Ferenczy, Lajos

Rezk, Malak R.

English

Rezk Malak R.

Ferenczy Lajos

University of Szeged

A c t a Bio logica Szeged 15 (1—4), pp. 71—78 (1969) GROWTH INHIBITING ACTIVITY OF SOME STEROID GLYCOALKALOIDS ON HIGHER PLANTS M A L A K R. REZK a n d L. FERENCZY Botany Department , Facul ty of Science, Univers i ty of A lexandr ia , A lexandr ia , U. A. R. Depar tment of P l a n t Phys io logy and Microbiology, At t i la József Univers i ty , Szeged (Rece ived N o v e m b e r 20, 1968) The relations between the fungicidal activity and the molecular structure of some steriod glycoalkaloids have recently been established ( F e r e n c z y and K e v e i , 1967a,b; K e v e i , 1968). The antifungal acti-vity of these compounds is strictly proportional to their ability of com-plex formation with ergosterol. Their primary acting site is the ergo-sterol-containing cell membrane system. The release of compounds of low molecular weight (e. g. amino acids) is very characteristic. In the present study w e intended to obtain data on the effects of some steroid glyalkaloids of plant origin on higher plants. Materials and Methods T h e compounds used w e r e chromatographica l ly pure and crysta l l ine tomatin , so laradix in , so lamargin , solasonin, and ^-so lamarg in . Al l c o m p o u n d s w e r e isolated f r o m plants by var ious methods. ( K e v e i , 1968.) Mel t ing points : tomat in (I) 272— 274°C (decomp.), so laradix in (II) 264°C (decomp.), so lamargin (III) 301—303°C (decomp.), so lasonin (IV) 292—294°C (decomp.) and / í - so lamargin (V) 225—226°C. T h e concentrat ions general ly appl ied w e r e 1 0 ~ \ 1 0 - 5 , 1 0 - 8 and 10~ 7 M. Each compound at ea'ch concentrat ion w a s used at t w o p H - v a l u e s (5 and 7) us ing M/300 phosphate buffer . Pre l iminary tests were m a d e w i t h four plant species: Cucumis sativus "Kínai Kígyó", Papaver somniferum "Fertődi kék", Sinapis alba and Solanum laciniatum. A s the four species g a v e s imi lar responses , detai led w o r k w a s carried out w i t h Cucumis sativus. The seeds w e r e of the 1967 crop. S e e d g e r m i n a t i o n t e s t . — T w o - l a y e r f i l ter papers w e r e placed into 9 c m Petr i -d i shes and w e t t e d w i t h 6 ml of dist i l led w a t e r (control) or w i t h the test so lut ions at pH 5 and 7. Four replicates, each w i t h 50 seeds w e r e prepared, incubated in the dark at 25°C for 36 hours, then e x a m i n e d for the percentage of germinat ion . A t four-hour in terva l s the results w e r e checked again. G r o w t h t e s t s . — A f t e r germinat ion o n f i l ter papers w e t t e d w i t h dist i l led water , the seed l ings hav ing u n i f o r m radic les of 15 m m l e n g t h w e r e se lec ted and transferred in to the test solutions. P i e c e s of bobbin net w e r e s tretched on 50 ml beakers f i l led w i t h the so lu -tions. T h e seed l ings (10 in each beaker) w e r e placed on the net w i t h their radic les d ipp ing into the solution. T h e controls w e r e buf fers a t pH 5 and 7. T h e beakers w e r e placed ins ide basins covered w i t h glass plates to ma in ta in a h u m i d a t m o s -phere. 7 2 A f t e r 72 hours of dark incubat ion at 25°C the seed l ings w e r e r e m o v e d , measured, and the d i f f erences b e t w e e n the init ial a n d f ina l l e n g t h s of roots a n d hypocoty l s w e r e calculated. G l y c o s i d e a b s o r p t i o n , t r a n s l o c a t i o n a n d a m i n o a c i d r e -l e a s e . — To s tudy the absorpt ion and translocat ion so lamarg in has b e e n chosen o w i n g to its pronounced biological act iv i ty and sens i t i v i ty to H a n s e n a n d D a m reagent ( H a n s e n and D a m , 1957) modi f ied by V a h o u n y e t al (V a h o u n y, B o r j a a n d W e e r s i n g , 1963). These e x p e r i m e n t s w e r e carried out under asept ic condit ions to avo id the interference of micro-organi sms . T h e w e t t e d f i l ter papers in P e t r i -d ishes , the g la s sware and the so lut ions w e r e s ter i l ized by auto -c lav ing and the seeds ware dis infected by 0,5 % b r o m i n e water . Af ter germinat ion , the s e e d l i n g s w e r e cul t ivated in buf fer solution (pH 7) at 25°C for three days , then 40 u n i f o r m plants (the total w e i g h t of roots corresponded to 55 ± 3 m g dry we ight ) w e r e transferred to 50 ml so lamargin so lut ion (10—* M) at the s a m e pH value . S a m p l e s w e r e taken f r o m the solution in every second hour, of incubat ion and ana lysed for so lamarg in and a m i n o acids. Also, p lant s a m p l e s w e r e t a k e n in the s a m e periods. The plants w e r e extracted w i t h m e t h a n o l for 30 m i n u t e s u n d e r ref lux, and the methano l i c so lut ions w e r e concentrated and appl ied to c h r o m a t o -plates in var ious amounts . The quant i ty of so lomarg in in the solut ions , roots and hypocoty l s w a s e s t imated by using th in- layer chromatography as p r e v i o u s l y descr ibed ( F e r e n c z y and K e v e 1, 1967a). S i l i ca gel G ( M e r c k, nach S t a h 1), ac t ivated at 110° C for one hour, w a s used as adsorbent and m e t h a n o l - b e n z e n e -- a m m o n i a (25%) (10:20:1) as so lvent system. Modi f i ed H a n s e n — D a m r e a g e n t w a s appl i ed (100 m g FeCl3 d i sso lved in 100 ml g lac ia l ace t i c acid + 100 m l conc. HiSOO as colour reagent . In s o m e e x p e r i m e n t s w i t h ^-so lamargin , the t rea tment of roots a n d the quant i ta t ive th in- layer chromatography w a s carried out as in case of so lamarg in . The total quant i ty of a m i n o acids w a s d e t e r m i n e d as prev ious ly 'descr ibed ( F e r e n c z y and K e v e i , 1967a) us ing 0,1 ml of e a c h solut ion, app ly ing o n c h r o -matographic paper, dry ing and deve lop ing with n inhydrin . T h e spots w e r e e luted by 50 % methanol and the colour intensi ty w a s m e a s u r e d fo tometr ica l ly at 520 nm. T h e ex t inc t ion v a l u e s w e r e referred to a s tandard cal ibrat ion c u r v e m a d e by alanin. T h e determinat ion of indiv idual a m i n o acids f r o m both so lut ions a n d root ex tracts w a s carried out w i t h paper chromatography ( K e v e i , 1968). B u t a n o l — glacial acet ic a c i d - w a t e r (2:2:1) w a s used as s o l v e n t s y s t e m a n d S c h l e i c h e r — S c h U11 2043b as paper. For the sake of comparison , a m i x t u r e of 20 a m i n o ac ids w a s run s imul taneous ly . When translocat ion of so lamargin f r o m l e a v e s to other organs w a s s tudied , 0.1 ml so lamargin so lut ion ( 2 X 1 0 - 3 M . pH 7 conta in ing 0.1 % T w e e n 80 and 0.1 % po lye thy leng lyco l ) w a s appl ied to the upper sur face (17 ,8+1 ,3 cm 2 ) of o n e leaf per plant and dispersed. Be fore treatment , Cucumis plants w e r e g r o w n in w a t e r c u l -ture for t w o weeks , a f ter treatment for 24 hours (one group in l ight at 6000 lux , and another in the dark) at 25°C, then cut, the ident ica l parts col lected a n d e x -tracted. T h e extracts w e r e concentrated and a n a l y s e d as m e n t i o n e d before. S t a t i s t i c s . T h e s tandard errors w e r e ca lculated in the usual m a n n e r ( C a v a l l i - S f o r z a , 1965.) Every exper iment w a s carried out in four repl icates . Results and discussion S e e d g e r m i n a t i o n t e s t . The steroid glycoalkaloids used did not inhibit the germination of Cucumis seeds even at the highest con-centration used (10 4 M). There was only a slight retardation (UD to 19 %) in germination rate in case of tomatin. solasonin, and solamarein. The germination rate lagged behind that of the control only in the f ' r s t 36 hours, but four hours later it reached the control value (98 %V The radicles treated with the above mentioned compounds and wi th ft-solamargin were deformed, shorter and thicker than those of the controls. 7 3 Fig. 1. T h e comopounds tested. I : tomatin, II:solaradixin, III:solamargin, IV: solasonin, V:/?-solamargin. This morphological aberration was characteristic (Fig. 2). In an earlier work ( K e v e i , 1968) it was also found that filamentous fungi growing in culture media containing the same compounds showed similar features (short, thick and disterted hyphae). G r o w t h t e s t . — The applied compounds except solaradixin exert a strong inhibitory effect on roots (Fig. 3 and 4). As a consequence, the hypocotyls were also inhibited (Fig. 5 and 6). The inhibition was proportional to the concentrations and was more pronounced at pH 7. In some cases lower concentrations caused slight promotions of the root and hypocotyl growth. Beside the retardation of growth of the main roots, there occurred also characteristic deformations (Fig. 7). Comparing these results with those of the earlier antifungal tests ( F e r e n c z y and K e v e i , 1967a,b; K e v e i , 1968), it can be established that there is a close similarity in t h e sequence of activity of the com-pounds examined. Both on higher plants and on fungi the sequence of activity was as follows: tomatin ) solamargin ) solasonin ) /9-solamargin ) solaradixin. The activity of /J-solamargin is higher in Cucumis growth test than in the microbiological tests. The more pronounced activity is not attributable to the alteration of y?-solamargin molecule in higher plants as was thought. The quantitative thin-layer 74 Fig. 3. G r o w t h inhib i t ing e f f e c t s on roots of Cucumis s eed l ings at pH 5. Vert ical l ines represent the s tandard errors. chromatography for /9-soIamargin and for hypothetical derivatives indi-cated a more intensive absorption of the compound into the roots than into fungi, but no change in the molecular composition. Solaradixin proved to be inactive both in case of higher plants and of micro-organisms. As reported previously, the activity of these compounds is strictly related to the sugar components of the molecules. E.g. the great differ-ence in activity between tomatin and solaradixin (both having four sugar molecules) cannot be attributed to the difference of stereo-position of ring-F or to the absence or presence of double bond in ring-B but to the difference in composition and sequence of the sugar molecules. 7 5 G l y c o s i d e a b s o r p t i o n , t r a n s l o c a t i o n a n d a m i n o a c i d r e l e a s e . Examining both the solutions and the roots for the amount of solamargin, it was foud that 40 + 5% of the quantity of solamargin was absorbed by the roots f rom the solution. It was also proved that there was no decomposition or other change of the solamargin was absodbed by the rots f rom the solution. It was also proved that there was no decomposition or other change of the sola-margin molecule in plants or in the solution. fomarln solarodixin solomorgin solasonin (5-solomorgin Fig. 4. G r o w t h inhib i t ing e f f ec t s on roots of Cucumis seed l ings at p H 7. Vert ical l ines represent the s tandard errors. The results obtained by chromatography of plant extracts show no translocation of the compound from roots to hypocotyls or from treated leaves to other leaves, stems or roots. The data of the release of amino acids from roots are shown in Fig. 8. The typical effect of solamargin is the release of amino acids from the roots into the external solution in the first two hours. The sub-sequent increase in the amount of released amino acids is slight. 7 6 It was also found that the release of amino acids was not selective. All kinds of f ree amino acids to be present in the pool of plant cells were released and their proportion in the external solution was the same as in the cells. The proportoin of amino acid did not show any variation with time. Concerning the release of amino acids, very similar data were obtained earlier with certain microorganisms too ( F e r e n c z y and Fig. 5. G r o w t h inhib i t ing e f f ec t s o n hypocoty ls of Cucumis s eed l ings a t p H 5. Vert ical l ines represent the s tandard errors. Fig. 6. G r o w t h inhibi t ing e f f e c t s on hypocoty l s of Cucumis s eed l ing at pH 7. Vert ical l ines represent the s tandard errors . K e v e i , 1967a,b; K e v e i , 1968). In experiments with various yeast and filamentous fungi it was established that the mode of action of these compounds is the destruc-tion of the ergosterol-containing cell membrane. From the data reported here it may be concluded with fair probability, tha t the growth inhibit-ing activity of the steroid glycoallkaloids on roots of higher plants is attributable to the disorganization of the /3-sitosterol-containing mem-brane system. T h i s research w o r k w a s carried out dur ing a scho larsh ip granted b y the Hungar ian G o v e r n m e n t to o n e of us (M. R. R e z k ) for w h i c h he o f f er s h i s s incere gratitude. Fig. 2. Dis tort ing e f f e c t of tomat in (10—* M); u p p e r r o w : t r e a t e d seeds. L o w e r r o w c o n t r o l s . Fig. 7. G r o w t h inhib i t ing e f f e c t of so lamargin at pH 7. Concentrat ions f r o m le f t to r i g h t : 1 0 - 4 , 1 0 - 5 , 1 0 - 8 , 1 0 - 7 M a n d control. Fig. 7 7 7 0 2 4 6 8 h o u r s Fig. 8. E f fec t of so lamargin on re lease of a m i n o ac ids f r o m roots of Cucumis seedl ings . Vert ical l ines represent the s tandard errors. Abstract Five steroid glyooalikaloids (tomatin, solaradixin, solamargin, sola-sonin, and /9-solamargin) isolated f rom plants were examined for growth inhibiting activity. It was found that these compounds had no inhibiting effect on germnaiton, but four of them caused characteristic root growth inhibition and distortion. There are close relations between the chemical structure and the biological activity. The sequence of activity is: tomatin > solamargin > solosonin > /?-solamargin > solaradixin (inactive). There was no sign of translocation of solamargin from the roots to the hypocotyls and from the leaves to other organs. This compound caused a quick and non-selective release of amino acids from the root cells. The mode of inhibitory action is probably the disorganization of the sterol-containing cellular membrane system. These data are in good agreement with the results of earlier work on fungi. 7 8 References C a v a l l i - S f o r z a , L. (1965): Grundbegr i f f e der Biometr ie . — Jena. F e r e n c z y , L. a n d K e v e i , F. (1967a): Studies on ant i funga l s teroid g lycos ides . I. Data o n the m o d e of act ion of s o m e ant i fungal s teroid g lycoalkalo ids . S y m -p o s i u m on M e c h a n i s m s of Act ion o n Fung ic ides a n d Antibiot ics . — Ber l in , pp. 59—68. F e r e n c z y , L. a n d K e v e i , F. (1967b): M e c h a n i s m of act ion of an t i funga l s tero id g lycoalkalo ids . — Acta Microbiol . Acad. Sci. Hung. 14, 127. H a n s e n , P. W. and D a m , H. (1957): Paper chromatography and co lor imetr i c de terminat ion of free and es ter i f ied choles tero l in very smal l a m o u n t s of blood. — Acta Chem. Scand. 11, 1658—1662. K e v e i , F. (1968): Hatásmódvizsgá la tok sze lekt ív an t i fungá l i s akt iv i tású szteroid g l ikoz idokkal (Studies on the m o d e of act ion steroid g lycos ides of s e l e c t i v e ant i fungal activity) . — Doctoral Dissertat ion, A. József Univers i ty , Szeged , Hungary . V a h o u n y, G. V., B o r j a , C. R. and W e e r s i n g , S. (1963): Rad ioac t ive and analyt ica l de terminat ion of free and es ter i f ied choles tero l f o l l o w i n g m i c r o th in- layer s i l is ic acid chromatography . — Anal . B iochem. 6, 555—559. P e r m a n e n t address of the authors : Dr. M a i a k R, R e z k Botany Department , F a c u l t y of S c i e n c e Univers i ty of A lexandr ia , M o h a r r a m Bey, A lexandr ia , U.A.R. Dr. L. F e r e n c z y D e p a r t m e n t of P lant P h y s i o l o g y a n d Microbio logy of the A. J. U n i v e r s i t y Szeged, Hungary 

Growth Inhibiting Activity of Some Steroid Glycoalkaloids on Higher Plants

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Growth Inhibiting Activity of Some Steroid Glycoalkaloids on Higher Plants

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