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Evelyn:3. Catli

racken fern is a tunic plant 3rorin3 in open fields
aAd along the efl3e of wooded area a. It is a poten5 source
of n thiamin dcstr 035. 3 f.etor ( hififlinn e) affectin3 mo.
nogcstric anirzls, it produces irrevorsihle hanntological
chan3cs inr m mats and cantaina a carcino 3onic factor
for both mono3nstric «nimuqa and rv;'m finnnbs. Cc m.i&ersble
work'has been agna with this fern because it 3.’ 0:5 in mm
fields, in difficult to ersaice we and.hns boen reperted to
be ea ton by man in 30:39 arena of the world.

Ihgre appears to be dl““"“CbJflnu concerni113 the chemi.
0:1 nature and mafia of action of the thiamin Gastroyin3
In tor in bracken fern. Reporta indicate that this facts?
is thccnulv'iTe or a} acnogumblc or that two factsrs are
1:: 13639.33: .

.his stud uj aiaa écsimnad to xter d LAVuQLiUULisns on
brackon thiaminnfie particul r13 on “ha h~--ous of enzyme
:xtraction and pres aervrtT LOH c: amz”ua rctiviv T in th.e fern

"rom.the time of ervauv ta i? itintion cf anrlys 3. Since

'3

'3estiens Esvo bps- méfic that v“*~;e“ £0133 IL yba toxic

3-- .L -.. .rc . rm. ,\ .. ~. '~ .‘ . — - 3*
only sq car min Lwi'fifi 0; fig dufvl p out, :;#nbm collected

k4}

Evelyn.G, Catli
at different SL2: es of maturity were analyzed for thiamin2se
tivity.

Keceurement of thi2minaee activity :28 carried out
using the proce2ur devised by Kontenl whicEL 2L3 b2 ecr on
the forrnojt i.on of 2 pyrimidinea2mihe ptoduet. Pyridine is
2 Live in the treJ efer reaction catalyzed by breeken thia-
min2se le2ding to the formation of one mole of heteropyri-
thiarin (HP?) per mole of thi2xin.deetroyed. Heteropyriu
thi2min is convertea to 2 fluoreecing subst2nce which 02n
be men outed in 2 phot ofluOr nee er. ThicLimL ee activity was
expressed 23 the quhntity of thinmin in micrograms inncti-

61 by 10 milligrems of fern (213 x-reic ght basis).

Ehe result show that there are several factors in~
fluencing the V2Lioue steps involved in the aeeey of br2cken
hiam: LDL- so. The prep2 2LLon of br Lacken homogen2te was found
to be influenced by the pH and Lhe kind o:L nO‘O”ODL%LP”

medium.uecd; the optimum.nK 225 about 8.0 2nd anwng the

2L1 fore ht solutions tested for hoho"enizin3 the Lern, Tris
buffer plus cysteine 22c ?”To1JJ buL:L r "ere the best. Com~
plate extr2ction of tlLe enzyme was not 2chieved when br2ckee
fern wo s homo3enir 0L1 with Twig buffer plus cvsteiLe; 7p per
cent of the tot2l thiaminese activitf 2ppe2red in the aque-
ous ph2se anti 25 per cent in the residue. For this re2son,
hraeken ho.:10,m 022Lee were used 23 2 souL” ce of Lhianinase. This
homogenate was stable for 2% hours at 503 2ne its activity
U23 destroyed by he2tin3.

Time of LI Helb Lion during the essay procedute influenced

the

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brass-2.311.-

11.11213313-

tc: {51112.0 19.239113th £3111.
oval} drying, £114.

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11:.blc £935

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m :13 tier-:1 22:13 team-rated haven-2.155.011 with

‘1" " 1 . 3 In - TT.;.? -‘ ‘5
~11“ £3113 1‘0:- 11“:- “.31.:1'": Jul-.1.. .* 1 v “’3

"1': .1.! ' .-. , . a- . :3": . .9.
02.-....1 2.9.2.1 to 1.1.43 21.210.119.13c2ng

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this-111.121 1:136 91101121 the €111.16 the 1213213353 hrrvcsted

511.3 15:1.3 started, fresh 1211210111121 fem 1. 22.1.9. brought

21.22.123.21 50:1 to either 2.11.12- errinr __, vscmm

1711:1010-c2723, r’efri;1ez.1.1tion 2.1.1.151

an coat-.1213 p 21:410'.11 t11c--.t:1101 1:: resulted in

.l

1111053101: 021? 11:21.11: awe-1:11:23. Bracken £011..

‘20 the laboratory in a ball 0:21? 3.111.111.3111 1211101111 2.21;:

1. 2 . 2.
Gus 1. :13 f0: sac-.1.:

3.11211 to be the boat; J 22.3

quay: 1R4 1n"-
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for (191%. the (:0: 1c r111 F1 (121‘ 15:11.1 32:11:13,139 in

' w . “I Q- ,-. 40 a C; y! I“. g '1!
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2-3—19 b1 age and 3:323:21 172-2111. 11.0%: in the roots 511d 5:23-129.

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Page
3T"”DUCPICH o o . . o . . . . . . a . n . ¢ . e 1
53-! .:.3 0? “311;; LIl‘m'5T’113 no. a u o o 0 o a v u c 14
Eistorical Background . . . . . . . . . h
gang of Bra Jc {on Thiauiunna
fictivity . a u o . o a a o c o O c n v i 8
”he khrraiolmicnl Si",nif1csnce
OfmiflrilinfiSeainlgiaunncoo 11!-
Importanca and Possible 'Tsas
of 2.13minese . . . . . . . . . . . . . 15
“..I':JRI‘T" 3";3'73313211033 . . .~ . . . . . . . . . . 17
Plant nflterifll q 0 o o o 0 o 0 o o o n o 17
Preparation 0? Ernckenlnomogenate . . . 20
easuro ment of fibiaminnse fictivicy . . . 20
:55 77.1173 $27.1) DISCUSSION . . . . . . .~ . . . . . . 2?.
?actora In.fluencing the “asay of
Brz—xcl-zen ’l‘hinminase o ,- u 0 0, i, .0 I i o O 22
"tability of Bracken Ehiaminasa . . . . 36
Distribution of Thiaminase in
Bracken E'OI‘II 0 v a t o o a a o o o a i 6 38
STEEPEClio-QOQOOtOCOIOI-Diti 14-8
LI‘HMTTUXE CITES . . ¢ . o u u a o . . o o o . . 50

1v

21>;-

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Special Rangonts s o s c o a . . o . a
Thimrfizmse ’ssny . o o o I o O ‘0 o a 0

Fit andord Iietempyrithiazfin .- . . a .. ¢

*3

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59

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3,- L4) 14.}
1 n ~33 “10 ‘” "no 'u-fs-t ‘7 '51 g"
:JGQCJ-«L ‘01: n OJ. 3—1-8 “6“..qu £04.11 Ci.“
Eifferent Lt;,3a of Zfaturit' . . . . . . .

2 Definition Oi TO?ZHS o c o 0 i 9 Q a O o 0

3 Thiaminaae fictlvoi J of'flrn: Ran
fern Homogcn'ta versus the rfinoua
Phase rnd Residue of the Home~cna a
and a Eiecozmizmtion of the 1-aL-- or 11:0 . .

h Influence of Different Buffers on
”hiaminnae fictivity of Bracken Fern . . .

5 Influence of Varying Time and
Temperatura on fiodiumfifiyiroxide
Incubation on Destructicn of
(3113-1111109900090000000...

6 Stabilit, of Heterop Tithisw*n
to Cormonarwo "3.1m - JILI’mLiLJ
Suring Incuaatian fer Varying
19113451133421an tonooooocidoo

7 Stabili3y of yfigéwlpfiflo
" .

s
in the Wrr sh .uxte and if‘
‘L‘reatz‘aents n n o o a O O

8 fihfigna fictiviiy of fifinckon ”roman
khan qubjected to Vsrious Kinds of Sr entxen
fiber HC'.1"V0$ L111,o o o c a n o a i 9 ¢ 0 o

9 Vrriatior n3 of Thimminnse ‘ctivity
of Lrsckcn; Bufis ( 3r3a 1 of fievolo.mcnt)
(hen.Collcctod on the Same Pay or on
Different z)_¢ys. Ihia ?ern 331$ 33 in
a Bumbor of i: onIOpmant~1 Stages
'fiul‘ing the Splfiin: . g g . . . g g Q . g .

vi

‘1)

téwvtw?;t
4L ‘ .;.).‘...‘.'.J I,“ 0.}. .9?)
10 bdififii nae Tctivity a? Biffer nt
Fhrts f M*.c”un Fern Tea?
(:tngo 8 of Qevol.pmcnt) , . . . s o . a 9 ha
‘1 Dis t: 1L uuien.of Th; nin.ae
ctivitv in Erachan :ara
at 50150430 J (31.:WGVC‘“"“""*"3"V o n o o c o a c 41-}-

. o as .
1° ”hxfimlnr" ‘ctiviay of urncgcn
3,.

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Lern at u ;c:enu mtfigea c$
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“Elwin“: Page

1 3‘fect of Varying bracke: form
concentrnfiion on amount of tliamin
destroyed; 2000?’ vitar‘n.31 in

93’011tubeoo¢taciowuot‘ooqA 30
2 fiffact at varying the tins of

incubation on tne amount of

thiamin destroyed using two levels

of air-dried 33 brackoz farm,

hemfifieflfltea o ,i a Q a o. a o o p o a o 32
3 Relationship of dry veight of

brackon fern to it: tniamin

dGStI‘QyiK'; flCtiVityo . g 1 . Q g g o 9 1+6

viii

To- -..‘ hflfi'i‘ ‘1Th‘.
“all. A n’K'JL-bL-‘a‘

7'2526‘12 f 2.222. 11:23 been descri‘ 0'1 by {3:221:11 (13129;..‘3;Q)
:33 :2. 2.231;. '2'21133‘3 121222424222, (:22. rec-311.21% of its. 0711:21210'303713u

.9 - I... _ 4n :1 . _.
1.3:: r:o..1:m.a 0;. (3222......1z23'251 developm..- 22." am! as (2.21 :2..__2:23.cu1a

. J- ., .- .. Ls.-. ,. an
1: 12:1 past; because: 1., {:22 -..3 1:1 27132:; 325.342.1293 and is (15.4.4.1-
"‘ ‘0 - . VJ- -'_~ 'I .‘. ' ‘
cult of) crs.r..n.cn.to. .2.» 2.43221; 2.2-. 0'1 the inter-ecu of 012.32 . 2.03.0.

gig-’33 2.211071 001;..." 3022.63, 2.2.5.011 12:22:23.9.136 1210152122; and 11152132221012.

'. J

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"n insac.3 v0-6 icclt.cl 2?

.'.:m- the 32:21.2 {23.32.1120 (”I-’5:

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23.3.13, 6. r--., 3.9.27}. ..-. ":1 yo 3.2::er are 3.2.0 veterincmitm's

 

2' .g, .’.. .1.. 2.. .. .. ,. . . . ".2
r.:::1€::1 2311 v3.1 enuuriuioniw ‘s con-.0223: .2 cut the 1.3...-m
~~ h‘ '0 2-15- AWL
magnum: 0.. “.’.-.3 pl. .2.
J. a . ,.. 1, _ . .5‘ ~..‘
1311:: 7351:. 22102.2 :3 21.32.1252: c... 12222. 9.3:: 39'2“.-. 2.1:... L.~c-..h 2.22.3113.»

0 It
2'23 mo $1122.22": and Exitinrz2t3 12's beam 12317103629: by 51:333-

7.2922... (2.22:1 ”£112.” (1955?). It

‘-d"

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0,. ‘r‘ "-3. , n. ' Luv up. '2‘
rec.,-.._-.-2:‘...:2 1:312 12:23.: 2': first: 3:: :- .MJ 3 $110321? in the ration.
,1 u A. an 3‘ .’ 1“ ,A- .- J- v- .u. a may
an... 12.5.3 13 12::.pe.-.::.a20 fer-.2 p.222: antiwar. see :1 in 2.1-1-3.... atria

.r

' .. : . z. 2' . 4.2 .. .2. ,3 1 . -
23.2.2313. 352.23 is no: mm «2.23:: .'....1 37223122."..12'vn..3 :‘I‘o: 0125:3129..-

2" ~ m- n u- 1. ~ 1-.- -v’_.- -'\ -- ,‘.I- _3 1-,. n ‘ m

.0112. -ou dry- elo'g 12.2202.r;.«.2c4.9131.c.2 - 22; 2c 1 (3.2.2. 2303 and
-k W“. L.- ~ '. O I ‘4‘

m: 32:22:. an s..1...22.-.._2.n:2.a 5.3 .31.",- n to .221 c:.:.:-...e the co litio...

In recent years, attention has 1.2150 been focused. on a
certain tozzinhz) in you. '3, actively (2220' 5'22 13:23:03 :1 fez-2.
which :20 produces 2m .'zr'r': 01‘ the 9.3: “act. a of raaiation; these
5.: 1cluc‘e 5.22.1: cs‘inr' .1 :2.c....-oc'2.:ci .osm in P333 and Japanese cur-.51,

l

2

pulmonary adenom.s in 2.2.1:: :3, and h----..::.‘-.:u121:3 and biz-2' dcr tumors

4‘. . .. . , r... P _
in :13 53. 1743.35 012.1 CLHJSQ 4-1“? T.“ («.1179 C"- Lilli-S ti...“ (3; 13

n c 1 a" - ,q I z
r L-‘I'..0 a‘C\L U4: 'v'V' .4 ‘3 \3.k" J

g...

“Y “-1: >’ "v;» ‘ 3‘ :- ‘g‘nn .- .2.- 13 2‘ A A“ ’I" a.” .. 1,0" I n1-‘-=‘-- -. 1”
“.0 9-3:. “in m... p.': r311“, .a. 3.. 3:.::‘ 3.3+...C... £2..- ‘ .L 2.33.1 .L3 0..

u". ‘ I ‘ 4' . . .~‘ 2' 2 .'.“ '.N , c l‘ . ' 1 s." '. ' .1
infirm-.'.: no; .'.-323.; 3223.130 .219 12.3... 3.3 0.1.321 1.3,; {22:31-2

1:11-1:22. 9:12:23 5.3 £33.33: Inc-1.353;: :23: 9:23;: {1'33} 5:12:2'. a in 87.10
33:.3‘;;..':3 1311': 2.3.3:: 1253.31.23: it is mm}. P. 2 ‘.. 3.32.2.1: via/.331.
.'.‘71331'5593. 722.111 21.5.3321 (12.73} I‘C'a'::";-.:-..l 513.215.: :1 v If. if.“ {:23 23115.3
€332 :2. is 0.7-3327. ",3"; $3.3 J;-;>;2.r.:_-.30. 2212.36.21.33 .1 2.21:1 '3 23:32:11 1(1’3- :32)
33.3.1231 1371:: 1.5.3 Jr: 353.3322: (.212 .122: 1.2.22.3" r3? 3.}?1‘61‘i-2331 33:2

"‘ .5

v .‘V D ’ 'n’ .g ‘- '; . . .l- 2‘ . I . . . . 5‘ -~. . "I‘v u- ,' 'I‘ V ’ $.- on
12335. .‘.. .2..:'o::..1 2.1-... c.3323... 3 1.2.1:. 3...} 22...: .0 .’.. 2.....2: .33. Lao 7:33:33

States. This 121.13; also be: true in other parts of 2.31:5: 1'13}...

'm' \--c 1 3 q " '3' 11 ffi r- «2' 'fl :rvnrs ~29 p ry'nfi 4-;27232'3'? 22. 2-2 {2123 c'f‘lpf‘wifi “.1
*3Agl.- J. a. .— a. .a. ”.'....“ J J t—‘J ,0 ......o\..u “Luann”: A.” -.~ .4 .4, 4--
In". 232.230 22:31.1 :2':3C.43 of victim: .31"? 'tI;.:3 1.11:5 .3: (L3: 5:12.313 33:13 "3032.
1-333. 12:: 23 be :2 13.2.13- "..t 1:311 :3 ESL-.6330? 13 citl;:.3.2 t..‘.'..:','.2;;:;3-u
1.273313 :22: " 1:233: £25113 :2:- "'.;‘.2.:'.’; 432:.) I :3"; 3:3 .13: ~ 2722' :10 .t i.

‘vvp

1"" . v - ~.-1. . haw“. 4.. 3.1. .. ... - .. .L .. .A .. .‘. J... 2
"‘1” I)-‘-'Cr(:'::'«£w "i‘v'ajas. r. 1:, , s» 4: flew“ . .‘ m, “‘5 u ‘ h gfi

o"- .‘.1: p..- :14 v“—.: as opc r1 “-2-: fig- '7”? s... ‘p‘auul '14...‘ I“ 75.1—! 1‘ _A;(:rg AA ”1‘15 ~-:‘~.‘a

b'nhn" w-r-v
. ‘ll..L-a. uuk'aq'u i...- o—J ‘4

v-3.v-s L -‘1U'W‘

I
5 r1 AWE?“ 3‘ ', (2.3" r~ an rw-w M" vhf-.2 :3 r-‘p “ 1J1 W2 J'- i"! 1’1‘2’3 P.3m
‘4' I’M-.0 i ..'. Jw‘l'ma \i-‘n (I'd-L- _v‘ v ‘5‘! .’.-‘9 #4. 'v 42'. 1'45“} I’v '

7‘- vuq U, ‘n y. ’1 1 - “a v - 3,4 : n. "p ‘1! .n A " "‘9 h“ . ~ ‘3’! n 1 f
.. .90. .'. 9A.; .3 1.1.4.4 :3 9.6. 0 3.3.4. .4 43. ):A u ... I. J -‘ JLJ x 0.5. VAL“-.. . .44. u .
IV R . . -1 .fl 5 Q- - I ‘5
n . r 'I‘ ‘ ”'1 C") v :1 P «an: g. ‘ rug ‘ . II . . J‘ N . f . r.“ ‘nm ’1 .. O '1. ,1 ’ I‘
9' 15.2.le .Jo- :u'aops::‘ oh. 141““ I“; tl‘u'akfi IA.“ *- (i‘Ju’ Dan‘- in: 0‘240 .‘./I) "I‘M/uh L’ ('I I “we
. .’. 4'9“," ‘n A i“ . ‘w s, ‘ Q .‘~ : '9 {-15% p- , a "I p. O. A G“ l"; 3’“: §-'1.-p ’. H ”'51 1 '1‘“
003': «AC! bu“. Lu #1:..-d- Do.- 4 .2... -AC... G .133 aw...ijusd Lawn.- ““‘J 3i- H “2"— 0"th

. . . . , ..' .n -3. ~ .3. . l 1 .. . “.2.
- ' a N914 fl .‘. 1*. int“ * . w- ('5 .H , I2). #2: '5.
1‘13 c .‘.).CS (u . .017... 1' .'. J1... \.3.'. 3 1......_,,;-,._..- , Lu ll -.-. I; 4.2} 4’ v A? f- L} :3 kr'u'. «J ‘
"'2 . . . .- . . .- -- .-., 0 . a .0 .9. O . r .. .-.. .I 1.9.. 1
_ . ~_ ‘ ,3 v, I n .«rfiA ._ . _ ., ,1 \, . q .. x -L . a «5 N r._ '{2 ~
-.. .1” E" ”-6 1C .‘L K. v.‘ n‘ ‘L‘ K} ‘nlb 4'- . .L-n'nl If. ’1’ .‘. 's).1 b-t- (3 :1 .L '. ‘ 'l‘h’nv JO ‘H’ Jun-31 0.19 ‘7' «g. J “Jr 1.1

n}: J. p . A) Q ’. .51...’ 3.. . "~‘ .‘.
”"216 c.1153 012913.. p..:12t3 0:. the 1032.1, 5.1-. 2..'.-....:..;...: .1 2.3.2 .3

3

classified mule? we came 3‘5"}: of matur ty mail at different

strgjes of develogzrxcnt.

g", “rt-Tow - (‘1 r-fiw‘f fl 1- "‘Q‘T"lf". \ V‘y—t-r- .<«
.'....Jb .'..LJ'. ‘2‘" .'..4...«J lJfiJ.-.J-¥h -.-- win. -1

nho- 1- ‘ n.- -u~.‘ ' .-J¢

-‘. ' ‘ vii.
3 ~ at 1...,“ $1: {h r- '1 !-' p. .s'f-’~-;.",‘—, 11"} i‘
' -’--" {HA-«4‘4 ‘ Lu- J x .2. ,w‘.

5v". ‘ H.’ 1’ fl ' {—5 A -‘~-‘_ . f..1--'-v 5.. ‘ I" '0 'a “-2. a. 1, .‘ uh ("I. 0‘ 1‘ \ q. 3 m ’ '
.2226 {290220.289 0.2. :9. 22:12:22.2}..‘1 ucSLrug’au- " .2. Hi, 2’02." .422”). £332).

w -’ _ 3- 4 .1-5-‘13 - 3 l: ‘5‘: u an. f, ‘ ,->‘ tr .1- Thug 2-. ' ‘ n;
123.3 f2?3 u I‘Cgut .104. 23:)? 1.2.13.1 2332, L. 12.1"]. Sun €2.23. .48 ; $51.3 (J. {2.1.1}

4. . L1». ' .. l4.- . , v. " I -~ 1 f. w p -. p '1 b...- .‘..
f2." H2 2.02.23 scrvu 225.3 :1 2.1 19,23 02 :2 123.2‘...5.;z.2.22;; (2-39.236

i: foxes: 02': :1 r7219}: at": 1:251 133. 33:2. J. :3. "Hana near Glen:-
coo, .‘.'i.-.:1csct5:. 12:20:: demons grate-:1 that tl'za (22392-239 2.2111022
11:23 33-2321 :20; erred 2’23 :23 "£L‘-";:2..:t“‘=: pa..::.1ysis" was :2. 2.1,.-.212‘2
103 of raw er frozen
$15.1. 1.212.: flcficicztcy 0:30 “2233. not Loczrxae the 21:10:; 22:23 3.0::
in "EH1 "325..., but 12523122372 1222032230 the fish 2.:...:.;;2:,J cc". the 22111 2:.-
rli '1. -2213 obsal’vzmian :-::23 c 11.311216; (21253.22: 12": 3.12:2 1*
M25: 0212222123 (3.. ”12" ) 312:3. :1 :2 1:22:22; 2222:9022 .z'mnr. 2:12;}: faxes by
{2120021, :32; :21. (193.22}.
m

2.222330311332133 002.1113 0:: by Toollcy (3.93.21) 8320:2021 1211.21;
the ac Hi ve 12221210 .Lule in cart: (322.233.22.24 2.3.3 not: dialyzzsblo,
un 3:21:10 to has-t 222:5. 121:2“: 223.6 {2222:2212} umurzxv2ng; 2:04-31:22).

22:: :.-.:....2:2cu.2. mimnc of tho 92...-y"“2c nature

(h
{it

2:22.: :20
of 22116 reaction 32.25332 .223. 1:22:22; the 123219222121 flestroyifig
factor is an 921:~..r2.c 22:23 achicvod by 852.112.2223.: .2228. coat—:orlccrs
(1934.3) cmd by :""2:2*: is: 322:1 ‘..':20119;;. (3. “3234.). Literature on

O

tho (1.-..3'tc: 1252222132123 fine .022 h:- 3 been reviews: 312372;" 2233::

(1.91:9) 122231 a there (75:2 .3232!” 19-123}.

’2

5

2.5215232522125113 51:12.22 322532222 22.12.“. 0:111:22. 1:23:25 the fimion

the me2a52jlcn..qu-- 2:33: ._-222:,r.1-: 12333322 12-32215 of." t12.2.1:2.:1..'22 5.22
tha presc..ce 0)? 00337551511 3.312.122.1321 (1:02.1902: 1.2.3.11 ~;;:r:..'.r5:3, 19'22;
filjita, 0’5 121., 3.93212; 'as'Qollagf, 19‘ 333. The Pena-{flan 5.22..
valves tho transfer cf 1520 1222225225. 5.: 22c 5:050:32 o1? 15325-232522
to the 1.2515223. Using a specific :2... 15.22 .:-2 :13 .222 ezzrxgle, the

reaction 03.5.2212 33:5 by 5325.1231252293 5.3 5320222 in 2322 a follow :23

equation

nmsa

    

3312 5. 3:225.
.

 

l
15?“? {3.111355% .2“ + 2?\ /
7: ([171 C1 \p :21
Lo sci- J
1'!
1372-5225. £55220 723225. 51.21.01 «:1 IgriJine
Is 353 Ioioty

1-11.- ..._,._r .
“(l%-”‘”1~“0-.12~210t2222r1 21:32:225123 C13"? -
3-1«(31-‘11-191 -ZI-MC:’:1 r1 m.1’"3fi-1~-7 11103—0‘-
«11.115122220121111 112.110.112.11

T- 3
3—31-3739 C=Coh£271.) .I
I I +
{3113.111 firfi..p¢iz\ / + 3.7
LEI—:25. 91 \0—3
5:
50":2"2r21222225 12‘ “22:15.11 Chintz-2:21:32
14'- (L2 «221.120-91.226.12321-r22'225222 :13"...

Some Wynnnyruiinlun 13123.: 225.513
5151120223. 1.1212 Species of 12-5172 .1253 and p1:2.nts 312022312522 22;;

thiafiurfie are 22... t maerous, t} 2.0 5222.22123er rev-5.105022 12:23 been

[3‘

he 22. 5.22 135311012, 52.521211255252023, bacteria: 1.2.22.5 Pterifioyhytcs
(:‘212‘25‘312, 1953.2, ).- In can 512.2232“; to this re:~:.c‘1:..sn, i’ujitn, 3036

2.22.25 512.1231: .225. (19;.-.) “he :05 1:52. 2': the bacterium 53129111213

' 6
aneurinolzticus Kimnra at Loynmm.produces thiaminase capable
cf catalysing the hydrolytic cleavage of thiamin according
to the equation
P .. c212 . 1"” «p 1-120 ”9 P . mean 4. 4. 11+

where P and T reprosant thy pyrimidine and thiazole com»
ponenta of thiamdn respectively.

The presence of an antithiamin factor in ferns was
first damonstrated experimentally by Ecswig, Freed and
Haas (l9h6) whan rats were fed a ration.ccntaining ho per
cent airadriad.bracken and adequate thiamin. ?hia work
was duplicated and confirmed by fivuna and Evans (1949b) who
suspected the tannin.fraction of bracken as antagonistic
to thiamin. In a similar study, Thomas and Ualker (1949)
showed that brackan contained a thermolabile factor ca able
of destroying thiamin and that this substance was not ab.
sorbod in tbs cut of the rat as indicated.by the relatively
high capacity for inactivation retained‘by thfl feces. Studies
by Roberts and othara (19h9) reproduced the bracken staggers
1n.horsea similar to thoaa reported by Hadven and.Bruce (1920,
1933) Which.they successfully treated.by thiamin therapy.
?urther experiments were conducted.by Carpenter and.othors
(1950) using rats, ponies and hoirara. It was observed that
the last group or animals did not respond to the administra-
tion of vitamin'al.

Evans, m. (1950) and Eaag’ and others (1950) were
able to axtrant tbs thiamin destroying factor from;&ir-dried

and frosh.bracken, respectively, and rcyortea it to be an

1:137:16. 13113 $017102: group h1..§.p:>rt ed tho’r finding; by 11:10:35,115;
chrorzzxmr'r I'13 31:15:22“: that 21113220112 2-1:: 1213 of the prod--

nets of.“ that: 1123-.-.-ic rc .ctian.

fit this time 5.1“ lemmmfi- 31;.13165 conc;‘-..¢‘.:ezl by ONT:
(193:9) in Switzerlmfl showed that than active *2; inc 31221-3 in
the errzi'ract cf 1::- :clzon "cm was “5311“”“1‘110 and ”2133395. :1
(liwljsrsizc r1~1101~"1n... 1"»..19’3fl'2131’ 33111151053 were 2:111:11 on the charli-
c2171 natz‘ra ("**:C***i {1:52;} .- ’ialflcr, 9-9-3 ml the 13311215101“:
(Keller 23.211 Sorth, 195.13 cf ‘63-: c thammstrbla fnctar

Jr}

vhlch 11' '13 31:15.11751 whi‘: ever}. by Sarita? 3.11:3. 33.1.3311 (19.67}

, , -. a . .12." ~ .. . ,
and. 1.13.11.41.12: as ,.I,.?~.:;.-:n:r 1:91: Trlrr'mw acid.
'.- - -'} . 3.; 3. - ‘
1121131112.; 1-3911" errlim flili-.§.1 3:: an 2:5: 3:11 3:212: cc of
_u ’ Q , 4,, . a h 1.
:1 them labile 1.3.1.:- 21 des:rc;:i..3 lactor in turn. men ram,

firms and Jen-’33 (193:3) cbsawmi t- at 1113.3."3513 Q's? hrs-"1:331
Grams-t 8n:.1*2.531‘3}f?3 1'1“”er 5.11332: 5-. ’c}*'.:1‘.*:‘.:>*'“2~‘:>lo “c-mfi‘Wto
1.1an a thrzrrmlrrbila "3.3013332. 2:22 ‘ 321-1311113211... 11:21:11.0}? 1.11.1112}: 211°.
requircrl for: 30:313-531.: 1:13:11 action on thicrir n. the 113.012-
125." uff of." the: cry-Em tor" in b.1311" 32:310.. 5:"- .53.: 111:. 11:33 11:23 s’maliod
by :.....Zcin 11.1.1.1! c132“ are: (19:33). 2.1 .31: renortc 3. the t smarrl
r2131; “ul‘bgti‘tlltgl z2r:*:.:1tic miner: could act :23 artificial
"ca-factors” to the 23117012312511: of bracket: and th* .’.: this
effect: 1.1.11.3 31:11:13: t0 312:: 6222:2113 533' 3r:- proscni‘. 12:: car?)
($031032: 3.11:3. Elwin, 192:9},3 116.135.5211 and lac-c 1:32:13}. thwamia
mac: (“22.55.33 3.21.5 Tashirc, 1953’ 2‘1. The: 1.13:1: :22? E‘ugizzsra and
3111.31.11 (1953) is 1*2 fi.::“*‘*1c::’:. with 1:311:21 finrlinj‘ts of 27:15:
(1953) :13 Show: 12:; {331-3er c‘.3:3r3"'*vr1.:icn that bras}: .1.:

~"*“‘ ‘1? t" ‘14.?“ “WWW" M‘ 1 4‘” r‘t".".‘ «w: 4‘ ,n4‘fififli
‘9}; nub .‘- htg '.. 0 Lu.“ ..e‘. a...“ 1:14: (3-3 or F‘-..- .VJ.1.‘,.::; “1‘, ,‘i :..}.tfl 14.x)... 5”

fi

hint-13121930 {1.21:1 9.. thommstnsle facial". 227111259 t':‘o 3.71:: JOE'S
"ore ch9.r:mc “1.".0d by .ujit9 hnl canvarkcra {:953).

In plants, thn bacwzcldbila and thermss T19 thi*"hln
deg blaring f:1.ctar are prosonb in 10-93 (“m 24””? .34 LatS"i,
3 and in cocLscomb (‘u31t9, 29134). C. tho 0th0? Knit
tha the.“ 3+9bls factor hhs boon.ahown.by'2h9;v:t 9ni Devi
(19444. W24b), i-ujit9 (1,543, ”1914* an 30m93Ti (1964)
anal unbwnouo aha Tujita (1° 36) t0 be pregame in certain

cereals, fruits n.9d vegetables.

1".

3999? e? nw-*“vfi ”h5*w 9*90 fictivlfiv

 

 

The prccc 39:0 for tha qurnT. Mt;tivc 9939y of thluminasa

activifiy appearing in.tho literature varies cansificrably.

ba-ceria and 91933 thiaminaacs (fujita, 10f M, this section
will deal nuiny w th.brcm .Ion thi-n£nn-c. lLi‘filhhsa acfiivifiy
from.othor sources will be 419019304 only to 0193139 certain
paints.

PfiO‘P‘rHr-Z 1031-, 1-3-5" thaw-~12 I‘l’SGfl c-M‘Hnnp-f‘

W

 

 

The imfio**9rce of pH and buffer used to prepar
brackon how gcnnto h9.s bean rep ort d.by various workers.
:9 ha *nd a uhers (19’0 M) nd “09:09 (lOSTb) shoucd.th9b Th9
active factor C991fl.3a 0:29: chad f:omfibrr.c1:on leaf with
chlor ‘ :9: 9 9.tor at ph 8. Q. Tujit., at :3 .(10 533 9190
fauna thia buffer to be effective 93 well 93 other soluu
Licna 111:6 pT Sphhte buffer at p5 7.0 an .10 per cent

acctano. n the o’:hcr hand, Fujiwara 94d Hatsui (1933)

'9
use& tratcr to prcpéro bracksn extrscta.

A

1L11310J ssu Jiau on L130 son Lhia: :3so‘havo J13’”

1H Lay-U
prim“? i y with cLaracLorization of La enzymstic activi .
Only two stul’L s have igcluied pH1J-131L103.prmc~Lure
far this enzyme. Iatkin 3nJ cauvork vs (192;) Pa ”L..cJ a
ncLhoi far purifyin3 the 3:3; which.can.bo prasorvcd in
the fracz-a‘“icd abate “kiln M :LLn (19;?b) describe& a
p 1Li3113 anlfiuJ.Lultminnso preprrttlog frar v1tor
3:1L3~¢ L3 01 ”"ioJ 1333 Jeeves xhish 31"& a 33 per C133 "101d
reproscntin3 a.purificut on of 3733L uL-: 1d.

:1" :3r (sf-1‘ ryn n 15‘" 3.11734 rn-11‘?¢m n a v, fi""?"7“l"f‘ 4- 3-;{111 {7.11: r0131? 11;"
- ‘4' ..| . —— . u 11- u -.;o\...- . ..\.a - t - ‘.. It. . . .' .1.-H. ‘ ' ,3.-." — A” .J'

€103Vfi30 of thiami: by brm n thinfiiwaso is infl”““cea
by difforozzt factor a. 1:030 inclm 3” of the rcnction

mixture, the i1wc153tion tennerature and.tho presence of

PI,-
1

(

O
(1;.-

iVatam rinhibitors.

in.)

) pfi
fleL M1135 of ownewiLcrLs conl1m teJ by'ldah at (191“)
W.
Shaved that onfiymntic ncLiviLj iNC?G?SOS fromjpx 2.0 {:0 39 5
or 6.5, th cans LL3:L11fi 331 nLL $13 of thL “affera usa'

{3.130(35111"; “33:10 (1.1.2:.12'xxzzic FFOC 13C; 3, L 1.- *1111’”l:}", 111.511,“ L "113. 331:},

'...-d 5.. J

21L ul (l9§"030113& thiamin ifl‘”"f Lian to be "“31Lcst

s)’

between 7 and C, Hujiua, 2* (19}; ) rL p3 6.0 333.K0nten

\4’ '5 5.. .
m
(1.! .:,... ,, .4
(1,1,; (.3) i '1 13113 216.1. ,LwJLJOLLL 01. {.p.
(2) Lezparatura
.' ’. ‘ " v1.31 1‘ 11- 'P‘ 1 7: " '4‘ .‘1 . . ' 1" 4“..-

491195.19330403 {1’411134161’153‘4ml‘8 5A; 3 8. Tl .. wane-3&1 C)? 35 0.1/3 1241‘.

.3... 6 ' 9 , 'in ’f' 1" .’. --. 31-9 nan." ' ‘1" 216 ‘ 3.5") .1 ‘1 '5’ 1' Q " 9' '-"'“
Tu..-v 1.3131 11 190. 1-13...” 1111-»). 09115.11. u J. 1011)) #1311111‘ 11110 0 L10

volvtl'

.1.: .... . ,. '1 .. .3. r1,1"?.-“' ...,1 . . . , , .11 - fl. .'~ “.5. J-
acLLVitg La be 103 a. a) L 331 133335591 0 UL“ bLL 3L

10

70° the activity decreased. Fujite. M. (1935) showed
that with a rise in temperature the decomposition of thiemin
became higher,- but at 50 and 60°C, decomposition remained
the same.
(3) Inhibitors

Several substances have been implicated as inhibitory
to the enzymatic reaction. imam these are figi‘s’03 (Hang,
.g§_g;.. 1950). Cu+*'end Mn+*'(?u3ita and others, 1955) and
Fe + +end 30+ +"(F‘ufjitzfi 19514).
(h) activators

'vfith the finding of Seelock and Livermnre (1914.9) that
mmuinobenzylcli-methylthiezolium chloride increased the
destruction of thiemin by carp thiemineee in contrast to
the menino analogue, they hypothesized that the activation
was due to the tonne): compound'e combination throws}: its
mine group with the Semethylene group or the pyimidine
moiety as it is split from thiamin in the course of the
enzyme reaction. In a subsequent study, Scales}: and Davis
(19’49) incubated desiccated carp viscera, thiamin end
m-nitroenilinc and isolated 31¢(2-methqu6omnino-npyrimidyl-
S—methylhm-nitroeanilinc. They concluded that amines enter
into the reaction at a stage which probably follows the
formation of the enzyme-substrate complex and are converted
to a similar pyrimidylmethyl derivative. They further stated
that because of the relatively small amount of enzyme inten-
tionally used in the experiment. the artificially added

reactor amine overwhelmed the naturally occurring one.

11

Ni th.brnoken t} c.1121naee, LVone and Jones (1952} showed
that m "*inocou, ic acid and S-onino~g .1icvlio acid
able to replace tile nstw o1 coenzyme or activate the epoe
enzyme produced e?ter dielytis of ‘rnclzon e twee Eujiwnre

“I! r’ .. 1:2,..91 . u. *- 3"? . .. .3 J. 4.1
5.1.11.1 11.9.12 M351 (1933‘) Ell-Ll "Vb-JALS.’ 47!:971. (19),) 3310-611 L119.» 1,116

 

eromntic amim il‘“e nni the heterocvelic amines pyridine
and quirnol ne pro.uoe i marked netiv;;i of enrynncie nee

-- 0 a p-!- V -
struotion of tn.~111n. gne pneoonoe of any of thee gcmnounds

. -. 2 - e. . ,1. - »~ -1-»? 11‘ 13-} n - .3 .. .'.
ie not sufficient to cease naming thinnin cestxu otion.buo

2 .'. ,,1 'Prh "71.1.1- :... 3...? .. .13 "I.
£13 inim-iC-fiizea by i1Ci.tGl'1 (1% ’7'?) L1..- GGIACQEbeq'CngOfl 0.3 13.7.13
1‘ ’ 4..- . -. ,. L- A G. v '-'P .1 u - ‘ 1. ' 1' 1‘," A.
activator is 91:10 inp<2rt...1.at. he meervod that t11aunase

activity increoeecl in.pyr:1di ne connem:rnti;n up to alsout
0.03 h and Penn mod on n. He even the ran;3e of O. 08» O 31
but 11th for the? 11* mc9e23e in c: ant rfiti , the one; rentic
activity iecrorocd.

The uulfhydhyl connound cysteine has eleo been shown

to inn lucn nee thi.o Izinnce activity by Rs 33, et el. (1950)

 

and Fujite end others (1955). Sodium tlaioerml ate on ndzflu

'2"?

acetonz/wmew anionpoml 3.er3 to move 5130 cited by -ju ita

(195k) ee rat‘v'tone.

h""“"::""1.”1 :n’; C?

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1.32.32.25vy22r2 no.3; 1:21;;

W w..-

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—--—t

 

 

The activity of LinnLl‘"G h.e bean etufiiod.by cotiu
mating t3 destructive action on thinnin. Cf the various
chemical methods 136d for the estimation of thi1min, the

colorimetric procedure of Kolnick and Pio].d (1939) hue

<~ r- -.—v- 9" r. v --- "‘ -- " ‘ .‘ -.- -. - a .- ' - - ~o
been'u ed o5 nealoch an; others (1913) and mndified bJ

12

I? vI-szits 32111500118" 9141.1) in their 1 .vost‘gs‘gozzs of
the 0‘1”"-.1‘5-.. istics of csrp 1311131111230. This 110111.021 svs

18.130? replace-:1 by ctl‘zsr prscodm'cs :..-2‘ tor 30.9.1031: ©2173. 2.20011:-
lrmd (191111) rep mt ed t1 at the 52...? an of.” erstsivs resulted
in interference 01 solar ("is 's layman‘s.
mother me had far 0.1.1.1... 11:: 13115. $15.21 remaking; after
cvmavntic: rsvction is by the thischrsno procczlurc.i 1-11.11
is csrzvorz‘tsd to :3. blue 11122222353132; su.‘-:~.t "1 1. 311.211.135.12 be
13.35131.er 111 a photsfluorsnstsr. To IZCSS‘E’G t1: 2111.11.27.23 no.

1

tit-'13:; in 1131),, 5113111220? and. etc: 7:33 (1913.) 111': (11?. the n thod

C‘-

dcscribed by 121313161331? (1.93.; .1) '- 11.1.19 3361111: '33: (19653) vcrkod
out t1". 30112312111 cedars eliminating: onsjms hz'm‘cljsis
9.22:1 bass 61:0}1931’39 tests: 123.

ihjits “1111.511ch (1972'?) 1:10 smilii‘i r3. Tonnes sy's
pr ocsduo (1935.1) to stulg' bracksn 221:1:‘111‘3a acti‘mty. 21101:?
nor: method 1:33 11:51 by the 5:;11911020 vorkcrs in th 11" subs-.0.
qusnt Btu; tics of thisri .:.s- Era-:1 vnx‘im‘s 53113303.

2319 fiis. dvsntstc of using ethics? menu: method vhcn
vo “1:21:15; with plant materi..ls was reporter! by Gm.) (1911.7).
Ha showed that th iochrsms am he minced by mam subs t “1608
presmt in ‘91 ants and converted ta :1 nan “lusseuc. :1; 111.1123) 1:: mo.
12:»; auctisn 1317.3 partic 11.1er Jr pwarsote :1 by 3.220133110111113, 33111::
0.6.121 , tannins, ascorbic said 9:13. c‘rstsinc This obs orvatisn
1-19.53 contimocl in a similar crqvorin . mnt condmted by Evans and
Elvsns (19319.3) .

fl ~J "t ~ ‘ - ‘Ia- -. ‘ ‘ .~ .1. “'5‘.
.2-.no..21:.‘21" 2210.11.01}. 1‘s: Ess.-.i.t‘.:.t1n;; tnim‘insso a tivitgr is

an.

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b; 13:13 use 0;. 1.17.9 b...so 6.1321.- :..;s rc'. cation. 11:3 use st....r.od

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f1:7...21:7 ...377<: .‘f-"I'on this 1272.: 07777777171
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in the 131733022ce of 17.111.727.117 .. II. .7317.
12.2.11 cmua extra-31:3 and. 137.1731: .1737
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13 133 1 3333121 3013 in the 3333133 cann 31.3 t. 51733

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3? 45339:...31‘ 13.113. “33*33 in 33r'tr-.:1.I st-I’...133 3;? fish 3.3

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3333.32.31 133.15.21.13? rat-o. P3331" (3.93.; 317.71 by 3311333.: 3.13»;

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1?), £33333 (19273) 33133133 (195’3) 3U;:3 3303

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b.3333 311d {33.13.3313 with 32:311.?1331, H. 3.1 3.216.. 3:31.33-.. thin.

saint-1 r1 e tclfr‘ntjfifl 5'; trig "qu ”:1. h 1 “thfifi’fl ..L: “2‘ t3... 1 C}? £713) fou‘fia 2'10

4“... mop-at'aaydfi nu v—rfit’

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33133133 3-. 311311 3 23333323331 1: 1:13.331233 1:13.. bmn’mvz 321......

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1313 review “-33.11. 1:323 “by 13311 921133. (19171;) In} 1.3 1’0““: by

”.'- ...? f) 9 ‘ o' 4'11q’fl ‘O, 'u I’ '7 3. I
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. 31.11 I I 1331.33. 3331-..3:I‘1I-) 1.2.3 3133 31.3.3.1 1.13.3.3 133313010

‘10:. *\ flaw-no Cu .0 -1 l..- ‘4‘ .-

’ ' a 'l‘cw ’ ' '- ‘ -§ ‘9” ‘ .
.315. 3.3 pL 3'33“" "VIII I313 333.11 33111:! 1:3 33311.1..33’313321 or; 3. LIIII..3~

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13

fihiamina 166. filthough.h6 does not consider this the mode of
bionrn‘ishesis of 1211666 00171901121118 65.11.66 the yields are 6:6-
tram 111 small, this 61,366t6 tho possibility of thim 1116666

:16 :1. 63111211661115.1143 enzyme.

T ‘- 'g 1"?!
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“.‘116 significance of! thimizin. 616 in 11 1111611 {1113. 6.115.111le
nutrition is still difficult to evaluate at this time. It
11:13 been pointed out in tz-ro reviews (Eu <3.1:in, 1.9119; ZT’utr.
Rev” 19213) that this 111.111.1111 (1661;? 035.113 facts-1" may be
used in the preparation of 133.116.171.111 £1166 diets as :1 subs.
t 1311126 for prolonged autoclnving. Evidence of success in
the production of B1 avitn.r1inosia experhmnt 611:7 11:16 been
shown by Spitzor and. others (1911.1) in chicks, 311111.11 and
Pro-111:1: (192114.) in cats 1.11:1 by {2-1-6621 621:1 613116113 (193.11) in
f .1265, by mixing; r11. 1.: carp or 1161121111; in the d16...”it...dn
131 rloficicncy symptoms due to 66113166161011 of brfECIIOnl rams
1.6116 do.:1o..s:;ratod by 02.11116 1.13611 and (so-workers (3.950) in
rats and ponies and. by Roberts and others (1969) in 116 .3366-

376661111131 , great interest 11:16 been given ‘66 6. 6611131111

61165.6. 1101' "36.11.. in ""116 66111111611 qurghog, nercennnig,111.6...661161’1

 

(.-.o;;yeli, 195.311; 3616116611, 1963.1). Studies by 3115...,6 (1956,1613)
6.6116116 mated that EDT/2 mice 111366665. with Erica-:1 lemzcrfia
virus 611:1 maintainofi on :1 {31:15.11 £1166 (15.61. :: owed no 611115..
611 nuanifestotion of the (15.865180 :16 601111.9r1rcd with 6116 con-
trols maintained on :1 116117111 63.61:. It 11:13 611113661160. that 6

11161215111166 1:11.31 b6 the modality of action of varimzs seafood.

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in a vooéod area at the 3030 13L fildl 33 prccirflrt
~tptiw near Bast >~nsing.
5 number of pruccl‘rcs rare tried in an o1fort to
preserve the en:3 rtic act iVity f?am tha time tb 0 Lora
9.3 harvosf ed until tho anslytical work coull be stnltod
in tha laboratory. Th.cse i”c]"‘e& (l) freul1J J out fern

trarm wrted in a dry = hata cr cht moia t 13h crushed ice,

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whim ton.r nutas after harvesting, the fern Laa made
1230 an aceizone PD”1C (L 3: n, 19 35) 01(3) transport 23
the iztnct err anr the s-m3m1ing earth.to tha laboratcyy.

Lt the labnrntsry, the i L~:ns mere car‘s ed 2000? diizg Lo

thair strga of r 1 p.3 t. 303 cript ion of the L’a.ious
st 303 of nnt :rity and definition of the @1330 30 mt parts

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divigcd into the blame (3) gnfl ctipa (3) which.rera treated
inflividuzlly. Sepwrstion or Lhasa tvo parfis was not possibla
for ferns at tLo first fem? stages of maturity because the

blade agyearcd as n.bud or as n :1: Wehend in n £13216 “haok'

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23

Table 3

Ehiaminaso Activity of Bracken Fern.Homofienato Versus
the Aqueous Phase and Residue of tho Homogenate
and a Recombination of tho Latter Two

v.—

Form othracken Fern Added ‘fi Thiamin f7? Dostrcyfd by 10

 

to the Assay Solution mg. Freeze-dried 78 Sample
ficmcgonate ** A“ H 1275 #A
Extract 750
Residue 225
Extract and.Residuc 1068

 

....

1 The number refers to the stage of development and
the letter indicates that the blade of the frond was used
for analysis.

21;

procedure apparently resulted in the destruction of some
activity since combining the resiiue and extract produced
only 85 per cent of the activity of the untreated homo.
senate. n1though.most studies appearing in the literature
have used tissue extracts to carry out the essay proceéurc,
the report of Eoolley (19h1) showed that the aqueous extract
of carp viscera use only 23 per cent as active as the car
suspension.

In Kenten's procevure (1957b) the time suggested for
homogenizing hrscken fern is 2.00 hours. in the present
study the enzyme was not completely extracted into the
supernatant after homogenizing the sample for the suggested
length of time. For this reason an experiment was conducted
to find out if there was s difference in the activity of
brackcn fern.homogcnized for 0.25, 0.50, 0.75, 1.00 and
2.00 hours. The results indicated that about the same amount
of thismin was destroyed by the homogenates prepared at
different lengths of time. ?rom.this obserVation, the home-
genates used in subsequent determinations were all prepared
for 15 minutes.

Since comylete extraction of the thismin destroying
rector in.bracken fern was not achieved, the activity of
this enzyme was studied when different solutions were used
for the preparation of the fern.homo3enstes. A comparison
of the various media for homogenizing the fern is shown in
Table A. For these experiments, the some batches of freezee

dried fern at stage 6 and another at stage 7 were used for

. I . . . . . [r

:.mmw o.mmm mmmm.chHv HmHm Aomm mm.monH meHm osHoHeso e quoHnu

o.mmm 0.3mm Amon.neHmv non Aoon.monA oeHm e uHcH
m.mmH o.mom Ame am no HA meeH AomoH.mmeH. omoH oeHesmse_¢ mHse
m.omH o.mmH Amen .QQOHA meHH AmmmH .ommH. mmHH ane
m.mmH .mo AommH.mem A HHHH Ammo .mmo new sums;
o.an 0.0m AemmH.om0HH mmHH Aam .ome A Ame er,A:. + «Hume
m.m0H o.emH AomoH.ome A use HommHAQQNH. memH ousemm
H2 0.90H .omH coo .Qam A see. Am MHH.mmHHA mmHH AwuHHA
2 38 0.8 Ame 5...... A SA... 33 .3. A mm...” 0..
m.mm o.ms Ammo .moe V om» QAA .omH A one che : QH
o.ooH 0.00H A.ee .Aoe A com Amso .00» A mmo HHospaoo

mm. , _ m. E. Mme Lt

”eHHH sassy

NV 13...: .....HAL. H3. ..w 95“.. 6th 3.6.00». H. 4 ...
EH Hmo a nu H ... 035 H -. Ho ..H as... H Hm. AA messes QHLHBAEGHA

O

seem. HHeuAeeeH. deHQIerAeewH..H umsn can he. Homemaker em GHQ... ......HHHM.

I." it

.A. we 09.5.3 mefi

.‘
OK. c . ......

Hashes HHoueHEHH so be Hwa. e... em,cH.AH..H.A...A.HHHm. so we.“ ac.H..__H . neon.)

s oHnsH

3x01»...a.....3>.30 A male A0 ......SA... on. MAKE n.9,... 33.3.: 3.0.. .... .. cu. wonmmnco mu

5 1Ifll‘

53.33 nag“..- TA 23 opium. Hobo...“ ”...: a. a...“ haAAfip 3AA 0A momma Hum 033...... .....A ...... .Hoo
m
.3... mo: Agnew An mmnwmumww m... A.

afidamAA... AH numcmwaA asuqum 60335 mom... can. own 90A. :5 Ac. no 3. ._...wocwmoo 903
20 Q
2

4.395 AoHHoo..... Ah mofihomofi m...

m
.833 33...... . an. ......a........?...... E wafinumov m... .A
.
53A... 8. ASA wenfi ..mfiohA o. A» we OJAHQ
as» REA uouwowwma .3»qu 33. fins ufiafiAoHeboJ A0 ondun 9.3. o» mAMAwh hogan 9.8.... m
.909?» .368 m9... £35.... Monufioo 3AA... Ed 3386 06 0...... vommfinwa mm
N

.flobfiu 09...“. movahobc
Ann 003...”; Hum..- www.mfiw 9...... Agéwofi Aw... 499.2.“ ...A 396 how. war." 93: mcpcfinfi...
H

27
12.11 21233-1373. the enzyme activity was apparently reduced :-:-hen
the: fern was suspended in 10 per cent saline salution. 21115
was the method used by {300115-32 (193.21) for extracting {3111:}.
mines-,6 from fish. 233,130:- haa been used by a mvfimr of Japanese
workers for attraction of the emmm frcm both braclcc‘n fern
(Fujimma ad Kata-211.1, 19.53; Fajita, at 21.1.,- 1955) and clam
viscera (F‘ujita 04-5. others, 193-52 21). E‘uji'ba, at 213... (1955)
extracted dried fern 2221:3311 water- and phosphate buffer at pH
7.0. This mmmct from S 222:3. of fern was reported to destroy
0.677 of thiamin :.zhen the solution contained. 1701‘ this
vitamin. If it is 23.32.212.2710-21 that $1.113 2.22:5 the ataxizmm activimr,
than the: 10 2.: 3. of Erica fem extract 2220:1121 destroy 1.314.701?
thismin. The present results Show that. 10 m3. freezeadried
fern 110221036211sz with water (pH 6.6} dcstrayod 9007’ of." thififi
min and in the case of 831213310 63, the ammmt of thiamin
destroyed liner-622.3951 to 112372-2121”). tha addition of 3:11.53! to
increase the pH of water to 8.0. Ehia observation agrees
with the ropo-r‘cs or Fujits... ct 221. (19533) and Kenten (1957b)
that exmaction of Israel-zen thinmizmso 1.3 maximum at H 7 to
8.

Kenton (1957b) extracted airodried ferns with chch-On
formwrcgter at. pH 8.0. after incubation :21th 2 2:35;. thiarfin
far 1 hour, about: 1-0007 thimzfin was 5.21:} tivatod by 13 1:33.
civic-:1 1321'. €22.13 is similar to the results in Table 1; using
the some medium.

“3110 use of other buffer-s 1 3316 "LP", 33:12.3, i‘ria and

T2213 127:3 for homogeniz: n3 braclzcn fern resulted in 111331101“

0221010100 01“ 11.11111 (1021 tray-01.1 002213100011 to 1:310 control
0011101012. .731” 2:021 £01m 1101110,; :0 212210.023. 1.11.3221 .’TZI"I‘I;3 21001;".

1:0th the greater-137:. 03:10112'22'. of 1232111222111. 6113010002: 0230132, this

0'
5:1
“a
o
1.1
0'
O
G
(J
O
1'1
<1

2111:1010 only toward 011.0 01213. of 1:110 02311032,
For this 002.0022, 9.1]. 01“ the 0002:3223 110220 02.102.25.06. out with
(3210 fern 111121120 :1? .011 in Tris plus 03:0001110.
C1‘S'tein0 212:0 been reperte: .to 00 1:112... 00 the enzmmtio
reacticnfia 3.11.2.7", 0.. 1., 19 ’13, Fujita 022d 031101-23, 19L2..)¢
‘xghon it 1-100 0000:1130 1:110 T010101 ”fer, it did increase
the 0112537130 activity by about a 131111111 01- more. IZo:-2ovo:2,
t1: 0 062611 01311 of 0:72.: 2:01.220 {:0 11110 T2113 3.3:: buffer had no
effect on the results.

1'30 (1000.. 20211.00 :."11003101? Tris, Tris plus cysteine and
33213 275.1 buffer 0 influenced the extraction of tho 0212' 31.10
or the assay procedure, 0110030120100 0120012011 fern 1.1.100
homogenized in 2'0t02'c-952120nia. solution at 3:11": 8.13:. The
33120012011 homogenn‘se 0.1113. 011.0 of the 2:30.000 1311330110 were 051.1021
to 1:110 1200001021. 1.11-...10 0:13. 10110 000 :1; 13.000032122211700 carried
out. 'i“10 12001.11 :0 1: 6.101.000 that the addition of 05.0.0? '1‘1'210
plus 032001120 or ”113:" ‘ 1311.10? .1.-31:10:1st the 0.22.01.11.11; of
1711101253 3000220320110 0.1001102}. to 0. 13011101011 1:12;} :..ut: 0...] of
0.10 2:11:00 22.021010 2201 buff .0. Tris 11:21 no effect on the
00021. yprocolm‘e. “251-0121 this observatio... it 01:10:: -0 that.
$721813) 1.2.3: .0100 be acting .0 an 000112213011 of! 1:110 012.2..- 21310
2009001011 in 0 12001011 0127211012 to 032.000.1210.

.110 0 011001: on the: 11:10:12 1210:; of the reaper-.200 pro-2121000

O

by "00120:! 02101.1. 212.13 02‘ braciccn $02.20,? . 110.2. to 10 1125;. 012' Can

29
airnariofi sample h0mogenizod in tho Tris plus cvstcino
colut on are incubated a th 11‘07’Lair.1 2. $h000 000 0
linear r0 ""1on.bct'ccn the amount of bra ken fern in tho
assny tube and the this: n ndestz eyed 00 shovn in figure 1.
2010 030000 with the obserV0tion of Kenton (19370).

Sever 01 reports h0.v0 indies ted that tb 1:31 r.000 in
bracken fern extract 10000 its activity slowly even vhon
preparations of the enzymo are stored at low t01ficrvtuwva.
to lock and 00-. orkorn (1,03 fauna that aqueous extracts
of fresh fish tissue lost up to 36 per cent of their thia.
minase activity in 10 days at 5°C. fhe fish thinzninasa ex.
tract prepared with 5 per cent potassium.chlorid0 by

Gnncdin 30? (196m1000 no enzymgtic activity d'win3 hours
of storage at 1°C but after an haurs at this ternoraturo,

a 1000 in activity of 0b0ut 10 per cent occurred. In this
0tu.y, expariszenta Vera confiucted to determine the stabili-
ty of thim -.inasa in bra ctcn fern h030300100d in Tris buffer
plus cysteine. The results were presented in Katerinls and
Hothoéa but it 10 well to point out that the homogenate

was found to be 0t0010 uildor refri3orntfion for$ "‘ haurs at
503 while free Hi 03 at .503 for the 00m0 number of hours
resulted in 0 10 per cent 1003 in activity. filthoufih no
comparison could be made 013.0'br: 0k n for n.hom030nized
nith.oth0r solutions, the ststbility of ti .0 brco'. :00.homo-
3000f 0 prcpared with.Tris buffe. plus cyst cine under refrig-
eration for one day may’have been due to the fact that the

lattcr solution 10 a reducing agent.

30

 

[.4
—L

 

 

700 '
600 _ ..
500 _ ‘ : , _
' 0
'd
0
p.
8
4;; uoo — _
o .
c:
)2
g 300 — O .1
...: .
,c:
E4
200 _. _.
O
100 _ ' _
0 l 1 1 ' n 'T

 

O 2 h 6 8 10
Air-dried SB Samples (mg.) '

Figure 1. Effect of varying bracken fern concentration on
amount of thiamin destroyed; 2000 Y vitamin B1
in 88.011 131.1133.

l‘ "" f" }“ ' o‘:‘ r.“ 7": ”71’1"”.(9 '1f3 4" 1‘9"? "."x' Y'f‘. . P1 (1
r’ L. Q :—-"Q I O‘- L. : U \.7'..‘ LII-513-!“ -.L|.....-.1o1 L53 ’ M ‘3 «I. .4 '1— ’JJ. ' l.) Lo;u‘-)O

 

 

 

(1:107:97 firm}- ‘. 111311 the }.;:‘.r.:;:77's.7.7~77710‘.'.s 2173.17.11, it 1.!) 13.17.7333?
{3.2737‘r13-7353. 71.313.72.311?” 7231.73- .i,7;::‘.:':1..s 57.73.1171 71:17.3 cb:cr**a‘7isr7.s ‘3‘
Thai: .- .m': “1 (1w?) 7: 2““7‘17'7wd *7717’7 7"77791'3 77‘77-7 F ““77"“ -
- “_Bb...' — . .a.’ ...J/‘, , «44-.»9 V-oJ-V‘ luxuu .u. aha. \.
”...-7,173.3» .z’fci 7:73 :17: 11:? (117.327; .5." 1:97.: 1:71.}. 77:27.3". i":- 7..7r.311<1:.110.l, III '1‘
1:33 2.7.2:; 1.11:1;77.:.-;:“‘..

7:.‘7'7‘7 f. 7 :77 r.‘ 2' 771.717 :.. :‘7'75’. :1- 177.77%

‘7.:-7:.77::.....Z:71:11-‘-wv:7:t::1 :en:“-.:e ‘71.: £.::-:~.7-:.:.1.:.‘:;::.i f:3::r.::3:’.:‘7.::3 721.7: the
3.71211337311 70 {11715111217143.3. 7'1: 77.1-17.9 3172.727 73...: (77.1.3: 7213;71:711-111., 3772.71.23.11:-
111711 3.313111132171716 117.1??? :1; £711? ..., 3, (I: r7711- 233;. 1-77. 0. 111.211.137.233
(1-. 117113 21.7.. 1.7.: 113-...7;.7?.1 were 11:13-, €331.67 72.77.773.322 the: ...-3:37.77. s'i‘tez‘
i::37-..‘::.7'.,:?.:~»:". the Ii...‘ 3"?“ '3 czar-eytoal to :‘7‘777‘7c--.7c:: 1.5.316.

'11:: I‘m-1114.73.57: {7.27:1 ”vesezm i2: S‘ih‘lsrr‘ £2. .7’7171121a.7.7:;;171 the rite
071‘ 13:91.7;7113‘. alt-37.7.1.1: 11.77.07.72- c 177123772171 {17.1 incve-ee cite? the
first 3.1.1.1113 the 13-17113 er‘.‘ the (37:97:17.1: i‘ 1.1-3.2177”? :1. inmti z-tci
r22: <2.-treb- i17‘7‘77.‘.‘:7:1ic:72- ‘73:.‘7 53:1 ‘31:; 3.3.7311“... {15.727151 to the 1.1771133
:.rI.’ Iv)."";-f‘“” 11:) 11:10 13..

1197119116" 7777-7717;,I.:Z.r;:7.13.:1 131.23.117.35. in 1,310 3;“ Kneeling rec-0711210,:
1.7....3 net (ice-'1 113:7,706. fixer-7.17.7; the 153?.ij853’5 27:21.12»: 51:31:21 gem 1-1.,
(7315:: 179.3 5.32.07: 33:01.1 07777" "771.13: Iii-5",? 72-73:: i.37:¢1.:‘;>:_‘.72m"-. for 01:77:}...
"min-”.119 pei‘iez-s. .310“ -. 511:.1377252118. 3011171110113 carried ti:1—7*:11::h

”11:: 3.11271"? *7-77‘ for. 1713,. r1213 1:: ezzisfirxtign 3373.301“ 77110 5.210132272110171
{21.x {:77’Lv'2‘77777r‘ue1.‘ "cedar-1 2:371:13}: var": 111: 3.71:: :13 the free}...-
13' tram? 7‘0 71 si;.~m~ln:z;.r.. This 37.133017 77:: their, III": formed. by;
bx'ncfngoz‘ {-32.73- 97:17-13: 10:): tug-:1 721;; 711110 3.373111112315121period 1.17.3 87213016.

..9 1037????

J

.1.. a. .t.‘ r1“
....) :1. DIN 7 17101.17: i213 1.12.22.0-

32

 

600 J T I I I l 1‘

C) 0.1 ml. fern homogenate equivalent
_ to 1 mg. air-dried sample.

500 ‘ £5 0.2 ml. fern homogenate equivalent 4
to 2 mg. air-dried sample.

 

 

 

 

 

'd
a 1:00
0
$4
4.)
U)
Q)
Q
So 300
1
or!
.c:
B. 200
100
O J l I l l L
0 L; 8 12 16 20 21;

Time (hr.)

Figure 2. Effect of varying the time of incubation on the
amount of thiamin destroyed using two levels of
air-dried 3S bracken fern homogenate.

...

l"

0222': the n .207 in 0:20 0:6-2'62510 aftrmetizclfiz 000222025 .5301" 0.2.2.315.-
0:25:00 put; 5212150213152 5.120 pros 2.11.2360 f2: :..-32.022 002': .
12015::‘05152‘53' 022' this 5300-53-12....

5.5.5500- the 5.2.6202“ 0505.021 per-5.015 5.5:. 022::m2 2-050 1620:2053 5:222
was 3'30321'2011 2:5011 2'37) p0? 0012*; :5:-2.05:;:2.-16350253320125. 0.05.2.5. 53253
2.3320 follozt'czl by {3120 adfiition 03'." 23 120‘: 003250 006252.21": hydra}:-
500 to 13152.0 acid 00121635022 012:5 22021122501022 at 3.703 for 0210
12022:. 5120 this 5:15.22 0:222:52“ -5262: in 53120 00122535012. after the
0:: ”201.550 r0r6ct50n 2.. :20 0026112101; 0:1 16:21:35; 120 (1005;002:051 05.32.00
K0225 011 (19:37:53) 51-202.: 05 that 522250022... 23.20 (fommd from 535150165112)
absorbs :2. cent-52102:..blo 12.522.226.525; 05‘ 02201233; :25: 3525 21111.2:56-5052 is
52220 ""VClO“"“‘-.h for 112:: 5:20.322 absorption of 2-m0bl'2361 pg -5-
016210525120 (5502* 50:! {2502225505515}. 51520 531252332512 12021205255123 5.6:: the
0011205011 can be 00001203100 by incubation with concentrated
sodium hydroxide. 5.0 012003: 0n the 00.210105002200221. 05' this-26.25
destruction and eta-2.0515523; 05.5 2.25”? 1:222:10: 5:120:20 00266: 55.235.02-22,
the following proccfluras rcre carried out:

(1) 57511050001509.5002 005.2. 5359123 05.26.2510:- to the 0220:: used
522 the :20 0:23 2-206’20 prearrcd 5:55.511. 235‘37’ 53.15 272511 in each 2312520
but no brad-:01: fern. 513.5210 :21. 0.25.‘ 20.011 0012.15; 50.2.2 2.206220 55202.1.
butcd 0t 370:? 1'0? 1 ham:- .—..-".":’:.02.6 125125.052. 1 1:23... 5.10311~1)}105”5J2?ic
005d :..-6.22.0 0:100:31. '20 2 2.62.1. 05’ 0:62.022 solut50.-, 1 111. of 20 per
002.5: 00:55:21: 2:53.20 0:25.25 0 21:30 ads-0:1. 3120 101356 022 00125550120 1.60160
5120211306305 5‘03? 90:50:13 up 550 2 520230. N; the 022:5 05' 53.520 512-
02253005012 120125.05, 0.55 201.. of alkali: .0 ferricy 0.16-5.00 26:23 22000:}:
the 0.10003 ferricyanida 2.2.22.3 fiestroycd by 0055.125; 1 2621.05 0.05

per ccnt 15231.: 3011 5:202:00: .0. 2110 result. 5.213 Solution 2::6

'31

,4.
623.12.23.22]. 2....21'311 water Lo 12332 ml. i"1m:{"1.2.2”~22co 01 L110 Lilautzl
solutioms 2.22:3 :.zmgmmoci 211: 3C 21,213.21 :2 “.1222“ I‘luorozimtor.
‘Zi'ho 120224.132: (3.112122 3‘3) 2211022 t11a2211cn :1 rorzoi: 3.0..- 2:13:11?
vifih Lginnin was lJCHFo M wi;h 23 per cont aofiiun hydroxide
379?, 111113113211 was 5121,2231. ion 222:: 0:21.23 2101; 12:11:12.1: 3’.) Lawton.

22111232212: doo’Tuo 2.2.021 ooouzTcri 22L 2: 121.02.121.22 Toto 23.1.2321 1312.0 5.22.
cvb "Lion Longorature was set at 2733.
(2) 23:0 331‘000311‘13 o:—:po1~i:1«.;nt :13 repooLmi (2:123:91: LhaL
1'21"? 2... at: 1233-32 at 2:220 Loyola (BOOY and 4007/1262? tube) in
place of thiamin and the incubation carried out only at
37°C.
T119 results presented in Tabla 6 22110:: that HP? 22223.3
1:31:12 to com: ontratod sodium hydrozzido during varying;
periods of incubation :21: 37°23. 1. us 2.21.211 1:116 ass-22.32 pron-n
(2323.10.20 used in this study, L113 11?”? .-. formoa by the 611:: 22:11:10
reaction was unaffected by a strong; baso "uri .23; the one
hour incubation period to destroy the residual 1311122111111.
’.E’his observation 3322:2223 with tho findings of Kenton (19571)).-
2‘. corsrporioon of tho g::.1v::.nomoto1~ rooéings at zero
til-110 for thiochromo (T913213 5') and Zaraotl‘ylp Tichro: 21110
Table 6) shows that 2"32007’L1113min had. 22 gimme; etc)?
129222112130? 65 1:... Lot: the 2:32: 2.2;! proooduro had been carried
through 2115.10 230711312, which is one tenth the thimain
concentration, had :2. regains; of 28. This imiicates that at
336 212,24. the fluorosc once of Borwthylpyriohrom no is about
Limos area"; or Lhom 11112231.: 2:110:22: by 1.1111001112012262. This 1210:2123

hat should 2.2my Lhiami I: remain 22112212 imubation 2. iLh L116

.u
Tabla 5
Influence of "-1 21231113 Time 21-21 Toz-zrpoz-atmre
of Cofiinfliryuroxiie I.cu.rtlon on

Lest-action oi Tninmin

 

 

 

 

Re 0121212 31211211119 - T311212 (hour)
with ZJJdv’Tlinmin Innu)~**Ofi Cn1V2n0moter“ m 3212:?
Incubated at: 1 01th 2113;} 3121137.
0 5 51.5
0n, 0. £300
27 ' 1.0 ’.o
2.0 1.5
o 56.0
1.0 1.0
2.0 1.0

 

Table 6

Ct shility of Rat aropyrithin: n to Concent 2ted
30:11.13; lijrdmrido luring 1'1 cubration for
Var3in3 Len3 tha of T-r

-- - 'A A... A

v v vr—wv w— “V h ...—w—

1. inc (hour) of

 

 

332“” “3'“ sure incubation with 6211;221:1111-6122? Reading;

’ 2’33 *2 at 3796‘

8 s gag

my 12-»: 130 2325

2.0 27,5

M 3 w 3 W. 5.300 w
(“7“ 0. 52,913,
[$3301 1—4; J. 1.3 331:.12

2.0 55.0

00111003113119.1503. 303.11.11.11 1113:1110:2:1.:‘:.e 11.011.135.911, it 1.15.11 have only

:1. 111111011 0313001: on 11:21.10 01.11:: 231-1111 11:: 31:11:11..

rfin‘q" 1 a 37*? (5 3‘ n?1fif\"'.’ffi‘1 (“11,1 (-1913 ”-11 (If!)
\ l Ir-y...,.... ‘4 .._-. .» -, - ,. . -.., .,..- _,.,,.., __. \.

 

There is little 1.1131111111131011 :21: the stability of this...
minase int: ‘10 intcc": 10.1.1.1? $110171 the 1:11:10 it is collected to
the 115.1 11:1 it 1:3 111191111163. 9111:1119: 1:11.133: carp 11:115.:1111111111111, 36:1.
lack, at 91. (l?u3) reports" that {“0n1r1bfion of acetona
1101.11.16? yield. :1 :1.: to: such tree: 1.1 021.11.: 91 11031061111? of 1:11
03:1 Lint-J. 9031111211. 3. Elva-:13 1.1.11.3. (so-1:11.12 503.13 (195,711) 2211.11.10.11 that
the antithianin factor was stable in the intact 163? after
321315.113 at room 11011110119, 1.1.1.110. 231101.11 their report, it is not

1:11.0 .111 to 1.11133 3133:1121. there 1:11.11 :1 change 5.11 act 51.111331

tho tin-:1 the 3119111: 11:11: harvests-.13. 1:111:11 1:1 11:.- 11:19 11.13.11.011 of

‘20 provide £111.? 3111111315-on on the preceding, $111.11 .1111 braclzcn
fem 1.1313 13111013111; to tho laborzztomr :..-here 1 batch 013' fern
1111:: 11119131103 5.12111 1011191501311 ..--1.11'..la nnotner batch from ’cha :1 .1110
31:11:39 of 1111151111 2:1: 1 :13 11111391121111 to one 01’th 31110131111111.1110:
11:1th in 2:13:10 7. {Such proceéuros :15 111.111 drying, Vacuum
oven drying; or fro:1.1:i~.,.9( .5053 for 1113 91:11) resulted in
con..1..-e1111ble alestruction of 0111131110 activity. .‘.:von freeze-
:‘irzring produced 11:13 12111311 10113 of activity :13 refrigeration
for 2‘1: 1011.113. 2’11. preparation of :1.. acetone 3101.1..01? even
:13th pwifyim; the acetone 11111-3. :1.-1:21.113 it 5.11 tbs chilled
st to (31.13. not 03311111210 the 1:11:- a 01? 9.130111. 1/11. of 3116 enzyme

activity. .'1.ctu.1ll:r, a greater 10.11.11 econ. 1111:103. in tha 111150110

 

 

 

 

 

(W .‘.g .. . ‘ us. a 3'".- .‘ . ’_ .f M ‘.‘ , to. "7 o 0'. ..
Mymu1M1if 0L Jhlxhgnfsc in urécMeM ~ in in bhd
"1. , (" - .1. . - n. .513- " I "‘ ”A... .._
rModh..L2ua cad .LMcr uaMMous M? a: he
MuiaA n 07) JqurflwOJ c” on.gMMe
m- 1* r”. """‘-*:1c " ‘ ¢ “ '
.-. ‘1 h --J --. .. ‘ m‘; m .'..
‘ Uh ‘Na 0: 3151c “ ‘3'“ WA' " aria-v? ”4-- H, "S" vs 0‘ -? (‘1 “(5’05 quC ti on
5‘.-.“ g_ A; -.U» 4UL-“ J. ' "U. JJILiiJ-Lb'.) .4
.M “iw " ~’ 0; atrync
‘ W *— .- c x- : ' 4-.
..,_ ,.. M ‘ ,3 . MJN’l
E.‘ POL-13.1 «POL?» 'u‘. "
. " . "2" r1 '7?” 6
.c.’ 11" P-‘I’i 0d ELEM 1 I ,‘3 9

1

1 Vegan: fven'LPio-i fi637 759 G7
63 from n (m ’*ys) 3283 1933 kl
83 ficotone Dwiod 6592 £356 97
63 fires: "Pied 3325 2322 23

J- nefr‘gcrr‘c (1d M 63 3L3? 23

 

, . M __—_A ...—._.. “._.
w

 

fl

3131:1111? 111*12::1:"11*c:3 1.11011 1:113 131*11 12:13 1111111311: into 1:31P 1511:1113-
tor-y in its 3131 0:? 621111221 (11":11113 8). 1.31011 that was (3.0110, the
metals parts? 1111.311 .1193. ‘11:..1'2dictely 8.11.1790. less 31m '31") 1191*
cent of? the activity of 1s 110:1 33114330 pr.pr:1*c<3.i‘ 1:21 ”1:113
froshly cut from} (21.3136 versus 33:7?7’t11121...n .1ss*o:;sl by
the rest as so simples). them-3,11 freezing as 1311;; been

cn:::.i..o‘ as :1 method 1.1303 to Pete... Pt. 0111:3219 activ1tjr, Jsslyn
(19g9) hfis reviewed sons of ha @130
tatsmz'atws on vogtjctz. ls tissues. 2.13 (Esta in the present
study is 11012on :....isd by the destm tier). of“ 23331.11; 1/11 of the
astiviir present. in 31116 fresh fem.

“.7313 results in "" his 53 111101;th the best 1191:11ch of
transporting 331* sake can fern 31°92: its scarce ta the lsborr...
tOPy is by ramming it in the ball 01‘ :1.-“:.. Pt 31.:1P10111'1113 1.11:3
the stern: and roots 83.1106 P (201131313 *31116 91.101.111.31 of activity
is lost from the time: tha fern is out to the {111219 it is
analysed. Kat-111:1; the f6? .0133: after 1.11:3;in duos net
£333. litgts rat 331111.13 its so ti ity but ones the lee-...? is 6.17,

the ssP'P'm so 3131:: to to stable.

 

 

FictPlbution P? .niPfiMPP.s
3911 "‘w n?{"fi'fl ’n't'an
In the apt s1 P, bP cksn ft Pu axis in various states

01‘ 6.0.1101 0132131141; 30119 from... buss 1P3 3.131; scum, tin-011331

1:313 surface of the grmmd 1113.13 other $911113 in the 5.1213.

{b

disco vie 1.11 t3; ere about 13 inc ms 3:311, The infiivifi .111

vrrrirt -011 111 13-....-1111 .10 cctivitjr 611.1 not 3P 11» “1331* relates

17316 3

{'31
1. I.

4!

Sn: yzm retivi c3 of ErP.ckcn :.‘eP11Tronas
thn Tuojectsd to Various “inks
of QPoPtfisuu fifter TPPvest‘ .3

M

fihiamin f7) Best 'Poyod.by 10 mg.
. ‘Lrtr‘ * 1s 1
Treatment :2.. ’13 (3173101 In D 8.3.)...
. 5’ "W
Stage 731: ttssa 8P
(1) 17'1‘653111', out {121.1 16.133; {133
roo.u tempcP- st1w me for 2
_ 1 raw». 7213 i:630 6753 +‘25
(2) freshly c 3t, kept moist
at ranm.temperat“Pe far
(3) Freshly cut, acetone
pcwdoP propsred
. meatsmlv 3173 1' 1-38 11.0313 : 7119
(4) LsP11 3P3 cuon.reuovod
to labasstory 1n ball
of Barth. 8877 i." 838

h; . ... - ._.... ....- .. - ... 4).; “A

 

 

A _.‘ AA ._..

....rw—v j.— W ._. Fvv—

1 :13ve tripnr'ti‘t o fern b1azm u'ors used. EPsh sleds or
fvond chsisteu 01 thes pPrts; one part fr011 arch 01 the
five bis-.303 "Pa t1ePtod 2.3 asteri'L1ed ch CF (1}, C.“ theP was
treated as described 3-t-er (2) and the thirl Ps described
{trail}. (3;. 111“.) V}..::..u{33 112131.511 ~1ng 41-13 (AVG? 5.1.1311.) £31. 3110 five
pPPts :i'fhe standsrd dcvistioz1.

3 Pne as i‘ostnots 1 steept the pPoceaures are dsscritad
after (1}, (3) Pad (1).

1:0

in the. 11::- 311111?0 in the ground or the 6:039:10 01? 3111111321111
filter. '11:: 1.111903. *‘1 the tree. Elia attempt 11:13 1.1.3.110 to 1301;
150111111113 fram tho 11:11:10 1111130113. ‘2‘; i3 1:313 be 0.5.1:?) (b.1111:
3111c: efcwn c..-13 :11: the £12310 33:15:13 0;}? maturity; 1:"ch usually
generated‘by a n fiber of feet.

To find out if there :11? :1 variwicns o1 t11i:11:.1.11:3
activity in bran}: n fern collected on 1:11- 311.1113 day or on

:1.:311‘feront (1037.11, braclwn buds (33:33:: 1 of d velopmcnt) 1:01:10

collected 11mm”: . 1:003:13 opp-rt 1111 cirudriod :11: room taupe-.-
r.-,.‘r‘3111?e 1‘31? 10 days. ’2'113 1:11:113*'.1;no content of drained

lcs 1.13.3 £01316. to be 2 per cent more thm the. oven

dried ferns. is 3110131 in 'L’..1ale 9, samples .n and 3 90113312911
on cliff-{11:01:22 11.1313, destroy-ed practically the 513.1113 3121011111;

015' thismzin. 2;“113 11:13 not the case for 53:13:13.3 C which 1.2::
collected on the 3:11:13 6.2;: 1:3 3M1; la 1‘1. Hammer, when several
form: were ground toqethor and finely: 3d, the 13311311111113.3111
netivi 3r 23:13 31:11.1 .1? to that of 3:323:133 _-'-. 1:111 13.

Since the results prescn ted in? able 9 .m- 151.113.1132} that
more are 1111;111:2113-1113). vwiations 1n the {11111331111333 activity
of 7320:1431 zen fern, t o ferns were brought; to the luoorrtory
in a ball ore 12:01:11 8116': each portion or the tripar .rtita 111:: 2:13,

stipe, atom and. roots were 1111:11'3011. The result a prosc..ted
in Table 10 31101-1 that there more no variation in :1 ‘tivity
3211:3113 the 811.1133 divisions of the fern blade b1: :13 indicatea
”fable 9, there 1:31:16 :3. - fercnces in :10 thrity 3.1110113 5.11111.
vi 11...:er $031113 clr'ssii‘icd 1.1111101? the 11:11:13 3113139 01"..-:~.'t11rity.

3321011311. f1: F238 1 3:11:31 2 in ii’able 10 had. about the-r1119 fr 3:111

kl

”510 9
"3" 0;».

' O J- .t. . V- v: "' a -.‘li? ’fi' 4'00".
(:72: 5 23191“... of 4.111"-ll"‘ se’0'u1viv'of1..r;..c3.on 31:13 {J.>‘G:...;",G 1
of 1:0ch}. “:rzfi“+‘) 7.5110118011631103. on 1:11 0 Emma 34333,? or on

’) W

’4. r”-. if! m . u": 1 h -4 n~1
3.163021“ bays. 1.1113 0921 111.393 in a. £13.: 01:- of
1‘. . “-f‘ ...- .'. 4,. ,1. a“. .‘.. I
bevolcgmcrL l J‘:.;ea ‘Lr-n; L19 ‘~”i“"

9 LI- .5...‘ J

“zfiule Uumber of flata Thiamin 07)lfieatr¢?od by
”‘ Earns used 001160131 10 mg. 11? Erica Lauzle

A A _._ H _A
W v— w.— 1 . ... ._.. _—

fi 010 5* 3

 

‘v—

3;.
L3
~J
\D

car"
I

1:?
1?

0
L110 5’2“ ’-() ‘-’

b3

1.3!.

3
C (.1310 502306 'J 9?
D

SQVG?31 5.25-63 13??

A ‘ ._....— A“

v ‘7 W...- . w W

‘11 frond bu1a “era air-dried. 5"“hles 1,3 pmd C v0, ?8
“.lvi;u*1 fron1 bu13~.in GfiCh case the encirsa frn nd ‘zs
used. Sample D” refircsentod nbo*t 7 of those fro Lbugl dfiidh
were ground and a portion was 333,331.

Hc.m

..N oHaMnm... @on H..me c.8333 .33. mo mphwm Mao...“

1 I‘ll I

80.8 5m.o hm.

fl.

8......9 3.3

 

Illl' E

Sum.“

 

0 mm.

53.. dOFOnMD m0?
Mifimv_ndfiuwgfi

a 2.6 85 no.8 35-8.85...
OH he 5 Mcfiumoaw

A .Mrycwx My vddfllw .H;

8.3 min 34. Man 5...... 89E

it: I." III E

 

 

 

 

 

.83 23. £3 38mm“ fimfl ... f 33......“ $3 3%? ”rid,“
QHOHW. 1:1 . ”Hg . .r Ill Ii
amuamflua Scwunom e3 cflm mo roammoa
N sham H. an
Mpfiowfiozmmbem .....d m and. my 780M ago...”
fioxuahm co mzham éfioaohmwm_mo Lufibwuo..omafififlwwflm

3 3%....

1L3

weight, the activity of the blade of fern 1 was obout twice
that of fern 2. The thiamin destroying activity of the etipe
appears to be proportional to the activity of its bleeo. Thia.
minaoe activity of the etipes of ferns 1 end 2 in relation

to their blades are h-S and 2.2 per cent respectively, The
etem.or rhizome inactiVnted 12.23 mg. while the roots do.
strayed 0.28 mg. thiemin.per 10 m3. sample on a dry weight
basis.

The results in Tables 9 and 10 indicating that differ.
emcee in thiaminoee activity exist among ferns classified
under the some stage of maturity may imply that either vorn
iatione really exist or perhaps the gross method of cleeeio
tying brooken fern in different developmental stages was
not adequate to detect more detailed.chengos that occur in
the plant as it matures which could be a.better index for
classification.

It appeared in Table 10 that there were differences in
thiamineee activity in the different parts of the bracken
leer. fine fern frond was taken and separated into differ.
out parts as indicated in Table 11. The greater portion
of the activity was found in the blade. Since the etipe has
two separate colors, a green.portion close to the blade and
a dork broun.portion close to the stem» it was suspected
that this differenoetmight be due to o fiifference in thie.
minase content. Results Show that the activity for both

portions were the eome.

1.3.4,}.

?oble 11

31331 “is ution of ii~nirzoe ’ctivitv in Srrokon
ern ”3 V39 3 0? "eveloI-meot

’111‘1’1 n (7’) occuojed

 

 

Port of Fresh Fern b; 1.3,“) . “WW-,0
NA. .3. I. 4.21.“, ...
'“ond $93
tipo, green portion close to the blade 169
Stipe, dork brown portion close to the at m 172

 

It is interesting; to note that the not rely gro:.'in:;
ports 03' he fern, the stem {1:51 the fr m1 blodee, 1-:ore
observed to possess the 1.1411633 thine-15.1mm activity. 3115.3
is in agreement 1: ith the observation of ammo and Evans
(191.; .952) in the r :13) vi Zr and animal foodie"; experiments.

2‘11ore are we *y fe reports on the thin miners ootivity
of broolzon 30.... at C1331. orent otzgoe of motor ty. Smith one.
T-‘enton (191-111), Shearer (1921.5)91111ivrne one fix-rams (19 9::.)
found a decreeeingg crude protein 11.11 inoreoeing or do fiber
with aging; or? this plmzt. co 1;, e___?_____:. e 1.. (1914.13) or ed Hang-3;
and others (1924.?) fed rote r. rewiod fem: colleei ed from
different lots and sat 31‘crer t 341' *ee 03 m-u..rwtv. {all
profiro o1 13111211311 do "ieionoy eyzzirptoam but no qua 'mti totive
date. on thisrmizrmo activity z-rer presented.

Ltudioo more conducted by irons and ovens (1915.91)
lining air-tonne}; brookoa loo W e herveetei 111511.131“ when the
plant was at full "ro‘mo one". still green, and those her-

rooted in {E‘ct‘ober then the plant 1.. re co**“le e“ ...y withered.

P-n'

14.14.
5 1
5

1.

.n1 y shered t12L the fermer‘anch e? ferns £22 2; we? ed 90

:3

23

er cent of the 19 Q’Lnienl 1121 to 2 seeey eeluLien
2222212123 0.5 gm. of fern. ?h13 batch of fern 2132 222201
Lexie "hen edded othe 22L ion of 2 Le 2L 2 20 per cent
level. ‘2 the other henfi, the letLer b2Lee 03 ferns in-
ncLiveted only 5 per cent of the 211 flz 213 .ed Le Lh essay
solution 221 223 net Lexie Le rate. Similar cesarV2tien
were 2130 2220 en.herees (Svmns 122 1vans, 19292; evens,
2L "1., 19L71). IL was peetulated by thee auteore Lnnt
‘arecken form 12 mere Lexie at cerL2in etsjee of growth.
Since the preceding reperLs iniicete the peseibility
e3 2 «diff crease in cencentretien of the L212nin deetreying
f2eL 23 bracken fern 212L1res, 322113 at different stages
of develepment 23 222221221 in'reb lo 1 Vere cellecL 21 221
anrlyzed for their Lhie:*n:e activity; Eben,the dry weijht
2nd thi“"**uue nativity xere pleL Lei en 2 graph 23 shown
in Figure 3, trere oppe:1red to be e relefiienfi m1) between
the L10. 1: the dry weight of the 1023 .22222222, the 2meunt
thiqmin deeLreyed.per 1223 212 o inere2eefi. Ehe values
plotted in 1:222 3 are charm T2ble 12 in L112 f1pondit.
2 2 2212222212 emeunt of enzyme 2 cLivi1Ly "23 lost when
the mature ferz dried up. 32121 fern 32021: were gethe re 2
in late Eevexber of 19$? in L32 same area where the pr eeenL
8232103 were cellecLed. ?he b1212 of the 21222222 fern de-
etr eyed 0.33 mg. th1flme.1hil free} fern bled 2t fullrme.
terity (SL132 8) inectiv.tel 6.69 mg. "Hein.‘fleth‘V2luea

9 Q

are expressed per 19 P3. M; 16 en 2 223 telght basic.

16

1h

12

10

8
é
£9

‘3 6
0
.4
a
0
0..
:3

u) h
0H
0
:3
E’
.n

2

0

M6

 

 

 

 

L. . O
" 0 Dry weight per leaf (gm.) ‘
F A Thiamin (mg.) destroyed '
_ per leaf (dry weight basis) _
P -
r— . c-
O
r— u
_ . -
O ‘ _
g o
. '. A ‘
fi’, 40 ‘ A A
v - _
In 1 l J. I l l I l
0 1 2 3 L1. 5 6 7 8

Stage of Development

16,000

ILL, 000

10,000

8,000

6,000

0,000

2,000

Figure 3. Relationship of dry weight of bracken fern to

its thiamin destroying activity.

,—

‘Thiamin (mg.) Destroyed Der Leaf (Dry Wt. Basi

:..” . ‘ v ~7~ ... -‘.- \ \t. I‘v. v1.4 1 q 9 J’, l —. , .’.. r
1110 I'LL-1,113 0:3 ()1 tan prosoz'z. 301.11%; one. that 3.11.11; Lapse
a ‘o - q, =-. ‘ . -' a. G ‘ ‘u, . , ,‘g 1- pi. ' It. a ”a u .. ' ,‘a 4.
is iOLLJLal in b91161..un LUZ‘I‘L So 211 stages 04. UGVQlUPL-lorlb’ 5.03

4- 4.. J . J... _. .'..‘1 . ":3
activity more. :sos a .11. (:11: 1 (3.1. ,111. per 101,; increase.

3

Lifhis 0:173:10 V0.2 07.21; omrozl to be 12.20:. 17:33.0 {13 soon as the
bier-:0 2021 lo 1:13 51,1.)"11‘001 L13 3221 its etc-2:1 '3. it- activity
was found to be 5.1“1'L101’100d by v rio com-1:1" 0:101" 51 in valved

in '110 11832}? proooa 3:0.

('1th 1r. pr
SJ ‘3‘“ 5‘. é‘ah

31‘212201'12'302383 were 00:: :.‘LL 10330-11 t0 (2""cr $02,215,300 02.: “0315.0.

1:513:00 21.0 tiv fin 12:30.05: 01:: 502323 13210050010313 0:: {2510 :21 1:520:20
05." 013233210 extraction and preservation 05' 02.12.3220 activi 1:3 in

the £02222. fro: the time of collection to initiation of 102.0.»
1305.3. 33002113 2.000 01:30 hC-‘I'VGS tci dsnalyn0d at different

A

dcvclcmz 21:; 3. st'..""s '50 0013233131011 '- -'hc 03.1012- 0 0.33.22.22.30 in 00:25....

4'

vity 0002-2... “rod :23 the plant Latuz'ed. 211103010 activity L303

1

:20'01. ed 103' the proceéarc 03'. 51011th (195371)).

In the 13230-002200 05' 02-20033 thi 2min, 1:11.LL....L1‘“..39 notivity
3:213 £02026- to 1:32.23; with the kind 013-3. 2911 013110 11:31:. 2'01 3:21.123;
m0rii0. {1013;212:200 0222:3330 czztrrc “2.5.0 2.20.0 1101:. 001401":- Contr '25..
fused 0.: :tr0.ct0 1300000000. high activity but 00:30 ref-3:32:03 in
the residue.

1315021513 5.110171270121013 :..-0.3 5‘03. '13. 120 be proportional to
the 0722. 02.1210 05:" 13130.0an 1103303021030 0000.. this 1‘: 201: 13030 033.00
:..-11.13 the e: iC-Lc 00 that the 22052.2. "220 factor is them-203.22.135.10
011333032 L; 03 L0 ILL-n... 33 cf." ether 2100.- '00:: that the principle
effecting; tit-5.1.3." 10:20 is an 0112232210.

'17" 22.5310. cctivity 2.2123 £02,033: to increase :23 the dry

L.7ci;;ht 03‘." the fresh 10:15." 113.creczscd. The act vity V731“ie <1 in

the 8.515. ‘Vz'cnt parts of tha p10.- 0‘5 0.. .3: 03:10:03 2.00310 at thc

021.1210 Etc; 0 of 01.303103. 0.3 00023 0:22 “the 1022;? 220.0 000023053021

1.52

[4|

.1.. , J... _.'_. . fl .
03 0000, 1000 0. 001

1 .1.? L...‘ “J.
3.35.35 (...-“7’ 9.113 3605.111...”

[4'

vitj; 1.2.0.13 01300211.?06. but: 01300

7000000 t0 be 000010.

I
‘5“:‘1

9.8.1

““y‘.~¢97‘11”1 ”‘P-‘V‘!

I‘LLJ.J~L $U;.L...J CJ-J. '1‘

v " .. " T 'u-I'.‘ ' q. ‘ m A ”1-1-10- '1
BQFJLCI", J. find. «I. . o:1:_,.;i 1967 ;,?;--..;.-.j 1:90 :33.

crlnnPP‘c rcig, an P“*i*hw~“~Pc factor of PCLAS.
"”HCPJ.» 1211a (3,1301‘ 23: 996.

”PPPJHP, K, and P. D ovi 19LLP In~cbivvtlvn of thfixui e
by cePtPin fnodac'ufizs and 01158923. 1: t I.
IP1iPn J. lied . Res. 32: 131.

EthVPt, K. and P. Devi 19Lh.b InPc LiVP Ption of thialfllne
bv cert in £003 311.13 an1 011303 a. IPPt II.
I£:.'\l.Lc;;.a‘L 01.711011. R03. 32- 139.

Braid, K. m. 19L9/193: 3?%Ck0flnda batsniat s playthim
and an agricultural Peat. 810$”ow Hatur. 16: 61.
(11%w13101. fibstr. 26: 22'7,19L2).

Carpenter,'K.J., n. T. Phillipson Pnd‘U. Thomson 19>0
“ 1 1:1 1h dried'br cken.(1tcris fqullina).
Brit. Vet. J. 106: 212.

Doutsch, H. K. and ‘. 3. Eagle? 19‘3 isgribuPPOn of
vitamin 31 desbructive enzyme in fish. Eroc. 300.
L111); 35.01. 1.4.0.31. 53 :63.

JV”fiu, 2. C. flfi& E. 3. R. fivr ns 19% 91 Ktudies on the
biochrmistrj of Pastime 231;:‘1133. The effects of
the inclus' ion of bracken Ptcris Pquilina) in

he aiets of rats, and the problam of brackan
poisoning in.farm animals. Brit. Vet. J. 135:
17S.

Xvans, K. T. R. Pna P. G. Evans 19L9b The effect of tha
iitclusion of brPckon (rtcris fiquilina) in the
uiet of rata. Eiochan. J. LL: ix.

Evans, 3. T. 3., H. C. “vans P1PQ.H. 3. Roberts 1951
Stu1ies 0n brec} zen fer n poisoning in the horse.
:31“ U. 9'01), J; 107: 3514., 399..

Evans, 1. A. 1958 WP radiomimctric na.tura of hrPcL an
toxin‘ CPmcer Rea, 2P: 2252.

5:1

25?:3110, 1.7. C. 0.111 2?. . 302100 193.12 21:11:11 1.313.211.1000.
“L;- n , 7'.» a. ..-... J’
010011011. 8‘. Ln}. ...-:1 i.
“ -.- ": . ,,. 1"? I1. "- .... » .f" ..
,;-\Jf't.3?.§3’ --I Go, 5.. :.. 1.1".” 23.03 9331 4'10 ".0 ..1' " ...-5:3 192:) '-':'?’~0
1510031110130 0.; 0123211.: 200111231 "C""V‘+“’ 0;? ‘00. 012011
-.--...u_ , In . '1"?
(. 03:12.3 L 0uili..r1. ). 111100110111. LE. 1.1.6: ...- 0:111:11.
,_ I ..- n
relgtcds 11500113 of 21140 0.11:: 051?. V1001. 1E3 cL‘E. by
9‘1 Pf
9. 2.". i.:€3;dq int-C r301. 34' E1). 1131.39, 1:: 1.. :72". 35:".
b91
H‘ O
O 1"! a! t". .‘ H a «'7‘- V a 5 . '*
£11341“ 1' ".'.:1. .6086, 5:. .1'..C‘.;.. 1.1”. 0.‘ :.LLSELELI’Q, I... (:0 asp-L 8.!" Ed
3. 13211000 19f" 1251 30126.1 0:3 «’11:: $115,011.00.

3 1.".16'33.’
vrtiqn 01‘ 0211011110 b10001 010 .01 by lawman c. 11:10::
51.01. Cl'l'Ellg 196: 2:3,}.

1771.35.00, E... ”if. 3‘:-“0:10 and 1.2.? 1100110111 1917334 2110 0000110. 11:, p0
of bacterial ”‘11....L111M J. virazinnl. ( 001:0)
1: l.

1"L:"it:~ , L., 1.. 2000, S. U; 00 1111-" J. ”oi~1.. 1952211 3011:1100
(2n “Ellwwiwzngo IL“ . d1"; 0011.0...” 01" #2100111: .
€116.31. 19L): 2.1-3.

$1135.00, .1., '2‘. 01:21:01.0 cm. 3”... 27.000 19:35 "ntiatELinszt'fi-‘sz
‘5

1‘ 't a. w “ L' P 0 !
10000113 0.? 1011-0. J. L’Ltcrmol. {0:21:10} .1 £24..

‘ujiuLL. "_. 01:: :L. Inmnro 195.72 Ltullcs on th.i:31*~*’:1000
III. 1‘1? 11:01:10 01‘ 00011120111000." 3. 13-101.. 31-001.
196': 3:1).

2055.2101'0, 12L. :1de ET. :..?sztzmi 19:33 s’nti-thicmina fact-003
of 01.10 fem. J. 33100110211. (301310) 13.0: 11.27,

..inaoLZ 11*ij.0 , IE. 11.19513 ...:110121‘111000 act vi"? in fish: an
1:: 000011011 11051:? m0ifl10:l.£"ish Ind... 0:3 . 2': SEE.

30210101, 1'3. 1975' 13003101000101". 01' bu?”0rs for use: in 011223-0310
burlies. 170:1.» ' azlaf 1:1 0110:7101 0331'. 1.701. 1 0:3. by .1. _P.
1

'33:! CHIC); ["311 ".C"15:*'-u TCPLi. 11’63’131301 :2.. Ya

13:10:11.,3. 3., ".‘.". E3. (301110011 0.113. C. 3‘1. 33.10213 19.: 3‘ defi-
ciency (110000 0 of 11:11:00 31110311003. by 300.1111" 11-91.
J. :E‘u'tr. 21: 20,3.

(7110021., If"... 3., 1?. 12?. 03:1 100:1. 11.21 C. .‘. ‘.'.iv:::::3 1925.2 The
5.1152003111101011 0;? vitr 51.12117 in 0.1.030 examining;
"JIM‘IG fine-Ll... Li. 1.21:1”... 6.3.511£:;.

5:2

"" ' ' i"! y; .'. ~ 1 "‘ -' ‘ ""‘ '. ~ 9 ~ ‘-
mrob, u. v. 19%? .LfiiLischc 191*L91*999hun3 999 Luiccnrunbofiflgs
‘- '14 5" . 1 a ‘I 'v - 'u s _ . '1:- - y» , «- ....
uni 39:90 br9uch7nrheit 999 fifiuBELnuCSuiflmwflfi 19
9 , ‘ ‘- ' , ,7. ,.. -.‘~ ...... ...." ‘P‘ .5- 5‘
1~Luli9hou'm9L09191. (EnJliLh.Lou.rJ) 1n“. 9.
1L9: .90? ch. 19: 179.

T? , " p} r' . v. x ; - '4' T 1 i 2. n 7‘} ; 1 1" . 3 "V ~ .‘1‘: .-"
:xvyg 9. 9., B. Ltenmzvh,: 9. 99591:, +H 9. 919.909 99&
o i. ) J. ' nr’.~ l ‘" Y ., u': ~

:2". . :- "9311119999 19 3:) 9911911319.: of L: 11.19.1910 99--
.. .... 1 ”H"?
eru tian‘by b9 9 Lon fer :.. 99d. iron. 9: 199.
*T. 1" T‘1 ‘2‘. ' - t7 .. 9 ‘99 z'. . .
2.1.3.118, 9). .‘.Lo, a. 0 I1. 03 13 find-1 W. J53. L'Eeed 1915 7 J-I:‘i..
e.» .. s. - ,3- v n. ‘t l .
thiamine 99L ivi J of 9999999 1099. .11. '1~oc.
6‘ O l;.\1:3.
I
7? 1,, Ag" ,. ~1 . , . :9: 4.
999919, N. 919 9. 9. Erase 1920 1h0 poinLfii.3 Who sea

‘
by the comnnn b99ckwn. Vet. J. 76: 93.

Lad 99, 2. and E. 9. Bruce 1933 The 901399 a: of hnrces
b7 “he common brackon U'i :oria fquilina 9.). get.
'1” C" Q - ‘1
I}. L'Qo 142.0.

HC?7011,0fi. 193% 19W9071uuro £979 enfienca of Lhn act v.9,

th 99 nL tumor fnc LOP in the common clam. Lcienc.

'nch'ngton} 11$ :77.

999.9.97, D. J. 19L1 Chemical mcLhois for L99 act rufln. ion
0.? Vit n 1112'}, 5.11 g .3. 12d . $9.;HTJJ93 Q 3113331. fiwnely't Q 14.111. 1 _D '3

215.

311999, D. E; 9nfi C. F. Peter 1996 intinthixmin activity
39‘9979‘1 fish, J. *utr. 89* 919
.'.-I. I». «Mn-Lana. a.- C {,- .

595179 L. 9. 19L9 :n93m9'9.ctiviL7 in.£rczen ve~et9ble
tissue. ’dvnncua in 99,7m91937 and 9919909 9:3;309Ls
o“ 91099.313993."vol. 9 90. b7 3. F. Lora. Intersci.
31.1.}. 1316., 9:. Yo EDP. 6.3.3.0,JCO

39930, J. R. 1990 InhgbiLion of effects 0L lauganogou_c
viruses in mice b7 e"L“9 Ls of narrfiw-“w:, 999999~9:;.
9999. 909. Lflp..ui-l. :99. 123: 299.

Jufije, J. R. 1953 :E3 9923 of thiamine écfiriv9‘i0n on friend
virus laulcenia inr mice. 3900. Soc. 93?. 3191. find.

annlnnia, J. 9., E. J Lhou.309, T. 3. Rohu1n9 999,3. In
“ 799 1907 Insect hormmnea: nlpha 00979099 and

3;?»199999711'9967991 a * *1 2199912011 339.11 :1. 519191-199

( r-

.L; ftOn) 1)7:1[+3£30

33
ii
if"
i

fjlr '9' 1"“ .‘J ‘ 3‘ .. .-' 5.9.1. .. 9‘
9.031 {30.1, 2.: . J. 1937:). 399919011 " .111 “11.1.1”. SC . 1:633 . 3:0 91113931: .
:..-3:}? a 74.5.5 3... p o 21...; .

53

ties of a thiamino from.brnckon Ptoriflium

agnilinnm.(i.) Kuhn . Siochom. J. «7: 2§.

Kenton, T; H. 1958 Kanomotric studios of brackon
3

 

Kenton, 3. R. 19373 The partial purifieatéon and proper-

 
 

(12...) Kuhzfl thi ominaoe.

 
 

H

L ochom. J.
Kingsbury, J. H. l96h Poisonous plants of the Enitod
States and Canada. Prentice.fiall, Inc., finglovood

Knobloch, I. X. and D. S. Corroll 1962 Ferns and fern
allies of Chihuahua, fiexico. Texas Research
fioundotion, Texas. p. 12.

Keller, A. and J. C. Somogyi 1961 Reitero voraucho zur
isolierung einos thermostobilen antionourinp
wirkstoffos nus adlerfornkrout. (onglish Summmny)
Int. 3. Vitominforooh. 31: 230.

Krompitz, L. O. and D. U. Voolloy 19hh The manner of
inactivation of thiamine of fish tissue. J. Biol.
Chem. 152: 9.

Kundis. H. and J. C. Somogyi 196u fintithiaminwirkstoffe
in pflonolichon nohrungomitteln. (onglish Summary)
Int. 3. Vitominforach. 3h: 135.

Helniok, D., H. Hochborg and B. L. Boer 19u5 Physiolo-
gical availability of the vitamins. II. The effect
of déotary thiaminnoo in fish.produots. J. Butr.
30: ‘1.

fiolnick, D. and H. Field. 1939 Chemical determination of
vitamin,81 II. Hothod for estimation of the thia.
mine content of biological materials mith.tho
diszotiood.puaminoaootophonono reagent. J. Biol.
Chem. 127: 515.

fiickolson, 0. and H. G. Yang 1966 Eaturally occurring
toxiconts in foods. Rod. free. 23: 100.

fioson, o. 1955 Extraction of soluble enzymes from.hi3hor
plants. fiethods in ezzymology. Vol. 1 ed. by S. P.
Colowick and E. 0. Koplan. fond. £ross, Ino.. E. Y.
pp. 62-63.

Roberts, fl. 3.. E. T. R. Evans and'fl. G. Evans l9h9 The
production of i'1'Jrooken staggers‘ in tho hora and
its troatment by vitamin.31 therapy. Vet. Rec.

6:“ : 514:9 a

5'3:

51.22232229-to,o. 21:21:33. Fujito 123276 i’twflioo in J1115.2 2:121:20

”locoozpooino; thomtaootrfolo footers II. Isolation

of thin nine (100011133 tho::22:112ooooblo factor
from o :oot po ototo lefivo (I. "v’itrg'zinol. {37:31}
2: 39¢

Eichoor, I11. 3‘:. 1961.; Grout}. miIfiIibitinr' agents from 2“:o.':'oo21'1'i§
ext? octo: chor i023: ond biologi cal pro; 20:21:10.3. ...:oio;i:oo
(.12 23111 Eton) 11131: L113.

‘eoloolc, Z3. It. anti 3?. C. Davis 19’39 Tho COLLVM'ZLP’" afloat
of m-nitroonilino on th 1:12.122. no (1022 truction by tho
run

Char-3‘:. 3:: paralysis 6:12-37:20. J. .‘..Zziol. :..-.5332. 17’? . 9C 7.

Soc-look, ET. Ti. and a". H”. Liverworo 19111;. The fomxotion
of." hydrogen ion in th 0 (flow-“o of thinraino by
the 31312. principle. 3.33101. Chem. 156: 379.

So: :310c ... 12. R. and.- . H. Iivommro 192: 9 The action of
amino bonovlthio ..oolium 23.1.1333 on thiammo dos: woo bion
by tho’ Jhosto-.. por2'1:7:3i3 foo tor. J. 13101.0 hon.

177:5 3H3.
:.t-oo.loo1-, T‘. :2., ’2. II. Tivomoro 22123: C. :‘2. llvozfio 19323

1-.

Thin: Tno innflc :ivz. ion by tho fresh-aria}: or Chno to}:-
paralysis f.-_s.ctor. .T. .222. Chom. I‘Zioo. 65: 935.

Conical-I, R. R. and 1.. I... ooocllond 191:}; Thinnino iI actin-
votion by tho Chas tot-32922717313 foo nor. Iohibition
of thimxino inactivotion. J. 32121. Chem. Soc. 66: 513-7.

:.ihooror, C3. D. 1924.9 Some observe. tions on the poisonous
properties of bro Icon (itoris ..quilin:). J. 302212.32.

Pathfll. 52: 3'31.

31211311, in. 2121'. and 133. '. I'on’oon 195,214 The (301122203113: ion 0...
hr T-:0n fronds and rhizomes at ”Li"1"oro:..t tor-loo
(3211' in; tho {31202-1213 semen. J. Soc. Chem. 1n... ,
L013: . J6 3 : 2.1.1.) .
3‘2" 1:11, 115‘. C. on C. If. I'routt 191;}. I ovolo opmo'zt of 131': amino

do: iciomv in t3 2o cot on 22. oi oi; 01.22....2 fir-211. l’roc.
30¢. 7:. 12061.56: 1.

3022'122321, J. C. 191:9 inoctivmfiion of onourin. b7 extracts
of animal and plant. Int. ‘21. Vitom'n: oioooh. 23.: “in .

So? 2123;" i, J. T}. and :5. 31201102: 19,739 {73:20:- c'iio chonioolo 22th
oinoo nntionem'im:irlcotof'foo 1:21 fornkroot. (..:::;:lioh
.1223:- 22:1: 1"?) int. 1.1;. ‘Jitrzminforoch. 2:9: 2:11.

3233

- . C 0 b- ‘~
'5 2 .. ... -- n-'—~i a ‘vvv Mrs-rt {1" " v21 .fl . ""
A1-» C ’ V2. {'3 «.1 0'»? +91 LIV-r «5 <3”: :0- .LtJAl. 1- J- :.l u 0:106 . «Jig-4‘ Q
o : 3(6).

I {1‘} 03‘1‘83 :’ C. {2'1" '___1VC1‘133:2 «wad . 15131110127?
0
J,

r’T.-‘ ..- .... L3 , 4-”? ..., 1.1 -~"‘ _1 .I ’5 "F- . Y" 2‘
..I:3.:2::l.:0 inC.CuA”-f3.u.ma.:1 of; I'm: .13.: 19.1,; ...:Lr. ....ov. 1: 13.33.

. --' n o ' _ ' ._ fl.
2310:1213, 33. 2121-31 13.3.”. 31250:. 19:39 The 3.312.633.1171:-
thiar'n ’03-" b 2 ' :1 (31:39:23. Iqu‘ lino) .
13320-111. .:'.n:3.., Eon/P. (215-: 6.

112322-3212, J. 33., 3:. J. 331202-193 and 3.”. C. 373210.22. 1933 1:30:131‘7
.'.-3‘ 1"}..0 2"‘1'1'm'1 “'00-: 0.0 3:02?” 03‘ broclrcn this 0215.12.12 so.
3-0423103. J. 322.3,: xiii.

5303'..— .3, P. 553., EL. 3':. EEI'CCKI {7.11151 3; R. EI“P.319}.L6 {Entifl
3.311.132.3220 activity 0.1-? 1521:2213: r122.tori:l. . J. Biol.
($110221. 10-): 737.

23001107, I). ’23. 193.33. Iiootmwt ion of tT 13.932.312.43 327 0. 32:31:23: 12200
I n= 1' l . -~ 2
in certs-.111 :1331. ... :3 01. (“She-:1. 1:11.- 99?.

..oollofgr, .1.). ‘12. 1933 ..Iioogntzhosio 0:2 11:3 e'mrmr 2::331'2'20rt
.. 1- 3'2 1203.10 125.02: of Moim' :..-2.13:3. I..t2.:..-2~0

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(1)

(2)
(3)

Special Reagents

1-Lu-am1n0¢2umothy1-pyrimidyi-S-methyl)npyridinium

bromide hydrobromido was prepared by the method of

‘Kenton (1957b) from.unamino~5obromamethyl-abmethyla

pyrtmidino hydrObromide.

Thiamine chloride hydrochloride 1

Buffers

(a) 0.2 M Tris Ehydroxymethvl) minomethana pH 8.0.

(b) 0.05 M MOPS 3 (Mbrpholine aultone) pH 8.0.

(c) 0.05 M THICIEEE 2 Eris (hydromethyl) methyl
glycine pH 8.0.

(a) 0.05 M'HSPPS 2 (Hydroxyethylpiperizine propane
Bulfonic acid) pH 8.0. _

(e) 0.05 H SEEP 2 (Sucoinyl hydroxyethylpiparizine)
PH 8.0.

1 Purchased from<§eneral Biochemicals, Chagrin Falls,

Ghio. This was equal to tha U. S. P. Thiamine Eydrochloride

Heferenca Standard.

2 Buffers furnished by Dr. Harman,Good of the Department

of BOtanV and Plant Pathology, filchignn state University. HOPE

and TRIS 113-133 are available from Calbiochem.

E‘rolinfinoly 0.2—:porir1ont s 1 are 0023111017061 to clotc Cl‘l’flinfl
the amount of brockon homogonnto that could ‘09 odfiod to
:1 1:102:11 amount of thiogfln chloride lynclrochlorldo 130 that
the rate of enzymatic reaction would be indoponflent of
the sabstrnto concentration.
-.. "‘nch agony involved the :2 iition of 0.1 :2". of fcro
honor-gnaw(3011121210111: to 1 1:11;. of chi ed fern) to a mix.
ture containing 22:3001thizmin. 21 ml. of 0.1 3': pyridine,
1 1:11. of 0.2 (E phagoh to bifi‘fer‘ (101' aori, 1935 at: 1211 7.5
and water to a tom-2.1 volume of 10 1:" . The 11332.)? nol ution
was prepared by trim; a 2 111. sample of the root: tion
ri2ttm10n or. incubating it for 1 hour at 370:3. .3132ch
activity was stopped with 1 ml. of 23 per 6:32:22; mote.-
phosphoric acid. The b13111? solutic :1 12:23 props-rod by taking;
2 1:11.. of the reaction mixture and aficlim; l 111. of the mobs...
12110331101216 acid at 230330 time. Both acid 121133111203 were:
centrifuged and. 2 ml. of the supernatant from each 5221113210
1-3118 incubated for 1 hour at 3703 1-21 :11 l r2 . of.“ 20 per cent
110-513 .tuu hflngj o to dostroy the res ...duol thinzun. $1.10

0.5 r." . o? ferricgmnido reagent E1. 111. of 1.4;? not‘. 1211.1. ‘1
forrlcycniao plus 6 1.21. of 23;.) sodium hydroxide 2:223 admit

£3111 after 15' minutes at room tome; othe 6320613

'.' r. .13
L r H.130; 01?.

2,
l‘
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1

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1 :rmxrgn 2.2....0 2 2:13 :20. 10v 21.1 0;; the o.u..1t....o.-
l' --. 1 ' .. h .?.l 4‘ a ‘- . . ‘ ' _1_‘a .9 gr tuft ‘u‘ "r. g! .'r“-4 . a 6."?
13090. .1153. .’W- .1. HOP uh‘Ql’C'qul nth—Lx&i.{i.;,;t’ U410 4L 3. 3111.14.16 fiC {3).'-‘r3 e 01- 9:18
.’ -'. fg‘gqn Y on 9.1 H "-1,.” a1
rAAJ‘.uL4.&. 3 Wm. ' 41.13.015.395; Gun.

if 2‘ n1_;..,m.1 I: 0130. .mm‘i 3111:: :11

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.131223 lo(zL-P:1...2:o—2-11o 21:71-33} 1'. 3.215.113]- 2.: 10:21.]. by; 2:122. 21.1.1.1

11:99:11,139 1""""”"‘J“O‘f do which is coax?” 3r re ”0:22:06. to 12:: lmimro.»

pyrifihimizin, 325:2.nd.-rda were: treated. in a 35.11113: 1112331161" 528

.1.

thinziflzmoe assay except bracken homoggonn“ 1:6 9.21:2.1321ir7:.1..n
zero omitted ”:30 .1 7:113 renc tion mixture. Tt'o levels of hetero-
pyrithiomin we; £1 u'socl 1n ench (16501211112135.1311. .‘ha P6212. 22.33
fro; the two levels of heteropyritbizminw re plot .5303. on a
graph. fem: this {gr-2:12.11, the 22221011112; of heteropyr 13h nrfiin
fommd from the $111 nminos o 9.311.523 using; a 22:31:; :11 122101141; of

brnoI-zcn fern was corgmtofl moor-£21113 to dilutions 1211.210.

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