. 171233., 2;,
17'}

mmmm, 2.11., 327., 11.73.1011 11m. Bibliographic Sat. 21.111. g (1:27-31).
relax-eon, 11.3., J. Biol. 62mm, 3:, 1117.1 (15313:).

inlet-son, 11.3., J. Biol. 61:213., :3 M737 (1915):

EJemzel, 13., kmlzirtz. 7ch. 2.70. och., 50:33:11., 110. 9, 7'6 (1 3757).

eczmmnor, 3., and .ur..-,,, 0., meta. Univ" Berna-Clearlactm‘ erg)
Dent. I. ban-.71.: 1.....111. , 1-13 1,713.9).

333131213 ’03., J. 1311?. 653?“. £976., 2;, 4- x 15(1317).

A"’1""O’fl,fi§:151., 611-1 1‘1111333, P.C., J. Sci. 5001.331. 1331311232) 2,
” );(1L3§2-.7 0

mu, :7... J. Sci. feed. 77-32., 5, 17:35 (15":5).
17.7.3101", 11.0., J. F761. 3169:. «PE. «0' 1);..1‘ ? £111.).

ED‘IOI :iivtyxi‘c'd, ‘70-50’ (:‘Egaz‘iffi‘m Itli;t. ificai. IEWI. {-::-(31., E.3.S.R.’
13’0“”) VOW—73y 1712:77777fi7. z:- , 1::. x ‘5 (17.3721).

I

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«_‘I

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Iit’~oo.h 131$}.C-71" LL4X$153353 0., 3:321": 4.275.311, 8., ‘3'"? 33 To}: hip-1’7"". 1.3., (1,: {11157.
Eda-117.1,...) , 2:; 211 1.1.3: 7 um ch:iC"’-:.t7n, _.:(!-,

1713;117:561311', G. , {‘31. E.':‘.JC310T'EW s ’02:. ’ mailml"; 710 :0 I ..'.-‘1‘ J 5:? (1.1) Q

70

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Bahama, (1., and P77713931.» 3.11., 13193311211. .11., ....Z: ,7...) (1,1531).
-7‘n.vlcr:so:1, 13.3., J. 3101.. 822.913., {'7'}, 1.35 (1:31;).

Flaunt, 17"., 011.1 Comftoia, 3., 8.21:1. Chin. M7117.” 3W, if”??? (17,275).

22360111123. 11.11.. am} 01:72.31", 11.3.; 319473377777‘"‘""o 1,1539%: £15?" (13;: )-

:3 .JJ' }:JQh-¢’ £70, 51.33 1319"" 1.7.1.1., :0, 602.2 7... 1211.51.11. BLOC..L5¢, EQVP'BL

hex—é.

COLL-$3.11., $31113. 2331351721130, n" i", ~7sz 0
2.001(3)", 30.30, J. 2101. 31393., yfi, 5.1 (2)3273)-
3.701611, 3., 1‘111. Pmc.,3_ " if} (‘ NI).

0.777.777.7617, 11.13., Cal-1.17.1.3 1.323., 313679731165, 171.11., 173.325, 8.0,, :17. .
:}J.2.....r.k...u, $17.11., 1771.71. r’rgc.’ ..., 1:!) (1J*#/).

11.7.2.732m171m, 3.11., 3:11 612.17.713.72135, 133., J. Biol. 611.13., V5.31"? (1,?" ),
as? 773.131 1, 6.11., 1111.71 Button, 11.3., J. 131.31. C1m:.,”‘. :3, 1.7.1 (15,7375).
Gleam, H.f:., Sela. ...c., 1."77'.__:, 37"1‘7; (1.31.1.7).

Ehrelcc-Ctmlmt, 11.5. , am} 11:77:21.1: , 11., (3522.77.13) £1.11, Soc. (31:12.2, 111.01.,

“$1.? """C' ((3.77‘" {)0

. 21.7 73011, LJ‘. ., 0.2.171 1.2. :c:.::, 6.8., 1”: .1. “me" 5, 2'7“} (1:33.70.
2:32:12.-.7:, .1.:..., J. .111. C71...” :____, ...; (1'7“).
Falch, J., J. 131.91.. 6117.72,, 331, :3; (3.3.9.3,7).

"1:"ij 173.302}, 31.13., $1.323- 11.755, 0.111., J. E;’7_‘i. infli‘ it"., 3""? w, 1.1:. (1:711).

Brackcriwféf, IL, 1:11;}. 3:111:11, C33,, J. 13.73.31, Che-:1” :‘7‘7, 1;;-7:117 (19.31).

“on.
71;“... Ker-...Vc ‘ ’OCC' muwm, IJQAz.’ Qty-2 c I‘VE:- £1.” 22.3., J. 81:31. GEES.’
.4, .7. (1...).
7:173: 355, .1... "., 1117-231: 17,, EZJL, 17.7.. 327.7111}, :..9., J. 13:01. Cains”? .'7"’,,

:13: ; (1,7,)v)1

"17:71:02 1., ..t.1.c.....12.1, 11.1%, 1;;: :1. running, 11.3., ...-3.. 13306., 1',

w. i (1;;. i7).
:27 «17.3.3, 11., 12.111171221923,...“, J. ...... {:2:-:12. $2.3m, :., 1351;?) (1.73733).

’ V '7 fir'fi "1 I" Q .,’ -1‘~ '11" .r‘ '13 1" 7.3 "‘1'“ 3‘37.4“.!‘7‘7 "1". ‘7 " :1 “" "' ' 1"" " ")
12-.L51t4-J, I... , ‘1th It" til-b .137 2‘!" ’ Ad . a Q , J. . ”An 9. 0 M nun... ..‘w-Cc ’ ':7 -! ’ ”'1'7“;"',7'.,-' ..., ,3; fl

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4 o

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Paths-'3, 3., 2223-3 K212312539, 5.?” J.‘ Biol. Chi-m" "’5- 2 ( 3,3).

g,

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Rifley, 5.3.3., O'h 1313.32.12.22», Eon-IO, JO fwr. rm. ISM?" “I“, “3
(1/’”)0 I

ntcimnm,)5.n.', aw! arm , 3.11., Ind and Eng. .21., final. £13., _E,
’3 (1933- .

ficizz‘xeisml, 31.13., am; .--.:::1ro1, 8., 2 91.2522: P" 22331231.,” 3;, , .222 (153‘ $75.
,.2.12-o:, 3., .22: p.222 2222 222,: 21.3., J. 13101. 0222222., 33,2. 3 (132571).

1:. 11cm, c.r:., .222: «222.223.2222, 22.3., J. Ann. chm. Sam, 11, 63:8 (1953:).
Janet, 3.3.1.3.. 22221 2122322291222, 1.21., 11am. are (102112331), 31, 93:) (1,22).
612232.32, 6., J. Cit-3:2. 302:" 2:;, 523'}, (19132).

G's-(:2:. uh: 1:23, 13.14., ”yam...“ on {21202212122an Lazrawh... .22.; ' 2.21. 13:11:02},

22.23.3153! (2122.222, 3., 12-52 , Vol. 1:, 9 5-9 (Baltimore! John 30:21am
{34:33.3 0

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lu‘uI '&!K

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.;T:"=.‘..-2 —: "3‘1 1‘2‘::"'t=>u%:s- 1:3 3:31.113 of 3.1.3.1111“..in

1::... J21... .c(C..-.) mm infiflal‘fii‘d 1'... 3‘?) :33..- .‘f 3:31:32; ......111 . rater ($001;
(“"0" ‘. ’1 t 1 .i a. ‘
,2‘-.2 u) in a 1 Later voluntz‘ic .:..:‘.:‘..~‘.. The 90.112393 1:113 0110:3021 to coo-
an." 3.53 1.11. 0.3 11.1 13“}:9“ ';'1" 0.310 £103. 1 312:; 11.3.3112} 51:11.11}; 1:12:11 5.1.1.4110 to
£3.13 393.011.1211. The. 501.....111 .3: as 000103 to. mom ...-.. 1.39.2020 1211.1 «...3." man}
to one lit-23:. 3:31:21: 1:131}?th 1...; Mg than 5011.1";1101113513 more: in an 016.3331:

ccaloml 1:03.313. ..‘.-.13 001131.011 £3 0:112:30. 2:0? 9:30 maths.

t5.3
*-

 

1-{f-’T‘E‘3';E{7‘e’,-E3 97:3..{11731 71‘ £11001: 1-35'311ei‘imn‘ Tilt-l 315.1151} of 11-12313.f'11(-__§0
1mm -.iiez;olm.l 2.11 5?; 2.11.. 0:?! coucmmmta .‘ Emirocifism‘ic acid in an 0:311:33
001010.} bottle. 1111.5: sciatica is a". :0 61; -- . for ““10 mama...

" .- 0 r F ’ ’l” a
9.21:»??? 91:. 1‘2? 9 c: ‘2' “ " 1‘ :7“ .13- To 01.0 L1. 08 1.10 saw: .13

 

chloz‘ilc 65:012.: 5013121011 1.3.23 0.2.3.0: 313:“ :21. 01' 113.231: .15... 1.33110: T2213 5:211:-
than 1‘22: 032w r235. 3‘; 3:1! 12.53.! to be aka-3:311:13} 53:3“ 3; ‘1”.

{35:303-293. 513-2031313312: schrt‘lm‘u- I... .111} mil}. 531.1. of {5.191304 in
11 cm 1:11:01: 1011;113:210 1313.123. 4221.3 311:1.“ to 1:011:10. 5:219 GENE: 302....11011

cost: .3213! 133 2.33.21. 05 92105922011133. Fiity £31. 11$ 3: 25.3 13011.1: 1:31: 1: as ...}...L03

3:) :21. to 0131..“‘in a 3.103.113. .1 001:1 311.113.3213 5 E’.?.’31. oz? pamgfimnm.

7?

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