Cyanogenic Glucosides In Linum Usitatissimum

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Pergamon PII] S9920Ð8311"86#99842Ð8

Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 48Ð52\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9920Ð8311:87:,Ðsee front matter

CYANOGENIC GLUCOSIDES IN LINUM USITATISSIMUM IRENA NIEDZłWIEDZł!SIEGIENł Institute of Biology\ University of Warsaw\ Branch in Bia
Key Word Index*Linum usitatissimum^ Linaceae^ ~ax^ HCN!potential^ cyanogenic glucosides^ linamarin^ linustatin^ lotaustralin^ neolinustatin[

Abstract*Cyanogenic glucosides were quanti_ed in di}erent organs of oil ~ax "Linum usitatissimum cv LCSD 199# plants at di}erent stages of development[ Monoglucosides "linamarin and lotaustralin# and diglucosides "linustatin and neolinustatin# appeared in developing embryos soon after anthesis\ but mature seeds accumu! lated only diglucosides[ Monoglucosides appeared again in germinating seeds and\ in young seedlings\ they were the only class of cyanogens[ High levels of linamarin and lotaustralin were found in leaves throughout the vegetation period\ but the highest amounts were in ~owers[ In contrast\ these glucosides occurred in relatively small amounts in roots and in stems[ The possible physiological roles of the changes are discussed[ Þ 0887 Elsevier Science Ltd[ All rights reserved

Linum usitatissimum is a cyanogenic plant of con! siderable economic importance[ The presence of large amounts of cyanogenic glucosides in seeds severely restricts the amount contained in animal feed[ The necessity to improve the commercial value of this spec! ies by reducing the content of cyanogenic glucosides is obvious[ Knowledge of their qualitative and quan! titative changes in di}erent parts of ~ax plants at di}erent stages of development is a condition for fur! ther research leading to modi_cation of cyanogen con! tent[

INTRODUCTION

The widespread distribution of cyanogenic glycosides in plant kingdom\ as well as some other evidence\ indicate that these compounds may play an important role in the primary metabolism of plants ð0\ 1Ł[ The content of cyanogens in plant tissues varies with age and developmental stage\ and can be greatly in~u! enced by seasonal\ nutritional and genetic factors ð2\ 3Ł[ Cyanogenic glycosides could function as nitrogen! containing precursors for amino acid and protein syn! thesis during seedling development ð4Ł or they could play a protective role against herbivores by liberation of free HCN from injured plant tissues ð5\ 6Ł[ Cyanide\ released from cyanogens\ may control seed devel! opment and germination in some species ð7\ 8Ł[ It can also induce the alternative respiratory pathway ð09Ł[ Moreover\ according to some evidence\ HCN controls nitrate reductase activity in Zea\ Sor`hum and Tri! ticum ð00Ł[ Fax plants "Linum usitatissimum# contain two major cyanogenic monoglucosides\ linamarin and lotaustralin\ aglycones of which are derived from L! valine and L!isoleucine\ respectively ð01\ 02Ł[ These two compounds\ without apparent exception\ always occur together in di}erent species\ although not necessarily in the same ratio ð03\ 04Ł[ The co!occur! rence of these two glucosides in linen ~ax is ascribed to the existence of a single set of biosynthetic enzymes ð05\ 06Ł[ Moreover\ ~ax seeds contain two cyanogenic diglucosides\ linustatin and neolinustatin\ the 5?!O! glucosides of linamarin and lotaustralin\ respectively ð07Ł[

RESULTS

The concentrations of particular cyanogenic glu! cosides were determined in seeds of oil ~ax "Linum usitatissimum cv[ LCSD 199# during germination\ in whole plants during growth and development\ and in di}erent separated organs[ In mature dormant seeds and during the _rst day following the start of imbibition only diglucosides "linustatin and neolinustatin# were detected "Fig[ 0A#\ the amount of linustatin exceeding that of neolinustatin 0[4 times[ The content of both diglucosides increased during the _rst 25 h of germination and then decreased to a nondetectable level on day 2 following germi! nation[ Monoglucosides "linamarin and lotaustralin# appeared in the seeds 25 h after germination and on day 1 they reached the same concentration as that of diglucosides "Fig[ 0A#^ subsequently they constituted the only class of cyanogens in growing seedlings and plants "Fig[ 0B#[ The amount of monoglucosides in the 2!day and 29!day old plants was 19 and 099 times 48

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Fig[ 1[ Concentrations of linamarin "# and lotaustralin "ž# in di}erent organs of ~ax plants at di}erent stages of development[ "**# leaves^ "Ð Ð Ð# stems^ "[ [ [# roots[

Fig[ 0[ Cyanogenic glucoside content in ~ax plants during ontogenesis] A\ in germinating embryos^ B\ at di}erent stages of growth[ Monoglucosides] " linamarin^ ž lotaustralin#[ Diglucosides] "D linustatin^ R neolinustatin#[

higher\ respectively\ than that of monoglucosides "and diglucosides# in 1!day!old plants "Fig[ 0#[ The con! centration of linamarin was twice as high as that of lotaustralin in all developmental stages and plant organs studied "Figs 0 and 1#[ The main site of accumulation of diglucosides in seeds are cotyledons\ in which the content of cyano! gens is 3 times greater than that in embryonic axes "Table 0#[ Nevertheless\ their concentration "cal! culated as the content per 099 g fr[ wt# was con! siderably higher in embryonic axes than in cotyledons[

Ratio of neolinustatin to linustatin was somewhat higher in cotyledons "9[5# than in axes "9[4#[ Leaves and cotyledons of 09!day!old ~ax seedlings contained the highest amounts of monoglucosides[ Their content in stems of the same seedlings was ca 3 times lower\ the lowest amounts being detected in the roots[ Similarly\ in 09!day!old seedlings\ the highest concentrations of cyanogens were found in leaves and stems exceeding several times those in roots "Table 1#[ Similar relationships were observed in the organs of mature plants during development "Fig[ 1#[ In leaves\ as well as in stems and roots\ both linamarin and lotaustralin were present^ however\ their con! centrations were di}erent\ depending on plant age and organ[ The highest concentration of monoglucosides was found in leaves\ slightly lower in stems "especially green ones# and the lowest in roots "Fig[ 1#[ The con! centration of cyanogens in young leaves of 09!day!old plants "2[9 mmol:099 g fr[ wt# was more than three times higher than that in leaves of 29! and 79!day!old plants "ca 0 mmol:099 g fr[ wt#\ and twice as high as that in leaves of 59!day!old plants[ Similarly\ stems of 09! and 59!day!old plants contained higher con! centrations of glucosides than those in stems of 29! and 59!day!old plants\ but the di}erences were less

Table 0[ Cyanogenic glucoside content in di}erent organs of ~ax embryos 13 h after germination Cyanogenic glucoside mmol:099 organs

mmol:099 g fr[ wt

Organ

Linustatin

Neolinustatin

Linustatin

Neolinustatin

Cotlyedons Embryonic axes

0[1929[00 9[2429[97

9[6229[98 9[0529[93

9[1029[91 9[3629[00

9[0229[91 9[1129[95

2s[d[ values indicated[

Cyanoglucosides in ~ax

50

Table 1[ Cyanogenic glucoside content in di}erent organs of 09!day!old ~ax seedlings Cyanogenic glucoside mmol:099 organs

mmol:099 g fr[ wt

Organ

Linamarin

Lotaustralin

Linamarin

Lotaustralin

Cotyledons¦leaves Stems Roots

53[028[5 01[822[9 4[220[2

14[625[6 7[029[5 1[329[5

1[0129[29 0[0629[17 9[1529[95

9[7729[11 9[6229[94 9[0329[92

2s[d[ values indicated[

Table 2[ Concentration of cyanogenic glucosides in ~owers and immature seeds of ~ax plants Cyanogenic glucoside mmol:099 g fr[ wt Organ

Linamarin

Lotaustralin

Linustatin

Neolinustatin

Flowers Immature seeds

09[1920[30 0[8929[44

3[1329[67 9[6229[08

9 9[5029[03

9 9[2229[98

2s[d[ values indicated[

signi_cant than for the leaves[ The concentration of linamarin and lotaustralin in roots of 59!day!old plants "9[72 mmol:099 g fr[ wt# was twice that of younger and older plants "Fig[ 1#[ Among the ~ax organs studied\ the highest con! centration of cyanogenic glucosides was found in the ~owers "03[3 mmol:099 g fr[ wt# "Table 2#[ This cor! responds to a concentration of 259 mmol:099 ~owers[ Already formed\ but still immature seeds\ extracted from fruits on day 03 following anthesis\ stored not only monoglucosides\ but also both diglucosides\ in lower amounts[ However\ the concentration of cyano! gens in such seeds was much less than that in ~owers "Table 2#[ DISCUSSION

The cyanogenic potential "capacity to produce HCN ð08Ł# changed during germination\ seedling development and subsequent growth of ~ax plants "Fig[ 2#[ The total amount of cyanogens in ger! minating seeds did not change signi_cantly but it increased markedly during early seedling growth\ especially at day 4\ when the young leaves began to expand[ After this stage\ the HCN!potential in ~ax tissues decreased\ and then it increased again before the onset of ~owering "Fig[ 2#[ These observations could indicate the involvement of cyanoglucosides in the control of some organogenetic processes[ The highest concentration of cyanogens at these extremely important stages of development may also indicate that these very sensitive stages are specially protected against pathogens and herbivores[ On the other hand\

Fig[ 2[ Cyanogenic potential "sum of cyanogenic glu! cosides*mmol:099 g fr[ wt# of whole ~ax plants at di}erent stages of growth and development[

according to some opinions\ cyanogenesis may be harmful to the plant\ impairing the action of other defence mechanisms ð19Ł[ Changes in cyanoglycoside content during ger! mination and seedling development of cyanogenic plants have also been reported earlier ð10\ 11Ł[ During germination of highly cyanogenic seeds\ the cyano! genic potential of seedlings may decrease rapidly\ as shown in Hevea ð1\ 08Ł\ or remain constant\ as dem! onstrated for lima beans "Phaseolus lunatus# ð10Ł[ On the other hand\ acyanogenic or weakly cyanogenic seeds\ like Sor`hum ð12Ł or Lotus corniculatus ð13Ł\

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rapidly synthetise cyanoglucosides during germi! nation[ Similar changes in cyanogen content during development have been observed in several varieties of white clover plants^ being highest at the start and end of the vegetative period ð14Ł[ As in cassava ð11\ 15Ł\ linamarin is the major mono! glucoside in Linum usitatissimum[ Lotaustralin\ which is the main glucoside in Trifolium repens ð04Ł\ occurs in ~ax in smaller amounts[ Although the amount of monoglucosides in ~ax tissues ~uctuates at di}erent stages of development "Figs 0 and 1#\ the ratio of linamarin to lotaustralin is fairly constant[ It may indicate\ that both monoglucosides play a similar role during growth and development of a ~ax plant[ The suggestion of Selmar et al[\ that diglucosides play the role of transport form of cyanogens ð4\ 16\ 17Ł does not appear to be con_rmed by the ~ax results[ Diglucosides are found only in ~ax seeds "Fig[ 0 and Table 2#[ This observation is in full agreement with the data of Smith et al[ ð07Ł\ who detected diglucosides only in ~ax seeds[ Similarly\ Oomah et al[ ð18Ł found diglucosides as the dominant cyanogens in seeds of 09 ~ax varieties and\ in some varieties low amounts of the monoglucoside\ linamarin[ The concentration of cyanogens in ~ax seeds of variety LCSD 199 in the present study is twice as low as that observed by Oomah et al[ The apparent absence of diglucosides in the vegetative parts of the plant\ and the observation that the concentration of cyanogens in seeds was much lower than that in other tissues "Figs 0 and 1# does not support the hypothesis that diglucosides are involved in the transport of cyanogens in ~ax seed[ Accumulation of diglucosides during ~ax seed matu! ration\ observed by Frehner et al[ ð29Ł and also in this paper "Table 2#\ concerns another developmental phase and is not involved in the long!distance trans! port of cyanogens\ postulated by Selmar et al[ ð16\ 20Ł[ On the other hand\ it has been demonstrated earlier that cotyledons are the only site of cyanogen biosyn! thesis in ~ax ð02Ł[ Similarly\ in emerging cassava seed! lings\ cyanogen biosynthetic activity is also con_ned to the cotyledons ð11Ł[ Therefore\ one can assume that cyanogenic diglucosides are transported throughout the plant\ and the absence of detectable amounts of diglucosides in parts of the ~ax plant\ other than seeds\ could be due to the activity of corresponding glu! cosidases in these organs[

old plants were grown in pots "03 cm diameter\ 1Ð2 seedlings per pot# on garden soil[ Plants were given H1O twice a week and\ alternatively\ with modi_ed Hoagland nutrient solution "pH 5[4#[ Extraction and TLC of cyano`enic `lucosides Fresh plant material "9[1Ð9[0 g# was frozen in liquid N1\ ground to a _ne powder in a mortal and pestle and extracted with 099 vols of boiling 79) aq[ EtOH for 4 min[ The slurry was _ltered under vacuum through Whatman No[ 0 paper "two layers\ glass Buchner funnel# and the solvent evapd in a rotary evaporator at 39> to yield a syrup[ The residue was redissolved in H1O and extracted with CHCl2 to remove lipids[ The aq[ phase was evapd in vacuo at 39> and the residue extracted with 1 ml of 09) aq[ iso!PrOH[ The extract containing cyanogenic glu! cosides was clari_ed by centrifugation\ if necessary\ and applied "09Ð59 ml# to TLC plates "cellulose MN299#[ Chromatograms were developed in H1O satd n!BuOH ð21Ł[ Rf values of linustatin and neo! linustatin were 9[01 and 9[10\ respectively^ linamarin and lotaustralin were 9[48 and 9[53\ respectively[ Cyanogenic glucosides were detected on chro! matograms by a sandwich technique with Feigl!Anger testpaper ð22Ł\ after spraying with soln of b!glu! cosidases in NaOAc bu}er "9[0 M\ pH 4[5#[ The mini! mum amount of HCN detectable was 09 nmol\ 3 times higher than that of the method of Ref[ ð22Ł[ Extraction of crude b!`lucoside Intact\ dry seeds were ground with 4 vols of dry iceÐ Me1CO\ in a Waring blender for 0 min[ The slurry was _ltered under vacuum through Whatman No[ 0 paper and the residue was re!extracted×2 with cold Me1CO[ The resulting Me1CO powder was left to air! dry for 0Ð1 h and then placed in a desiccator under vacuum to remove traces of Me1CO[ The Me1CO pow! der was resuspended in NaOAc bu}er "9[0 M\ pH 4[5# "0]09 w:v# and stirred in an ice bath for 0 h[ The slurry was spun o} at 09\999 ` for 19 min and the supernatant dialysed overnight against NaOAc bu}er "9[0 M\ pH 4[5#[ The resulting prepn "a crude source of b!glucosidase# was used for spraying chromatograms when analysing cyanogens ð22Ł and for hydrolysis for cyanogenic glucosides eluted from chromatograms[

EXPERIMENTAL

Quantitative analysis of cyano`enic `lucosides Plant material Seeds of oil ~ax "L[ usitatissimum L[# cv[ LCSD 199 harvested in 0881 were received from S[H[R[ Sobotka "Poland# and stored dry at 3> in darkness[ Seeds were sown in lots of 49 in 8 cm diameter Petri dishes lines with 1 discs of _lter paper moistened with 5 ml of H1O and then incubated for 4 days under the following conditions] 05 h photoperiod\ light:dark temp[ 14>:05>\ light intensity 049 mmol m−1 s−0[ Seven!day!

Cyanogenic glucosides were _rst separated by TLC and the zones of chromatograms corresponding with individual cyanogens were then removed and eluted with NaOAc bu}er "9[0 M\ pH 4[5#[ Glucosides were then hydrolysed with the b!glucosidase prepn[ Incu! bation was carried out at 26> for 2 h with 9[1 ml enzyme "equivalent 5 seeds#*for monoglucosides^ and for 10 h with 9[2 ml enzyme "equivalent 8 seeds#* for diglucosides[ The amount of glc liberated was

Cyanoglucosides in ~ax

determined in incubation mixts using the glucose oxi! dase "GOD# and peroxidase "POD# method ð23Ł\ against a non!incubated blank[ Each expt was per! formed using 1 to 2 replicates\ except for immature seeds "Table 2#\ where only one replicate was used[ Each value is a mean of at least 7 determinations[ Standard deviation "s[d[# was calculated and its range is shown in tables and _gures[ Acknowled`ements*The author wishes to thank Prof[ dr hab[ S[ Lewak "Warsaw University\ Poland# for helpful discussion and critical reading of the manu! script[ This research was\ in part\ _nancially sup! ported by Grant no[ 4 S290 929 95 from the State Committee for Scienti_c Research "Poland#[

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