RESEARCH CONCERNED WITH ALKALOIDS IN LUPINUS

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• Release Decision: RIPPUB
• Original Classification: C
• Document Page Count: 24
• Document Creation Date: December 23, 2016
• Document Release Date: September 6, 2013
• Sequence Number: 1
• Publication Date: March 26, 1963
• Content Type: REPORT

r". im
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INFORMATION REPORT INFORMATION REPORT
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C-0-N-F-I-D-E-N-T-I-A-L
COUNTRY
Poland
REPORT
50X1
SUBJECT Research Concerned with Alkaloids DATE DISTR.
in Lupinus
NO. PAGES
C, REFERENCES
\
DATE OF
INFO.
PLACE &
DATE ACQ.
26 mar 63
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THIS IS INFVAI HATFD INHIRMATION
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RESEARCH PROGRESS REPORT
Main College of Agriculture Department of Plant Physiology
Warsaw9 Poland
Name Of principal investigator: Prof .dr H.Birecka
Project title: Metabolism of alkaloids in Lupinus and phy-
iological role of these comm
mil pounds
Project numbers E21-CR-1
Grant number: PG-Po-130-61
Report period: from 1.11(.1961 to 31.X11.1962.
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fOR
SUMMARY
Duri g recent years lupin alkaloids - as well as alkaloids
of other species - have been investigated very int neely. The
results obtained yielded much valuable informations about their
chemical structure, patterns of biosynthesis and metabolism,
quantitative changes during plant growth etc. However up till
now the phy iological role of these compounds in lupins /or
the role of the processes leading to their synthesis or decom-
position/ is unknown. Therefore further investigation in the
field of: a/ identification of alkaloids occurring in plants
often in traces, b/ their interconversions and metabolism in
general and c/ their rate of biosynthesis during plant growth
and the influence of different factors on this process can be
of a great help.
Th se, problems are important especially in the ease of
lupinsx/ /fodder varieties/ because - among other things - the
toxicity of various alkaloids is different /sparteinea. lupani-
ne hydroxylupanine/.
In the reported experiments: 1/ transformations of alkaloids
characteristic .f bitter and fodder white lupin plants, 2/ iden-
tification of'some alkaloids occurring in bitter plants and
3/ the role of aerial parts in the alkaloid synthesis during
their greatest accumulation in the bitter plants were investi-
gated.
ad 1/ Enzymatic extracts from bitter as well as fodder plants
of white lapin are able to transform sparteine, lupanine, hydro-
xylupanine and angustifoline, i.e., alkaloids characteristic of
the species investigated. The transformations are catalysed by
dehydrogenases, present in extracts. The optima of temperature,
pH, substrate concentration etc. for the activity of extracts
from plants of both investigated varietiee are analogous. The
products of transformations of investigated lkaloids were the
same in extracts from fodder and bitter plants, i.e, dehydro
forms of the incubated bases. These facts show, that the ex-
tracted ctive enzymes from plants of both varieties are very
similar. Some alkal ids which were not till now found in white
lupin, like lupinine, 17-oxosparteine, 17-oxolupanine, did not
undergo any changes in the extracts from plants of both varie-
ties.
x/ We are working mainly with white lapin because the variabi-
lity of the populations investigated is not so great as in
Lupinus luteus or angustifolius. Therefore - among other
things - the differences in the plant weight, alkaloid con-
tent between the replications are not very great. We have
carried out Many experiments with Lupinus luteus, but because
of great differences between the replications we could not
draw any conclusions, statistically proved. Some other diffi-
culties occur with Lupinus angustifolius. It seems to us that
the new forms of L. luteus and angustifolius we have recently
received are less variable.
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In order to understand better the patterns of alkaloid
interconversion in white lupin labelled iradiactivi( sparteine,
lupanine and their dehydroforms obtained in vitro /thanks to
transformations by enzymatic extracts/were introduced into
plants of bitter and fodder varieties. It must be added that
the alkaloid spectrum of the two varieties /at least at the in-
vestigated stage of plant development/ differs somewhat from
each other mainly in the fraction whichx9hromatographically
behaves like a base called multiflorine ; this alkaloid occurs
in relatively large amount in bitter plants, but it could not
be found in the fodder ones.
The detailed analysis of the investigated plants 2$ 24 and
96 hours after injection - in addition to the results obtained
in experiments with enzymatic extracts - permit the assumption
that the general pattern of alkaloid transformation in white
lupin is as follows: ,
alkaloid ----; dehydroform ---3 another alkaloid
4
nonalkaloi.a compounds
and the interconversion of the investigated alkaloids proceeds:
sparteine
4 it x
dehydrosparteine multiflorinex I /bitter variety/
i
lupanine
4 1
dehydreolupanine
4
g(DICIhydroxylupanine .4 hydroxylupanine ester
OH
det ydrohydroxylupanine
This scheme does not include all alkaloids present in whits
lupin, alkaloids, which may play an important role in the inter-
conversion processes.
According to some hypothesis a reverse direction of intercon-
version was postulated. One of the main argument of this hypothe-
sis was the fact, that the occurence of sparteins in Inpinus album,
/as well as in angustifolius/ wee not fully proved.
ad 2/ Therefore we tried ta_vpamine the base occurring in vege-
tative parts of white lupin'', which behaves chromatographiu
cally like sparteine. It was proved by Infrared spectrometry ana-
lyses that this base is identical with sparteine /not only in
bitter but, also in, fodder white lupin plants/. Its occurrence in
small amounts especially in bitter plants can be a result of a
very high rate of transformation of this alkaloid. In addition
x/ 0
xx/ it is possible that this is not the only way of multiflo-
rine formatior 'in bitter plants/
xxx/ we could not find this base in seeds.
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[1111011.
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we showed that s / another alkaloid, present in bitter white
lupin is hydroxylupanine ester identical with hydroxylupanine
ester occurring in fodder plantsto b/ hydroxylupanine in side
shoots of bitter white lupin is identical with hydroxylupani-
ne, isolated from seed f lapinus angustifolium /in our
previous investigations we have proved that, 4ydroxylupanine
in bitter white lupin seeds is identic 1 with the hydroxylu-
panine from Loangustifolius/. How ver this hydroxylupanine
seems to differ somewhat from the hydroxylupanine9 isolated
from 'vegetatiVe organs of fodder plants f lupisus albus
/investigations are going on/.
In our previous as well as in the reported investigations
we have based our conclusions on dat showing transformations
of alkaloids introduced into plants. But these data could have
been considered as n t.fully certain becauSe they reflected
mainly changes of compounds introduc d and not exactly the,
changes sf alkaloids formed.in.situ.Theref re we tried to
obtain some informations .about theiint rpenversion of investi-
gated:alkaloids on :the basis 1/ of alkaloid content changes
in plants during their, development and 2/ of the total and
specific eotivity of,some alkaloids in plant exposed to la-
belled /Ci4/CO20 The results obtained rather c nfitmed sur
assumption that :the interconversion of investigated compoUnds
pr eeeded fr*m lower to higher levels of oxidation.
ad 3/ It as shown in our previous experiments that the
greatest total alkaloid accumulation in bitter white lupin
plants /similarly t fodder ones/ ocours during a relatively
eh .rt time after the beginning of sid shoot flowering.
According to the results of the reported investigations a/ t
this time the side shoota are mainly responsible for the alka-
loid synthesi /70-90% of the alkaloid increment/. b/ at the
later stages of growth the total alkaloid content in plants
does not undergo any marked changes. This i in all probabi-
lity the result of inhibition of synthesis as well as of de-
composition of the investigated c mpoaads. c/ the ageing
process of leaves brings about a diminishing in the rate of
alkaloid synthesis and d/ the pod .are t able to synthesia
alkaloids the stem of the main shoot sho this capacity but
to-a very small degree.
These results draw our attention mainly to the side shoots
of the investigated plante. The alkaloid precursors and pro-
ducts f their metabolism will be investigated with special
reference to these plant parts. The results obtained may be
of s me importance especially for plant breeding works
DETAILED REPORT
I. Enzymatic transformation of lupin alkaloids /Lupinum
albus/.
Several years ago only two alkaloids characteristic in
the bitter forms of lupinus albus'and angustif lius were
known /identified/. 10e. lupanine and hydroxylupanine.
During the last years great progress was mad in investiga-
tions on the alkaloid content in the two species. In seeds
cm) mom! [Mr nmt
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of bitter white lupin - besides the two aforementioned bases
angustifoline, multiflorine, albine, hydroxymultiflorine,
dehydrohydroxymultiflorine were identified; the latter three
alkaloids occur in seeds only in traces.
At out laboratory s a/ in extracts from aerial parts of,
bitter whita lupin multiflorine9 hydroxylupanine esterx/s
sparteine' and an alkaloid, which in all probability is
dehydrolupanine were identified; b/ in extracts from aerial
parts of a fodder population of white lupin sparteine, lupa-
nine, hydroxylupanine? hydroxylupanine ester, angustifoline
were identified; the occurrence of three other alkaloids
was established; the first one is probably dehydrolupanine,
the second behaves chromatographically like multiflorine
but differs from the latter in UV; the third one has a double
bound Cue and can be reduced to sparteine, but it is not 4Nr...
dehydrosparteine.
According to the hypothesis of Shapf /1951/, Mothes /1955/,
Hegnauer /1958/ the interconversion of lupin alkaloids proceeds
from lower to higher levels of oxidation /e.g. lupanine
hydroxylupanine Wiewi6rowski and Reifer /1961, 1962/ postu-
lated a reverse direction in this interconverslon /angusti-
foline...--u.hydroxylupanine...--u sparteine/.
It must be stressed that the toxicity of various alkaloids
is different /sparteine> lupanine;?. hydroxylupanine
Up till now lupanine has been considered as the main /domi-
nant quantitatively/ alkaloid in bitter as well as fodder
white lupin. If the hydroxylupanine ester is taken into account
the total amount of hydroxylupanine in plants may sometimes
even be equal to the content of lupanine /the total content
of alkaloids is till now the main criterion for t'sweetness"/.
Our previous investigations on alkaloid metabolism in
fodder and bitter forms of lupinus album indicated, that spar-
teine /injected into the plants/ could be converted - among
other alkaloids - to lupanine? and lupanine to hydroxylupanine.
But 1/ the identification of these compounds was based only on
paper chromatography results; 2/ the intermediary /dehydro/
products could not be isolated. The main aims of the reported
investigations were a/ to obtain enzymatic extracts from
bitter and sweet populations of white lupin, extracts which
might be able to transform lupin alkaloids; b/ to isolate and
identify the products of enzymatic transformation in vitro of
some alkaloids characteristic of the investigated species,
3/ to investigate the patterns of transformation of sparteine
and lupanine /and of the products of their transformation is
vitro/ in plants of bitter and fodder white lupine, d/ to
compare these two forms as regards the character of alkaloid
interconversion in vitro and in vivo,
x/ multiflorine called by us previously ax
hydroxylupanine ester - called by us az or /oxylupanine ? /;
xx/ previously identified only chromatographically. Sparteine
and hydroxylupanine ester will be discussed latter.
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or ;11,1
f0t r-g
f.ji011, .1
E erimentai roeeduress A. Enzymatic extrects were btained
fro er a par s o -4 weeks old plants of fodder /var.
Przebedowska/ and bitter /var.Czeehnicka/ population of lupi-
nus albus. Among viue applied moth ds of extract purific
tion the method Of extraction of aceton po der with ph phate
buffer followed by fractio ated protein sedimentati n with
/NH4/004 was eh sen for further experiments. The extracts
showed after dialysis the highest enzymatic activity - 3.6eaM
of transformed sparteine per 1 mg of protein per h / xtract
obtained in other ways - 0.13 - 0.22 uld per 1 mg of protein
per hour/. The rate of transformation of lupanine abo t
)0 and of hydroxylupanine about 60% higher. The re ult were
the same for extracts from bitter as well as from fodder plants.
The influe ce of temperature9 pH, of inhibitors and ctiva-
torso of the concentrati n gf substrates wa investigated;
temperature - from 0" to 60"C9 pH - fr m 5 t 99 the concen-
? tration /of pa tin, lupanine9 hydroxylupanine/ from 0.1 to
13 uM in 1 ml; inhibitors and activaters /sparteine or lupe-
nine/schloridess NO, Ag+, Ca, Mg449 Me+p Co449 ZW140 Cur44o
He+ and B; N F0 C Pp H202, N82A802, KN, NaN3_in nc.
10-5to lenfi. The influence of ATP, ADP and DPN w-s also invee
stigated.
Substratwfs sp rteiney 1upanine0 hydroxylup ine0 angusti-
foline9 me1eiflorine0 17-oxosparteine0 17-oxc1upanine0 hydro-
xylupanine enter nd lupinine.
B. 1/ The transformation of sparoeine0 lueani e and hydroxylu-
panine by un urified en z matic extract from bitter and fodder
plants of rup nusib0 y roxy TiTifine was applied On lebelled
form /r14/ after 16 hrs of iiicubaici with buffered extracts0
the so1ve-4t w a evaporated9 the renidue dieeolved in 75% ethanol
and chromatographied /paper chromatographei with,n-b tanol s
HCl* toluen. The paper was cut in stripe cof 2 to 4 mm in width,
e1usted9 th radioactivity aed alkalvid content in each eluate
as determined; 2/ unpus,,ified enzymatie extracta ere sbtained
from aerial parts /5eg/ ofaPhaseolue velg rise Vicia faba0 Nico-
tiana glauc g Zee may 0 Spic e oleraoe ad Lupinus lut us -
their activity /sparteine as substrate/ w a comp red with the
activity of an analogous extract obtained from plants of foddez.
white lupin.
C. Identification of produete of enzymatic transformation i
vitro of some lupin alk loide. Spartein /300 mg/ was incubated
with a purified enzym tie extract from bitter white lupin; the
product f transf rmation after separ tion on a celulose column
/cton i HC1/ wa investigated chromattgraphical1y9 in IR
-
spectrometer, crystallized as picrates /1/9 m.p0 determined;
reduced with H2 /Pt02 in n HC1/. 10 mg of the base /picrates/
obtikd from sparteine incubated with an enzymatic extract
from fodder white lupin was added to 10 mg of picrate and
the depression of the m.p.? was investigated. To 1 mg of T-label-
led product of enzym tic transformation of sparteine /in extract
fran fodder plants/ 10 mg of unlabelled product9 obtained after
incubation with an extract from bitter form9 was added. After
crystallization /picratts/ the specific radioactiVity was deter-
min d feryetalle ad solation/. ?
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A similar procedure was applied in the attempts of identifi-
cation of products of enzymatic transformation in vitro of lu-
panine /labelled with 014/, hydroxylupanine and angustifoline.
Products of enzymatic transformations of lupanine, multiflorine,
17-oxosparteine, 17-oxolupanine and of hydroxylupanine ester
were investigated only chromatographically and in UV.
D. Biosynthesis of C14 and T-labelled lupin alkaloid0: 0.1 M
water solution of 1-C14-cadaverine /total activity 1 mc/ was
introduced to 10 plants of the bitter variety /0.5 ml per plant/
at the stage of bud formation. In the same way 5 mg of HJ-spar-
teine /total activity 687.54uc/ was introduced to other 10 plants.
After 10 days the alkaloids were extracted, purified, separated
/column chromatography!, and sublimated or distilled in vacuo
at 10-,T.
Lupanine - twice crystallized from n-hexane; to 1 mg of C14-
1upanine 19 mg unlabelled lupanine was-added, twice crystallized,
specific radioactivity determined.
Hydroxylupanine fraction - in order to eliminate dehydrolupanine,
this fraction was sublimated four times at 145?C and 10-'11
before sublimation 100/ug was reduced with H2 and the dehydrolu-
panine content was determined /hydroxylupanine gives hydroxyspar-
teine, dehydrolupanine sparteine/.
Multiflorine fraction /which may contain dehydrosparteine/was
distilled between 145 to 166'C 0 10-3T; the specific radioacti-
vity of particular fractions /5'C differences in distillation
temperature/ was determined.
Angustifoline: twice distilled to 170?C at 10-3T.
Hydroxylupanine ester - twice distilled to 165?C
Sparteine - /only T-labelled/ - after elution from the celulose
column - was dilutgd with 15 mg of unlabelled sparteine. After
distillation at 98'C radioactivity determined. From T-sparteine
after incubation with an enzymatic extract from fo44er plants
T-dehydrosparteine was obtained. In the same way C'4-dehydrolu-
panine was also prepared.
2 1r gas flow counter /frn the firm Frieseke and Hoepfner/ was
used. Its efficiency for C14 was 35 and for T 2.2%.
E. Transformation of sparteine and lupanine in vivo.
1/ Identification of dehydrosparteine and dehydrolupanine in
plants after injection of sparteine and lupanine.
a/ Dehydrosparteine. 50 plants of the fodder population
received 15 mg of sparteine /per plant/ as sulphate
After 14 days alkaloids were extracted. The fraction of Ri of
dehydrosparteine was chromatographied /on celulos2 columd/ kth
acetons in 1101; the alkaloid was distilled at 120'C and 10-3T
and compared in IR spectrometer, chromatographically and chemi-
cally with the product of enzymatic transfortation in vitro.
A part of the fraction
drosparteine /181/ and
b/ 241. E9441141
troduce3 1no p an s
killed /-60 C/ after 2
graphically separated.
was crystallized as picrates with T-dehy-
the specific activity was determined.
. 2 mg of C14-labelled lupanine were in-
of the fodder variety. The plants were
hours. Alkaloids extracted, chromato-
The fraction of Rf of dehydrolupanine
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MAL "lia
/the same Rf am the Rf of hydroxylupanine/ was eluted adio-
*
activity measured; reduction with H2/2n HC1 and Pt02/0 Pr ducts
of the reduction - after addition of a small amount of unlabel-
led sparteine - separated chromatographically and their radio-
activity determined. ?
2/ Products of tranzformation of T-sparteine9 C14 lupanine and
their dehydroforms in plants. Into 49 days old plants of the
bitter and fodder population of Lupinus albus HJ-sparteine9
?
C14 lupanine and their labelled dehydroforms ob ined enzyme-
tically in vitro 'were introduced. The amount of njected alka-
loid varied between 0.1 to 0.5 mg per plant in the f.dder and
0.3 - 3 mg in the bitter variety.
The plants were analyzed after 29 249 96 hrs. The alkaloids
extracted 9 separated on paper9 eluted 9 radioactivity and con-
tent determined. The fraction of Rf of dehydrosparteine./the
same Rf as the Rf of multiflorine in bitter and ax in fodder
var./ transferred into chloroform9 heated f?i? 3 min to bop?
After evaporation of chliroform the alkaloids were sublimated
to 14509 activity and quantity determined. The differences
between the results before and after, eublimation indicate the
amount and the activity of dehydrosparteine. The fractions of
radioactive lupanine /T-Iabelled/ was investigated by means of
isotope dilution with unlabelled lupanine. The fractions of Rf
f dehydr lupantne from plants9 which were injected with C14-
alkaloid as redueed with H2.
F. 1/ Synthesis of 1-C1 '4
-eadaverine
1C14-eadaverite was obtained /at micro-scale/ as follows
14 NW 0114 H904 14 CH3ON
I BaC 03 --a. KC N -ea.> HC N ----
K
II NaC14N Br.CH2.0H2.0H2 CN NaBr C14NCH2.0H2.0H2.0N
He
1 14_
III C4N.CH2.CH2.CH2 CN Tre- 0Le
e, CH CH H H NH
? 42? 2? '2 2? 2 2
.Lvy4
NaC14N
the obtained cadaverine was purified; crystallyzed; the m.p4 and
activity determined.
2/ Preparation of H3 - sparteine.
25 mg, of unlali)elled sparteine /chloride/ reacted with 0.5 ml
THO /with Adams red.catala at 100?C for 48 hrs. THO having been
distilled9 1 ml H20 was added and afterwards distilled9 this
procedure was repeated four timeS. The residue was dissolved ine
5 ml 0.5 n Na0H9 filtrated through glassefiber and heated at 95'C
for /0 min. The solution was cooled to eC9 sparteine extracted
with chlorofo-em9 which afterwards was distilled off? 5 ml 0.5 n
HC1 was added and again heated. H3-sparteine was extracted with
chloroform evaporated; 5 mi 0.5 n NaOH added and the
described above procedure w-=o repeated five times9 the radioacti-
vity of each chloroform extract having been determined. T-spar-
teine was twice crystallized as-picrates9 afterwards converted
into chlorides. TO, 0.5 ma of labelled sparteine 25 mg of unlabel- ,
led sparteine was added, crystallized and the radioactivity deter-
mined.
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Results: I.Enzymatic transformations of alkaloids in vitro.
The optimum temperature for the enzymatic activity of extracts
from both investigated populations lies for sparteine beIween
30-450C, for lupanine and hydroXylupanine 25-400C. At 57'C the
activity accounted only for 25% of its maximum.
The optimum of pH ranges between 6.6 - 7.7 for the three inve-
stigated substrates, the extracts from bitter plants reacted
to the changes of pH similarly as the extracts from the fodder
ones. Under optimal conditions of temperature and pH the in-
fluence of alkaloid concentrations was investigated. The opti-
mum of sparteine concentration was 2 - 4.5 of lupanine conc.
2 - 704 of hydroxylupanine conc. 3 - 8,11:M/m1 - in both kinds
of enzymatic extracts. In further investigations /quantitative
in character/ 31t0/m1 of particular alkaloids was used.
Inhibitors and activatust Na+ and Ca++ - no influence on the
++
enzymatic actlyityA_Mgmtyg, end Zn4+-increased the activity
about 20%; Co',CU", Ea"' Ag' in conc. 10-3U - inhibition
about 40%, in conc. 10-2 - 100%. Versene had no influence,
NaAs02 /10-3/ - did not cause any inhibition, 11202 at any con-
centrations did not influence the activity of extracts. ATP at
conc.2.10-4 to 3.10-3 M - increased the activity about 30%, but
at 10-2M caged a marked inhibition. ADP . no influence; DPN
at 2.5 . 10-111 increased the activity about 25% The addition
of activators or inhibitors to extracts previously boiled did
not cause any changes in the substrates applied. The results
obtained with both kinds of extracts were the same.
Table 1 and 2 represent the results obtained with purified
and unpurified extracts from fodder and bitter white lupins:
Table 1
)UM of the transformed substrate in 1 ml of enzymatic
extracts per hour
Extracts before
from plants spar eine
fodder 1 0.8
bitter 7 0.06
sic after dial sis
upanine spar e ne upan ne
0.09 I 0.5
1.2
1.0
The unpurified extracts from bitter lupin showed a much
lower activity than analogous extracts from fodder lupin.
Dialysis of "bitter" extracts increased their activity to the
level, observed in the "fodder" extracts.
Table 2
The influence of juice from fresh bitter plants on the
activity of extracts from fodder plants /substrate:sparteine/
ml of juice
added
activity of the extract/5/ after
addition of
fresh juice
0.0
0.1
0.2
0.3
0.4
0.5
100
100
100
85
80
70
fri
boiled julce
100
100
90
82
68
50
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rim avliAll!, unLY
- 9 -
The activity of extracts from fodder plants was lowered
not only by the fresh but also by the boiled juice obtained
from bitter plants. The addition of a mixture of alkaloids
/characteristic of the bitter population/ to the investigated
extract did not have any influence on its activity.
In the case of sparteine and lupanine the total amount of
alkaloid introdaced the product of its transformation in
unpUrified as well as in purified extracts was the same as
,the amount applied. However in the case of hydroxylupanine
the total amount of both bases after 16 hrs of incubation
with unpurified extracts.was about 50% lower than the amount
applied /in purified exacts there was no difference/.
Additional tests with C14-hydroxylupanine showed that about
50% of the radioactivity applied could' be found in compounds
which did not react with Dragendorffs reagent. .
The activity of extracts from fodder white lupin plant
/100%/ was compared with the activity of extracts from other
species /substrate: sparteine/: Nicotiana glauca 09 Zola
mays - 0, Phaseolus'vulgaris - 15, Viola faba 200 Spinacea
oleracea SO, Lupinus luteus /fodder/ - 60%.
Fractionation of proteins of the extract from fodder whit
lupin on hydroxylapatite column showed that only fractions
elated with buffer solution from 0614 to 0.22 M are 'active.
The maximum of activity was manifested by the fractio eluted
with 0.16 M /11.5seM of transformed substrate per 1 mg of
protein per hour/.
The identification of product of alkaloid transformations
in vitro gave the following results:
1/ sparteine - in extracts from both forms of hits lupin -
yielded dehydrosparteine /the only product/, m.papicrates/
204-206?C; this dehydrofor% however is not D5-dehydrosparteine
/m40-picrates - 189 - 190C/.
2/ lupanins Yloslded /in both extracts/ dehydrolupanine /onlY/9
but it is not D"-dehydrolupanine or ,'ehydrolupanine found by
Rink and SchAfer /1954/ in seeds of Luoinus perennis.
3/ hydroxylupanine - yielded dehydroxylupanine.
4/ angustifoline dehydroangustifoline.
Lupinite, 17-oxosparteine0 17- xolupanine, hydroxylupanine
ester and multiflorine /the latter investigated in extracts
only from fodder plants/ - did not change during incubation with
enzymatic extracts.
II. Transformation of alkaloids in vivo.
The degree of incorporation of C14-cadaverine into alkaloids
in plants of bitter lupin after 10 days accounted only for 0.7%,
while the incorporation of T-sparteine amounted to 50%. In both
cases - all isolated alkaloids were radioactive, the distribution
of the radioactivity between the investigated bases was similar,
and the quantitative proportions betweem them were in -e-leral
analogous to the proportions for radioactivity, except Lor the
multiflorine0 whose participation in the total alkaloideoacti-
vity was higher than i the total alkaloid content. These facts
show - among other things - that T-sparteine after chemical re-
moval of easily exchangeable hydrogen - can be used with good
FOR OFFICIAL USE OM
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- 10 -
results for labelling other lupin alkaloids in spite of the
fact that hydrogen atoms bound with the sparteine skeleton
are "attacked" by enzymes more easily than carbon in this
skeleton.
An intense transformation of sparteine into other alkaloids
was found also in plants of the fodder variety.
The isolated fraction, whose Rf value was the same as the
Af of dehydrosparteine after distillation to 120'e yielded
an oily substance. The chemical analysis, paper chromatogra-
phy and IR spectrum showed that it was dehydrosparteine iden-
tical with the dehydrosparteine obtained enzymatically in
vitro. The fraction, in which besides hydroxylupanine dehydro-
-xylupanine may also be present, was isolated from plants,
which received 014-lupanine and two hours after injection were
killed.
Table 3
Radioactivity of the fraction investigates c p m
Plants
1111?1????114101,
e ore re uc on
er re uc on
rac ion 1 o y ro- rac on
xylupanine and dehy- Rf of
drolupanine sparteine
= =
rac ion
RF hydroxy-
=spa/stein% .
1
2 300
2 100
40
2
1 450
1 520
0
3
2 880
2 640
0
The results obtained after reduction with H2 /tab.3/ indicate
that the product of lupanine transformation in vivo is dehydro-
lupanine. This conclusion was confirmed by other analysis.
Results obtained in the experiment with bitter lupin plants
2, 24 and 96 hours after injection of alkaloids:
1/ During the time under investigation the plants did not change
their total radioactivity. A part of the activity could be found
in nonalkaloid compounds. This part was always higher in plants,
which received dehydroforms of the investigated bases /35 and
29% as compared to 11 and 12 per cent, after 96 hours/.
2/ All injected alkaloids almost immediately were transformed
partially into other alkaloids. The degree of transformation
of dehydroforms especially of dehydrolupanine was much higher
as compared to the saturated bases. The rate of transformation
was increasing during the time under investigation. After 96
hrs: a/ the injected sparteine was transformed into: dehydro-
sparteine 12%, lupanine 9%, hydrolupanine + hydroxylupanine
ester - 3% and multiflorine - 3%; b/ the injected dehydrospar-
teine: lupanine - 17%, hydroxylupanine + its ester - 10%, multi-
florine - 10%. 8S-:, 'the radioactivity applied could be found also
in sparteine; c/ the ipjected lupanine: dehydrolgpanine 4%,
hydroxylupanine + its ester - 18%cmultiflorine4 6%, sparteine
did not show any radioactivltY; d/ dehydrolupanines hydroxylu-
panine + its ester - 31%, multiflorine 10%; 10% of the radio-
activity applied could be found in lupanine./About the same
4, -teevalueVwes obtained 2 bpp 'after injection/; no radiactive spar-
. teine was found.
In experiment with fodder lupin plants:
1/ the total radipctivity of plants during 96 lire did not
change. The radioactivity found in non-alkaloid compound$:
x/ not very well purified
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VU Lt Ulia ?
after a/ sparteine 30%9 b/ dehydrosparteine - 44%9 c/ lupe-
nine - 20%s d/ dehydrolupanine 38%.
2/ the transformations of injected alkaloids into other bases
investigated after 96 hrss a/ sparteines dehydrosparteine
21%9 lupanine 2%9 hydroxylupanine + its ester - 1%. Rf
value the same as the Rf of multiflorine 1%; b/ deLydfio-
sparteines lupanine - 4%, hydroxylupanine + its ester - 3%9
ay - 0.3%. 7% c uld be found in sparteine /the same value was
obtained 2 hrs after injection and did not change afterwards&
c/ lupaninesdehydrolupanine - 8%9 hydroxylupanine +1t8 ester -
9%; y 0.3% /sparteine did not show any radiactivity/0
d/ deHydrolupanine hydroxylupanine + its ester - 37%; a,
14% 9% was found in lupanine. No radioactive sparteine was
found
DISCUSSION
The results concerning the purified enzymatic extracts from
bitter and fodder plants of Laoinus albus show that they are
very similar in their reaction to changes in temperature, pH,
the kind'of s bstratess their concentrations and to inhibitors
and activators investigated. The similarity of these extracts
was also revealed in the facts that their enzymatic activity
led to the formation of only one products which did not undergo
further transformation; this product was always a dehydrofor
of the alkaloid applied. Thus it can be assumed that enzymes,
which ceased these transformetiane, wer lehydrogena es of ana-
logous characteristics in both investig ted for:se of white
lupin.
The investigated extracts from both: kinds of plants showed
some differences in their activity depenAinie on the substrate
used. But thi fact is n t yet proof that the extracts con-
tained several dehydrogenases a It is keewns that same dehydro
-
geneses of a very little specificity oey show different activi-
' ty depending on the kind of the eubetsste applied. The enzyma-
tic activity of extracts fror other spscies indicates s that de-
hydrogenases, which have affinity to the investigated compeunds
are rather widespread. The behaviour Ktf hydroxylupanine in un
purified extracts indicate that this OR loid as compared to
sparteine and lupanine is much more labile /this was confirmed
In our other investigational.
The results obtained in vivo show that the interconversion
of alkaloids 'proceeds mainly from a lower to a higher level of
oxidation. The facts of transf rmation of dehydrospartein
into sparteine and of dehydr lupanine into lupanine shows that
revers direction of interconversion is possible; these facts
how ver may be an "artefact" resulting from the technique
applied /injections ?translocation n one hand - localisation
ortransformations in intact plants on the other hand/.
, It is possible th t the low content of alkaloids in fodder
white lupin results not only from a very low rate of synthesis
from nonalkaloid precursors /this was established in our pre-
vious works/ but also from the low rate of interconversion of
the alkaloid themselveso It must be added that the transfor-
mation of introduced alkaloids into nonalkaloid compounds in
fodder plants was greater than in the bitter ones /but the
difference in isotope dilution could also play a role in this
phenoman n/. om?.
UOr Aiis
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- 12 -
The results obtained in vivo and in vitro permit the
assumption that the general pattern of alkaloid transforma-
tion in white lupin is as follows:
alkaloid /introduced/ dehydroform -.N. another alkaloid
nonalkaloid compounds
The interconversion of the investigated alkaloids:
sparteine
dehydrosparteine multiflorine /bitter variety/
lupanine
dehydrolupanine
hydroxyl upanine hydroxylupanine ester
dehydrohydroxylupanine
CONCLUSIONS
1/ Enzymatic extracts from bitter as well as fodder popu-
lations of white lupin are able to transform sparteine, lupa-
nine, hydroxylupanine and angustifoline i.e. alkaloids charac-
teristic of the species investigated. The transformations are
catalyzed by dehydrogenases present in the extracts. The best
method of enzymatic extract preparation was protein sedimen-
tation from aceton powder with /NH4/2SO4 /33-66% of satura-
tion/.
2/ The highest enzymatic activity in purified extracts from
fodder plants was found in the protein fraction, eluted from
a hydroxylapatite column with 0.16 - 0.18 M phosphate buffer.
The amount of this fraction accounted for less than 3% of the
total protein content in the extracts investigated.
3/ The optima of temperature, pH and of substrate concen-
tration /sparteine, lupanine and hydroxylupanine/ for extracts
from plants of both investigated varieties are analogous.
4/ Sparteine, lupanine? hydroxylupanine and angustifoline
were transformed by purified enzymatic extracts - from bitter
as well as fodder plants - into their dehydroforms.
In unpurified extracts hydroxylupanine only underwent trans-
formations 91so to other compounds, which do not react With
Dragendorff's reagent.
5/ Sparteine and lupanine after injection into plants of
bitter and fodder lupinus albus are transformed into other
alkaloids: sparteine is transformed mainly into dehydrospartei-
ne, lupanine and hydroxylupanine; lupanine : into dehydrolupa-
nine and hydroxylupanine; dehydrosparteine and dohydrolupanine
after injection show w similar pattern of transformation, but
they can be partially converted also into sparteine and lupa-
nine respectively. Lupanine and dehydrolupanine were not trans-
formed in any case into sparteine.
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t.;
t
FOR MAL USE MY
6/ One of the products of sparteine and dehydrosparteine
transformation - after injection into bitter plants - is also
multiflorine.
7/ The character and the rate of transformations of spartei-
ne, lupanine and their dehydroforms indicate, that the inter-
conversion of these alkaloids in plants of bitter as well as
fodder white lupin proceeds mainly from lower to higher le-
vels of oxidation.
On the basis of the results obtained in the described expe-
riments it seems worthwhile to continue investigations 1/ on
the dehydrogenases from,bitter and fodder plants, 2/ on trans-
formations of other alkaloids /besides sparteine and lupanine/
characteristic of the species investigated, 3/ on the alkaloid
a, in fodder lupin. /We must interrupt the investigations men-
tTioned in p.1 for 8-10 months, because Mr Nalborezyk went
abroad/.
.II.. The synthesisof alkaloidsA.n bitter white lupine.
.Our previous investigations /195859/ on bitter white lupine.
./var.Czechnickaishowed, that the greatest increase of the alka-
,
bid in plants occurred during a. relatively short time, in the
period of. side shoot flowering and pod formation on the main
shoot. During the subsequent period of growth the. alkaloid con-
tent did not change. Very similar facts were observed in the
fodder variety /Pretbedowski/.
The aims of the reported investigations were:
a/ to determine the role of partinuTar aerial parts in the bio-
synthesis of alkaloids at the time of the-greateet alkaloid in-
crement. /the insignificant role of roots in this pease was
Shown in our earlier investigations/r.
b/ to identify two alkaloids whose chromategraphie behaviour
MEW similar to 1/ sparteine, 2/ oxolupazine-/ealled previously
by us az or /oxylupanine
Cl to examine the total and specific radioactivity of particu-
lar alkaloids in order to undeetand tbeir interconversion better.
In.1960 pot experiments were carried out: on the 54th day
of growth /beginning of side shoot flowering/ following treat-
ments were applied: 1/ control; 2/ 5 upper leaves removed;.
3/ side shoot ? leaves removed; 4/ pods from the main shoot re-
moved. On the same day a part of the plants investigated /from
treat,10 3 and 4/ were exposed to C1402 for one and two weeks
in aslexiglass chamber /750 1 vol./. The plants in the green-
house were harvested several times at weekly intervals. Aeeor-.
ding to the results obtained the contributions of side shoots
to the accumulation of alkaloids amounted to 57-70%. The redia-
ctivity of lupanine accounted for about 80% of. the total alka-
loid activity. But in this experiment the removal of particular
_plant starts brought about a reaction of the remaining vegeta-
tiVe organs, consisting in a more intense growth and in a grea-
ter rate of alkaloid synthesis. The greatest alkaloid accumula-
tion, in control plants was observed not between. 56 and 70 days
of growth /as in the experiment 1958/ but 10-14 days later.
Therefore in experiments 1961 and 1962 the technique was changed.
Experimental procedure! 1/ experiments 1961 seeds o bitter
white lupin were sown.in pots'on april 24. On the 62n day of
growth the plants were divided into several eroups. In the
FOR CMCIAL USE n'ILY
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first group some plants'were deprived of 1/ side shoots or
2/ p ds. They were harvested together with controls after
7 days. On the day of the harvest the eame treatments were
applied in the second group. The next harvest was compl ted
also after 7 days. On the 76th day of plant growth the expe-
riment was repeated in the same way. The last harvest as
completed at full maturity. On the 66th and 75th day of
growth analogous treatments were applied and the plants were
exposed to C14020 After 24 hrs they were killed /-600C and
analysed. In the first experiment with labelled carbon ex-
cised stem and pods were also investigated.
2/ experiment 19628 bitter white lupin was sown on February
23. On the 63rd day of growth plants were exposed to C1402
for 3 hrs; than a part of them was harvested; from the re-
maining plants pods and stems were cut off and exposed to
01402 for 21 hrs.
In the plants harvested in the greenhouse total alkaloid
content only was determined. Alkaloids extracted from th
radioactive plants were separated on a celulose columntin-
butano18 0.In Hal/ and distilled /oresublimated/ at 104".5T;
total and specific radioactivity was determined?
From sid shoots of lupin investigated in 1960 alkaloids
were extracted and separated on a celulose column. Fractions
corresponding chromatographically to sparteine hydroxylupanine
and 17-oxolupanine were isolated s purified s distilled in
vacuo and analysed in IR-spectrometer.
RESULTS AND DISCUSSION
I. Identification of three alkaloid fractions
a/ The amcunt of the fraction whose Rf value was similar to the
Rf of sparteine accounted for only about 0.5% of the total
content in the investigated side shoots; after distillation at
90-950C it yielded an oily substances whose IR absorption spec-
trum was identical with the IR spectrum of sparteine prod,
aEFARM and with the IR-spectrum of this bases published by
Leonard and Beyler /1950/.
b/ The alkaloid fractiqns whose Rf value was similar to the
Rf of hydrexylupaninex/ was sublimated at 130-140?O. Its IR- tti
spectrum showed the same maxima of absorption /-0H at 3500 cm 9
-a00 group in lactamat 1630 amtl/ and the same vibrations in
the "finger print" region as hydroxylupanine isolated from
seed, of bitter lueinus angustifolius /the hydroxylupanine
fr4m the fodder variety differs somewhat in the"finger printn/t
c/ The IR absorption -spectrum of the purified alkaloid fraction
whose Rf ws similar to the Rf of 17-oxolupanines, was analogous
to the IR spectrum of an alkaloid isolated from vegetative
organ of fodder plants of lupinus albus and identified as hy-
droxylupanine, ester. The investigated fraction after hydroly-
sis in Hal yielded hydroxylupanine,
x/ This fraction contains - in very small amounts - a base
which in all probability is dehydrolupanines but the puri-
fication procedure applied causes its decomposition.
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:
- 15 -
II. According to the results presented in tab.40 the alka-
loid increment during the first week amounted to 37% of the
content, found on the 62na day of plant growth. During the
subsequent period /till full maturity/ there were no signifi-
cant changes in the total content of the investigated bases.
After the first week of investigation the reaction of the
plants to pod removal can be clearly seen /a more intense
growth of side shoots!. But the same treatment applied a week
later did not cause any marked changes 4 The removal of side
shoots in the first period brought about a very small alkaloid
increment whereas the removal of pods had no influence on the
total increase of these compounds.
The results of the first experiment with 01402 /table 5/
show that g 1/ the contribution of side shoots to the total
carbon assimilated was about 70%, 2/ the plants deprived of
pods were less radioactive than the controls /the same pheno-
menon was observed in the experiment 1960 and in an additional
experiment in 1962; the diffgrenee - in comparison with the
control - in the amount of C1-4 translocated to the roots could
not havel-been considered as the main cause of the difference
in the total radioactivity of the aerial parts!; 3/ the amount
of CO2 assimilated only by pods accounted for 1.5% and assimi-
lated by the stem - 0.3% of the total.
Similar results concerning the side shoot contribution to
the total CO2 assimilation were obtained in the experiment .
carried out one week later /tab.6/. However it must be added,
that the rate of photosynthesis was less /002 eontent and
activity, conditions of temperature and light were very similar
in both experiments/.
The total alkaloid activity amounted in the first experiment
to about 0.29%, in the second one to about c'e03 per cent of the
total plant activity. If the difference in the rate of ph tosyn-
thesis is taken into account, thus the Alkaloid synthesis in
the second experiment can be considered as very low, less than
10% of the synthesis rate found one week earlier?
The side shoots were the main organs, in which alkaloid syn-
thesis occurred during the period under investigation; in the
first week their contribution to the C14 alkaloid accumulation
was about 90%. Although the total alkaloid activity in plants
deprived of pods was less than in the controls, but it amounted
also to about 0.30 per cent of the total plant activity.
According to the results obtained in the experiment 1962 /table
7/ it may be Concluded that g 1/ pods are not able to synthesize
alkaloids /from nonalkaloid compounds/at least.ct the investi-
gated stage of their development, 2/ the stem ofvhain shoot
show this capacity, but the participation of alkaloids - formed
after 21 hrs - in the total stem activity /about 0.03 per cent/
is very low in comparison with their participation in stems of
intacts plants /tables 2 and 3/. Therefore it can be assumed
that in the latter the most part of Cl4alkaloid found in Otems
resulted from translocation from other organs, mainly frioin the
side shoots.
The alkaloid content in pitticular plant parts, of 0 and
69 days old.plants /table If and the'lletribution oi alkaloid
activity among these parts /table 3/ acv, ,L,ett the riew formed
rn1 pin
lUil? dr 4
Fat&
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alkaloids /at least those derived from the products of current
phetosynthesis/ had been translocated during 24 lire fran side
shoots and leaves of the main shoot at a greater rate than
the alkaloids earlier accumulated in these plant organs.
The radioactivity of side shoot alkaloids in particular
in plants deprived of pods can give seme informatiOns about
the relationships between the alkaloids investigated 1/ the
specific /per mg/ activity of lupanine was 3-5 times higher
than that of hydroxylupanine /whose content accounted only
for 1/3 of the lupanine content/ and multiflorine. In parts
that show a mall capacity of alkaloiel,synthesis, e.g0 leaves
of the main shoot or in parts whose 0'4 alkaloids derived
mainly from other organs e.g0 the stem the specific activity
of the hydroxylupanine ester /which included also the activi-
ty of the bound organic acid/ was very high and sometimes -
even greater than the specific activity of lupanine.
It is possible that a part of hydroxylupanine was bound
in the stem with an organic acid of a relatively high radio-.
activity.
The results concerning lunanine and hydroxylupanine /con-
tent and activity/ indicate that the interconversion of these
two alkaloids,- under conditions, where no injection of an
alkaloid takes place - would proceed from the lower to the
higher level of oxidation. The reverse 'direction would have
been probable only in the cas when the rats of'Cl4 -hydroxy-
lupanine transformation into lupanine had been so high that
the specific activity of hydrexylupanine in,the side shOots
oculd not have been signifioant0 It is difficult to accept
this possibility, when the quantitative relationships between
the two alkaloids /not only in this experiment but also in
ee,x previous investigations/ are borne in mind.
CONCLUSIONS /ineluding the results of the first experiment/0
1/ The greatest alkaloid ccumulation in titter white lupin
occurs during two to four weeks after the beginning of side
shoot flowering /this feet confirms our previously obtained
results/.
2/ At this time the side shoots are mainly responsible'
for the alkaloid synthesis. At the first stage of accumulation
their contribution accounted for 70 and in the later period
for about 90 per cent of the alkaloid increment.
3/ At the late stages of growth the total alkaloid content
in plants does not underg any marked changes. This is in a/1
probability the result of inhibition of synthesis as well as
of decomposition of these compounds.
4/ The ageing process of leaves on the main as well as on
the side shots brings about a diminishing in the rate of alka-
laid synthesis.
5/ The pods were not able to syntheze alkaloids, the stem
of the main shoot showed this capacity but to a very mall
degree.
6/ At the early stage of pod development the translocation
f new formed alkaloids from leaves of the main shoot as well
as from side shoots is greater than the translocation of alka-
loids earlier accumulated?
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FOR OFF 0111110E W111
7/ On the basis of the infra red spectra of investigated
alkaloids a/ the occurrence of sparteine in side shoots /in
very small amount/ could be established, b/ the alkaloid of
the Rf value similar to the Rf value of 17-oxolupanine was
identified as an ester of,hydroxylupanine and an organic acid,
Cl the hydroxylupanine occurring in the side shoots was shown
tn be identical with the hydroxylupanine from seeds of Lupi
flue angustifollus;'
The interconversion between lupanine and hydroxylupanite
seems to proceed from the lower to the higher level of oxi-
dation.
? It seems worthwhile to repeat the experiment with older
bitter lupin plants,, when the total'alkalgid content does
not change, and to examine the rate of C04fincorporation int
alkaloids /an analogous course of alkaloid accumulation -
but different as regards the total amounts - was observed
previously in fodder white lupin/.
III. The described experiments drew our attenticm mainly
to the side shoots of the investigated plants. That s why in
the experiments with bitter and fodder homozygotes of lupinus
at the beginning of side shoots flowering the pods were re-
moved from the main shoots and the plants were allowed to grow
for another week in the greenhouse. Afterwards they were put
into a plexiglass chamber and exposed to C0402 for 4 hours.
A part of them was killed /-600C/ impediately and the remaining
plants were exposed for 20 he to 2 71'n they were killed
and have been analysized. We are now determinie6 "(le alkaloid
and amino-anid content as well as their specific activy. We
pay great attention to arginine.
IV. The plants /bitter and fodder homeeygotes/ grown in the
greenhouse were harvested several time- during the vegetation
periodp till full maturitye The weaV-)-r conditions were unu-
sual this year and perhaps this is tA- maiel,reason why a very'
early fall of leaves from the thajn as -Nell as from the side
shoots was observed? We are net contee with the size and
number of seeds we have obtained /the" are to be investigated
next year/. The harvested plants are analysed /alkaloids and
nitrogen/.
? Arnrimncv
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FOR OFFICIAL USE ONV
RESULTS
The dry weight and alkaloid content /per plant/
Table 4
f
Plant-
parts
Ae of plants /das/
62
,,,- 76
,
,-
Con
trol
don t pkarits depriVed:
, 'lents deprived
con
1 sIde 1 I -
trol, shoot34 pods trol
1 si e
4
shoots
.pods
dry
.weight - g dern. per
slant
!main
I shoot
!side
shoots
podsx/
total
3.01
1.62
0.66
3.06 3.08
1,70L
1.62 ' 1.12
3,03
2.28
-
3617
1.80
-.i.96
2.86
-
2400
3.16
1.85
5.29 6.38, 4.20
j
alkal.id content- m.:,
5.31
r slant
6.93
- _
/4.86
- - -
5.01
main
shoot
side
, shoots
ipods
!total
L
10.3
18.1
7.6
11.3 10.4
.
14.3 -
2347 111.6
f,
11.2
32.9
-
1.4
10.9
-30.0 ,29.3
6.6
12.3
14..2
36.0 49,3 i 22.0?
11JL
44.1 152.3
3569
4
,
26.5
x/ of the main shoots
xxi the total dry weight at the stage of full maturity was
7.46 g per plant.
t! U,
rto.ow' nITT. VV., 1 0,1 0
?
neclassified in Part - Sanitized Copy Approved for Release 2013/09/06: CIA-RDP80T00246A021000010001-6 .
Declassified in Part - Sanitized Copy Approved for Release 2013/09/06: CIA-RDP80T00246A021000010001-6
Treatment
plant
parts
alkaloid
content
mg
Radioactivity
'
of 69 day,psid plants and a
24 hr elrposure to 0'02 /per plant/ )-
m91--Le 5
Alkricid xx4.t1z:1_
t-6,1
plant -77.-PcJ7c_a6TTivi-17
ac'-ivity la hl rf -hl ],a
10-c m ostei
Control main shoot
leaves 2.2
stem ; 8.6
sids soot d 15.4
,pods 21.6
total
Side sho'',1-
Main sloot
removed leaves'
stem
Dods
-TbdS
remOvc(;'.
Main
, Main
I Pods
47.8
Eb5
499
1442
226'7
Vu
44C
(7,07
.56
66
125
165
Anq
625
116
15u
4873
2.0 746
8.2 251
20.3 5_58
93
62
19
90
63
19
121?
405
10
otrls after_
tota
mr I -hi
ester
250 8.4 71 18
446 2136 529 .575
223 .6282 179 10d
79 17')6 -1(4/ 572
10288 1316 1093
294
219
total
:0.) 115.5
maim shoot,
leaves r 2.1
stem , 9.0
side shoo L,4 18,0
total
stem excised
stem /on rcets/
ecised
758
787
2125
3670
15
12
72
180
238
(2.53
229
21
246
4.?
1075f,_
508 72
136 73
boo' 181
174,
30
227
yu
526
598
196
total
aihrloiet
activity
or totea
ninnt nctiv
203 0.03
3616 u.v2
7167 0,50
:508i 0.16
390
91
256
14v
494,
228 243
626 1()Y6
165 6762
8081
69
'43
662
1074
26
393
446
365
64
107U
-93
X/ In all experiments total plant radic activity vas determined
/mica window9 1.1 mg/cm 0 5 Luc,/ tht alkaloid activiy in a
Friesecke und npiner/ whose efiicieny was 3 L,Imes hihei
bias were divided by 0.
14Ut? 0.29
25V
810
46
0,t43
U.2
0.u8
150 0.10
402
2882.
3266
u.y5
0.37
0.79
0.31
a G,:iger-Liller coater
221 ;Sas-ziow count1,4._/:/
he data ot'uaincd Jur alLa-
la - lc anin.?9 hi - hydrc:cylanine, mi hl -ester-hydroxyl-epanine ester
/his fJac-wier ooe.atailis a minimal amount of an alkaloid or a similar Ri/5 the base -ay-
whin cr in laces was net aken intu.account anGustiioline -found in ielatively
verw small amount could no have been separated from Lupanine therefore its specific radio-
activity was net deuermined.
FOR OFFICIAL USE ONLY
Declassified in Part - Sanitized Copy Approved for Release 2013/09/06: CIA-RDP80T00246A021000010001-6
Declassified in Part - Sanitized Copy Approved for Release 2013/09/06: CIA-RDP80T00246A021000010001-6
01-1
VS "
Radioactivity of 74 days old plants and of their
alkaloids after 24 hrs exposure to C1402 /perplant/
Table 6
-
plant itotal total alkaloid acti
,. :alkaloid. an
parts plant L...___?11I?SLILII__
content : activityl.
io3c pm i sPepific total
1
A
'
alkaloid
activity
% of the
total plani
activity
Control main
Ishoot
Ileaves 0696 453
j stem ?9.03 197
side
1 shoots 14.37 697
pods 1179
total 52.86 2526
Side main
shoots shoot
removed
leaves 0.85
stem 8.98
pods 27.73
total 37.56
148 1 142 0.03
46 i. 415 0621
26 .374 0.05
8 228. 0.02
1159 0.04
392 152 137 0.03
62 9 81 0%313
'375 , 2 55 0.01
829
j 273-
0003
Pods main
removed stem
leaves
stem
side
shoots
0.90
13.20
12.63
total
?
?
=
26.73
[GI
729
543
882
2154
202 :182
53 1 700
35 F 442
1324
0.03
0.13 ,
0.05
0.06
Declassified in Part - Sanitized Copy Approved for Release 2013/09/06: CIA-RDP80T00246A021000010001-6
Declassified in Part - Sanitized Copy Approved for Release 2013/09/06 : CIA-RDP80T00246A021000010001-6
"It
FOR CFR AL USE ONLY
Radioactivity of alkaloids in the stem and pods
of the main shoot /per plant/
Table 7'
Time of
plant
fresh
weight
alkaloid
,
plant
alkalo?id a ctiy*
:harvesting
parts
g
content
mg
activity
,:103 c pm
,71E--2-2-41------
specific!
total
; After
main
3 hrs.
exposure
to C1402
shoot
' leaves + i
side
.
shoots -
10.18
1320
stem
4675
2693
282
9
26
: pods
1-0.65
11.78
183
0
0
total '
25.58
1785
?
,
After
subsequent
excised
21 hrs
exposure
stem
4.89
2-.88
263
40
115
' to C12?2
pods
11.10
12.49
192
0
0
= =
FOR CF
?
Declassified in Part - Sanitized Copy Approved for Release 2013/09/06: CIA-RDP80T00246A021000010001-6