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Izolovanje, strukturna karakterizacija i biološka aktivnost naftnih kiselina iz Vojvođanske nafte / Isolatin, structural characterization and biological activity of naphthenic acids from Vojvodina oil

<p>Grupno-strukturna analiza naftnih kiselina izolovanih iz srednjih uljnih&nbsp;komercijalnih frakcija vojvođanske nafte &bdquo;Velebit&rdquo; rađena je analizom IR-,&nbsp;<sup>1</sup>H- i&nbsp;<sup>13</sup>C NMR- i ESI-MS spektara niske rezolucije. Određeno je &scaron;est klasa&nbsp;karboksilnih kiselina op&scaron;te molekulske formule C<sub>n</sub>H<sub>2n-Z</sub>O<sub>2</sub>. To su karboksilne&nbsp;kiseline u opsegu masa 240-466 sa 15-31 C atoma u molekulu koje su&nbsp;grupisane u pet cikloalkil Z-serija: monociklična- (C<sub>n</sub>H<sub>2n-2</sub>O<sub>2</sub>, 14.72%),&nbsp;biciklična- (C<sub>n</sub>H<sub>2n-4</sub>O<sub>2</sub>, 34.63%), triciklična- (C<sub>n</sub>H<sub>2n-6</sub>O<sub>2</sub>, 25.03%), tetraciklična- (C<sub>n</sub>H<sub>2n-8</sub>O<sub>2</sub>, 10.04%), pentaciklična klasa karboksilnih kiselina (C<sub>n</sub>H<sub>2n-10</sub>O<sub>2</sub>, 4.99%) i jedna alkanska klasa kiselina sa alkil grupama otvorenog niza (C<sub>n</sub>H<sub>2n</sub>O<sub>2</sub>, 10.57%).</p><p>Razdvajanjem na bazi različite kiselosti i njihove rastvorljivosti u vodi na&nbsp;određenim pH vrednostima u rasponu od pH 2-10 dobijeno je devet užih&nbsp;frakcija kiselina. Ekstrakcijom sa etrom u vodi nerastvornih kiselina u rasponu&nbsp;od po jedne pH jedinice dobijena je sledeća distribucija masa: 9.57% (pH 10),&nbsp;12.69% (pH 9), 5.91% (pH 8), 4.85% (pH 7), 2.44% (pH 6), 11.71% (pH 5),&nbsp;23.94% (pH 4), 16.76% (pH 3) i 10.12% (pH 2). Razdvajanjem kiselina na bazi&nbsp;njihove kiselosti oko 50% mase kiselina ekstrahovano je od pH 2-4, znači u&nbsp;grupi jačih kiselina. Masenom spektrometrijom niske rezolucije ESI-MS na&nbsp;pH 4 vidi se da su u sme&scaron;i kiselina skoncentrisane kiseline sa vi&scaron;e prstenova u&nbsp;alkilnom delu sa maksimumom na tricikličnim kiselinama (36.50%), dok su&nbsp;pojedinačno najzastupljenije tetraciklične strukture kiselina C<sub>20</sub>H<sub>32</sub>O<sub>2</sub>(4.43%) i&nbsp;C<sub>21</sub>H<sub>34</sub>O<sub>2</sub>(4.56%). Na pH 8 biciklične- i triciklične strukture su zastupljene&nbsp;ukupno sa 65.02% sa maksimalno zastupljenim kiselinama sa 20-26 C atoma&nbsp;u molekulu, a izdvajaju se C<sub>22</sub>H<sub>38</sub>O<sub>2</sub> (5.26%)&nbsp;biciklične- i C<sub>24</sub>H<sub>42</sub>O<sub>2</sub>(7.01%)&nbsp;triciklične strukure, a od tetracikličnih struktura kiselina izdvaja se C<sub>24</sub>H<sub>40</sub>O<sub>2&nbsp;</sub>(4.77%). Na pH 10 najzastupljenije izolovane kiseline su aciklične, odnosno&nbsp;masne kiseline zastupljene sa 25.28%, a najdominantnije su strukture&nbsp;C<sub>21</sub>H<sub>42</sub>O<sub>2</sub> (4.83%), dok su ostale klase kiselina ujednačene po svom masenom&nbsp;udelu u odnosu na sastav u ukupnoj polaznoj sme&scaron;i kiselina.&nbsp;Drugi deo rada obuvata derivatizaciju izolovanih prirodnih naftnih kiselina.&nbsp;Sintetizovani su funkcionalni kiseonični metil-, etil-, n-butil-,&nbsp; terc-butil- i&nbsp;benzil-derivati naftnih kiselina kao i funkcionalni azotni derivati: amidi i anilidi&nbsp;naftnih kiselina. Sinteze navedenih estara rađene su klasičnim postupcima i&nbsp;modifikovanom metodom esterifikacije u kiselo-katalizovanim uslovima&nbsp;upotrebom mikrotalasa kao ko-katalizatora, &scaron;to je imalo za cilj skraćenje&nbsp;reakciong vremena i pobolj&scaron;anje ekolo&scaron;kih sintetskih uslova.<br />Modifikacijom sinteze metilnaftenata MT-zagrevanjem 48 puta je skraćeno&nbsp;vreme trajanja sinteze, prinosi ni promenom reakcionih parametara (vreme,&nbsp;snaga MT) nisu bitno promenjeni. Strukturnom analizom ESI-MS spektra&nbsp;polaznih kiselina i metilnaftenata dobijenih metilovanjem naftnih kiselina&nbsp;utvrđeno je da je odnos klasa u dobroj podudarnosti. Prinos estara u ovoj&nbsp;sintezi je 95.47%. Esterifikacija naftnih kiselina etil alkoholom rađena je u&nbsp;uslovima kisele katalize uz konvencionalno zagrevanje (91.76%), a&nbsp;modifikacijom metode MT-zagrevanjem (150 W) reakciono vreme je skaćeno&nbsp;96 puta (92.19%). Esterifikacija naftnih kiselina n-butil alkoholom rađena je uz&nbsp;sumpornu kiselinu kao katalizator i uz konvencionalno zagrevanje (94.24%), a&nbsp;u uslovima MT-zagrevanja (150 W) 72 puta je skraćeno reakciono vreme&nbsp;(61.15%). Sinteza terc-butilnaftenata rađena je prevođenjem naftnih kiselina u&nbsp;hloride a zatim reakcijom sa terc-butil alkoholom nastaju terc-butilnaftenati&nbsp;(80.17%). Modifikovanom metodom klasične reakcije esterifikacije naftnih&nbsp;kiselina terc-butil alkoholom katalizovanom sumpornom kiselinom u uslovima&nbsp;MT-zagrevanja (150 W) ostvaren je prinos od 85.49% a vreme trajanja reakcije&nbsp;je 5 minuta. Sinteza benzilnaftenata rađena je prevođenjem naftnih kiselina u&nbsp;hloride a zatim reakcijom sa benzil alkoholom nastaju benzilnaftenati&nbsp;(84.43%), a modifikovanom metodom klasične kiselo-katalizovane&nbsp;esterifikacije naftnih kiselina benzil alkoholom u uslovima MT-zagrevanja&nbsp;(150 W) ostvaren je prinos od 85.49% uz reakciono vreme od 5 minuta.&nbsp;Sinteza amida naftnih kiselina rađena je iz hlorida reakcijom sa amonijakom.&nbsp;Prinos čistih amida je 65.74%, a reakcija amidacije sa amonijakom trajala je&nbsp;15 minuta. Strukturnom analizom ESI-MS spektra amida naftnih kiselina&nbsp;utvrđen je grupno-strukturni sastav amida. Među strukturama amida naftenskih&nbsp;kiselina takođe su dominantne biciklične- i triciklične strukture, kao i u sme&scaron;i&nbsp;slobodnih kiselina. Sinteza anilida naftnih kiselina rađena je iz hlorida reakcijom sa anilinom. Prinos čistih anilida je 96.48%, a reakcija amidacije anilinom trajala je 30 minuta. Strukturnom analizom ESI-MS spektra anilida naftnih kiselina utvrđen je grupno-strukturni sastav proizvoda, anilida. Među strukturama anilida naftenskih kiselina takođe su dominantne biciklične- i triciklične strukture kao i u sme&scaron;i slobodnih kiselina.</p><p>U trećem delu ovog rada ispitivana je biolo&scaron;ka aktivnost naftnih kiselina&nbsp;auksinskog i giberelinskog tipa, njihov uticaj na ožiljavanje reznica,&nbsp;ukorenjivanje bočnih grana i mikroizdanaka biljaka, na aktivnost biljnih&nbsp;hormona, kao i na usvajanje metalnih jona kod biljaka.&nbsp;</p><p>Aktivnost naftenskih kiselina utvrđena je pomoću tri in vitro&nbsp; biolo&scaron;ka testa.&nbsp;&bdquo;Koleoptil test&rdquo;, rađen je na dva supstrata, odnosno na semenu ozime p&scaron;enice&nbsp;novosadske sorte Partizankai jare p&scaron;enice takođe novosadske sorte Venera.&nbsp;Referentna aktivnost u testu rađena je sa -naftilsirćetnom 3-indolsirćetnom&nbsp;kiselinom. U oba testa ustanovljen je približno isti odnos aktivnosti rastvora&nbsp;naftenskih kiselina i aktivnosti standardnih biljnih hormona. &bdquo;Test inhibicije&nbsp;klijanja&rdquo; semena rađen je sa semenom crne slačice, Brasscia nigra. Najveći&nbsp;uticaj naftenskih kiselina na inhibiciju klijanja postignut je u rasponu&nbsp;koncentracija kiselina od 10<sup>-7</sup>&nbsp;-10<sup>-8&nbsp;</sup>mol/L (0.05-0.01 mg/L). Testovi za &scaron;est užih&nbsp;frakcija dobijenih iz sme&scaron;e ukupnih kiselina razdvajanjem na bazi različite&nbsp;rastvorljivosti pri različitim pH vrednostima pokazuju istu aktivnost kao&nbsp;3-indolsirćetna kiselina (0.5 mg/L), a da je frakcija izdvojena iz vodenog&nbsp;rastvora na pH 7 takođe aktivna ali u poređenju sa 3-indolsirćetnom kiselinom&nbsp;to je 10 puta niža aktivnost. Hormonska aktivnost giberelenskog tipa ispitivanih&nbsp;kalijumovih soli naftenskih kiselina utvrđena je &bdquo;Endosperm testom&rdquo;, u kojem je&nbsp;određivana aktivnost amilaze spektrofotometrijskim praćenjem povećanja&nbsp;koncentracije redukujućih &scaron;ećera u endospermu semena tretiranog ječma i&nbsp;poređena sa aktivno&scaron;ću rastvora giberelinske kiseline (GA3). Rezultati ovih&nbsp;testova pokazuju da delovanjem vodenih rastvora užih frakcija naftenskih&nbsp;kiselina u koncentraciji 1.0 mg/L (3.5 x 10<sup>-6 </sup>mol/L)frakcija izolovana pri pH 8&nbsp;ima aktivnost koja je približno u opsegu aktivnosti giberelina koncentracije&nbsp;10<sup>-2</sup>-10<sup>-3&nbsp;</sup>mg/L.&nbsp;</p><p>Natrijum-naftenati u koncentraciji od 10<sup>-6&nbsp;</sup><br />do 10<sup>-8&nbsp;</sup>mol/L stimuli&scaron;u formiranje&nbsp;adventivnih korenova kod reznica suncokreta pa je broj korenova po biljci 40&nbsp;puta veći kod biljaka koje su bile potopljene u rastvor natrijum-naftenata u&nbsp;odnosu na one koje su bile potopljene u vodu, a sličan efekat utvrđen je i pri&nbsp;tretiranju bočnih grana suncokreta.&nbsp;</p><p>Ožiljavanje drvenastih biljaka rađeno je na reznicama bele topole (Populus&nbsp;alba) i reznicama američke crne topole (Populus deltoides). Uočen je jasan&nbsp;inhibitomi efekat na rast korenčića i izbojka u vodenom medijumu sa 10<sup>-4&nbsp;</sup>mol/L&nbsp;naftenskih kiselina, dok je tretman od 24 časa doprineo većem broju korenčića&nbsp;na donjih 5 cm reznice, kao i ukupnog broja korenčića nego kod kontrole.&nbsp;</p><p>U testu ukorenjivanja mikroizdanaka kod hrizantema najveći broj korenova&nbsp;dobijen je nakon tretmana sa rastvorom koji sadrži 10 &micro;mol/L ukupnih naftenata&nbsp;i tretmanom sa 50 &micro;mol/Lfrakcijom kiselina izolovanom pri pH 7. Oba rezultata&nbsp;su na nivou aktivnosti 3-indolbuterne kiseline koncentracije 10 &micro;mol/L&scaron;to znači&nbsp;da u ovom slučaju frakcionacija nije neophodna.&nbsp;</p><p>Efekti natrijum-naftenata na ukorenjivanje praćeni su merenjem nivoa totalnih&nbsp;peroksidaza i amilaze, &nbsp;kao i sadržaja redukujućih &scaron;ećera i ukupnih proteina u&nbsp;bazalnim delovima reznica bagrema(Rozaszin-AC).&nbsp; Nakon 1; 3 i 6 dana&nbsp;reznice su uzete za biohemijske analize. U svakom slučaju, aktivnosti&nbsp;IAA-oksidaze i amilaze su se povećavale do trećeg dana, &nbsp;a zatim smanjuje.&nbsp;Efekat je bio jače izražen posle tročasovnog tretmana sa natrijum-naftenatima&nbsp;u poređenju sa &scaron;estočasovnim tretmanom i kontrolom. Sadržaj rastvornih&nbsp;proteina je bio povećan jedan dan posle tretmana, smanjen trećeg i ponovo povećan &scaron;estog dana, osim za &scaron;estočasovni tretman natrijum-naftenatima, kada je efekat bio sasvim suprotan.</p><p>Test uticaja naftnih kiselina na nivo kadmijuma u biljkama pokazuje fiziolo&scaron;ko delovanje naftnih kiselina na snižavanje nivoa te&scaron;kih metala u biljci, u ovom slučaju kadmijuma. Ispitivan je efekat niske koncentracije natrijum-naftenata (10<sup>-7&nbsp;</sup>mol/L) na ukupan sadržaj Cd u pojedinim frakcijama interćelijskog prostora kao i unutar ćelija, kao i na neke fiziolo&scaron;ke i biohemijske parametre kod mladih biljaka soje koje su uzgajane u prisustvu kadmijum-hlorida koncentracije 1 mmol/L. Prisustvo naftenata smanjuje sadržaj ukupnog kadmijuma kako u korenu tako i u stablu i listovima u proseku za oko 40% i ublažava &scaron;tetne efekte kadmijuma na aktivnost nitrat-reduktaze kao i na sadržaje fotosintetskih pigmenata.</p><p>Tretman biljaka niskim koncentracijama natrijum-naftenata utiče na&nbsp;akumulaciju nekih esencijalnih elemenata kod mladih biljaka soje. Prisustvo&nbsp;naftenata (10<sup>-7 &nbsp;</sup>mol/L) značajno povećava sadržaj Mn, Fe, Zn i Ni u korenu, ali&nbsp;u stabljici i listovima samo sadržaj Fe i Mn. U korenu, sadržaj Mn je četiri puta&nbsp;veći a sadržaji Fe, Zn i Ni su povećani 17%, 60% i 68%, respektivno.&nbsp;</p><p>Ispitivanja na celeru i mrkvi su potvrdila da uticaj naftenata na mineralnu&nbsp;ishranu zavisi od primenjenog elementa i da je način preuzimanja određenih&nbsp;jona različit, &scaron;to ima za posledicu ili povećanje ili smanjenje sadržaja pojedinih&nbsp;jona u nekim delovima biljaka. Mlade biljke celera i mrkve, gajene u hranljivom&nbsp;medijumu, tretirane su natrijumovim solima naftenskih kiselina (10<sup>-7&nbsp;</sup>mol/L)&nbsp;folijarno ili prisustvom u hranljivom medijumu. Jedino je tretman putem&nbsp;prisustva naftenata u hranljivom medijumu smanjio svežu masu korena i&nbsp;nadzemnog dela oko 20% kod obe biljke.Oba tretmana uticala su na sadržaj&nbsp;Fe, Cu, Mn, Mg i Ca kod biljaka celera i Fe, Mn, Zn i Na kod biljaka mrkve.&nbsp;Kod biljaka celera do&scaron;lo je do smanjenja sadržaja navedenih nutrienata dok je&nbsp;u korenu biljaka mrkve do&scaron;lo do povećanja sadržaja Fe za 45%, Mn za 70% i&nbsp;Zn za 37%. Kod ovih biljaka prisustvo naftenata u hranljivom medijumu dovelo&nbsp;do smanjenja mase korenova i nadzemnih delova, &scaron;to je potpuno suprotno od&nbsp;rezultata dobijenih kod biljaka soje.&nbsp; Folijarnim tretmanom je značajno<br />povećana masa nadzemnog samo kod celera.</p> / <p>Group-structural analysis of naphthenic acids isolated from middle&nbsp;commercial fractions of Vojvodina oil &bdquo;Velebit&rdquo; was performed with the&nbsp;analysis of IR-,<br /><sup>1</sup>H- and&nbsp;<sup>13</sup>C NMR- and ESI-MS low resolution spectrums.&nbsp;Six classes of carboxylic acids of the general molecular formula C<sub>n</sub>H<sub>2n-Z</sub>O<sub>2&nbsp;</sub>were determined. These are carboxylic acids with mass range of 240-466&nbsp;with 15-31 C atoms in molecule which are grouped in five cycloalkyl&nbsp;<span style="font-size: 12px;">Z-series: monocyclic- (C<sub>n</sub>H<sub>2n-2</sub>O<sub>2</sub>, 14.72%), bicyclic- (C<sub>n</sub>H<sub>2n-4</sub>O<sub>2</sub>, 34.63%),&nbsp;</span><span style="font-size: 12px;">tricyclic- (C<sub>n</sub>H<sub>2n-6</sub>O<sub>2</sub>, 25.03%), tetracyclic- (C<sub>n</sub>H<sub>2n-8</sub>O<sub>2</sub>, &nbsp;10.04%), pentacyclic&nbsp;</span><span style="font-size: 12px;">class of carboxylic acids (C<sub>n</sub>H<sub>2n-10</sub>O<sub>2</sub>, 4.99%) and one alyphatic class of&nbsp;</span><span style="font-size: 12px;">acids with open chain alkyl groups (C<sub>n</sub>H<sub>2n</sub>O<sub>2</sub>, 10.57%).</span></p><p>By fractioning on the basis of different levels of acidity and their solubility in&nbsp;water with pH values ranging from pH 2-10 nine subfractions of acids were&nbsp;obtained. By extraction with ether in water undissolved acids in the range of&nbsp;one pH unit the following distribution of mass was performed: 9.57%&nbsp;(pH 10), 12.69% (pH 9), 5.91% (pH 8), 4.85% (pH 7), 2.44% (pH 6), 11.71%&nbsp;(pH 5), 23.94% (pH 4), 16.76% (pH 3) and 10.12% (pH 2). By fractioning&nbsp;acids on the basis of their acidity about 50% of acid &nbsp;mass was extracted at&nbsp;pH 2-4, i.e., in the group of stronger acids. Low resolution mass&nbsp;spectrometry ESI-MS shows that at pH 4 acids with more rings in the alkyl&nbsp;<br />section were concentrated in the mixture with maximum at tricyclic acids&nbsp;(36.50%), while individually tetracyclic acid structures C<sub>20</sub>H<sub>32</sub>O<sub>2</sub> (4.43%) and&nbsp;C<sub>21</sub>H<sub>34</sub>O<sub>2&nbsp;</sub>(4.56%) were most prominent. At pH 8 bicyclic- and tricyclic&nbsp;structures are represented &nbsp;together by 65.02% with acids with 20-26 C&nbsp;atoms in molecule being represented the most, and C<sub>22</sub>H<sub>38</sub>O<sub>2</sub>&nbsp; (5.26%)&nbsp;bicyclic- and C<sub>24</sub>H<sub>42</sub>O<sub>2</sub>(7.01%) tricyclic structures stand &nbsp;out, while with&nbsp;tertacyclic acid structures C<sub>24</sub>H<sub>40</sub>O<sub>2</sub> (4.77%) stands out. At pH 10 most&nbsp;represented isolated acids are acyclic, i.e., fatty acids which are&nbsp;represented by 25.28%, with the most dominant C<sub>21</sub>H<sub>42</sub>O<sub>2&nbsp;</sub>structures&nbsp;(4.83%), while the other classes &nbsp;of acids are well balanced in their mass&nbsp;share in relation to the composition of the overall initial acid mixture.</p><p>The second part of this work deals with derivatization of the isolated natural&nbsp;naphthenic acids. Functional oxygen methyl-, ethyl-, n-butyl-, tert-butyl and&nbsp;benzyl-derivatives of naphthenic acids as well as functional nitrogen&nbsp;derivatives: amides and anilides of naphthenic acids were synthesized. The&nbsp;syntheses of the above mentioned esters were performed by conventional&nbsp;methods and by a modified method of esterification in acid-catalyzed&nbsp;conditions using microwaves as a co-catalyst with the aim of shortening the&nbsp;reaction time and improving the ecological synthetic conditions.&nbsp;</p><p>By modification of methyl naphthenates synthesis using MT-heating the&nbsp;time period for synthesis is reduced 48 times and the yield does not&nbsp;significantly change even after the reaction parameters (time, MT power)&nbsp;have been altered. Using structural analysis of ESI-MS spectrum of initial&nbsp;carboxylic acids and the methyl naphthenates obtained through methylation&nbsp;of naphthenic acids it was establishedthat the class ratio shows great&nbsp;compatibility. The esters yield in this synthesis is 95.47%. The esterification&nbsp;of naphthenic acids with ethyl alcohol done under the conditions of acid&nbsp;catalysis with conventional heating (91.76%), and with the modified method&nbsp;MT-heating (150 W) the reaction time is reduced 96 times (92.19%). The&nbsp;esterification of naphthenic acids with n-butyl alcohol was performed using&nbsp;sulphuric acid as a catalyst with conventional heating (94.24%), and under&nbsp;MT-heating conditions (150 W) reaction time was 72 times&nbsp; shorter&nbsp;(61.15%). The synthesis of&nbsp; tert-butyl naphthenates was performed by&nbsp;conversion of naphthenic acids into chlorides, followed by the reaction with&nbsp;tert-butyl alcohol which resulted in tert-butyl naphthenates (80.17%). By the&nbsp;modified method of conventional reaction of esterification of naphthenic&nbsp;acids using tert-butyl alcohol catalysed with sulphuric acid in MT-heating&nbsp;conditions (150 W) the yield was 85.49% and reaction time was 5 minutes.&nbsp;The synthesis of benzyl naphthenates was performed by conversion of&nbsp;naphthenic acids into chlorides, followed by a reaction with benzyl alcohol&nbsp;which produces benzyl naphthenates (84.43%), whereas the modified&nbsp;method of conventional acid-catalysed esterification of naphthenic acids&nbsp;using benzyl alcohol under MT-heating (150 W) conditions the yield was&nbsp;85.49% with the reaction time of 5 minutes.&nbsp;</p><p>The synthesis of naphthenic acid amides was done from chlorides by&nbsp;reaction with ammonia. The yield of pure amides was 65.74%, and the&nbsp;reaction of amidation with ammonia lasted for 15 minutes. The structural&nbsp;analysis of ESI-MS spectrum of naphthenic acid amides determined the&nbsp;group-structural composition of amides. With the structures of amides of&nbsp;naphthenic acids bicyclic- and tricyclic structures are also dominant, as in&nbsp;the mixture of free acids. The synthesis of naphthenic acid anilides were&nbsp;performed from chloride by reacting with aniline. The yield of pure anilides&nbsp;<br />was 96.48%, and the reaction of amidatation lasted for 30 minutes. The&nbsp;structural analysis of ESI-MS spectrum of naphthenic acid anilides&nbsp;determined the group-structural composition of anilide products. With the&nbsp;structures of anilides of naphthenic &nbsp;acids bicyclic- and tricyclic structures&nbsp;are also dominant, as in the mixture of free acids.</p><p>The third part of this work tests the biological activity of naphthenic acids of&nbsp;auxine and gibberellinic type, their influence on the rooting of cuttings,&nbsp;lateral branches and microshoots of plants, on the activity of plant&nbsp;hormones as well as on the metal ions uptake by plants.&nbsp;</p><p>The activity of naphthenic acids was determined using three in vitro&nbsp;biological tests. &bdquo;Koleoptil test&rdquo;, was done on two substrates, namely the&nbsp;seed of winter wheat of the Partizankasort and the spring wheat of the&nbsp;Venerasort. The referential activity in the test was performed with&nbsp;&alpha;-naphthylacetic- and 3-indolacetic acid. Both tests showed approximately&nbsp;the same ratio of activity of naphthenic acid solutions and standard plant&nbsp;hormones. &bdquo;Germination Inhibition Test&rdquo; of the seed was performed using&nbsp;<br />Brasscia nigraseed. The greatest influence of naphthenic acids to&nbsp;germination inhibition was achieved in the acid concentration ranging from&nbsp;10<sup>-7</sup>-10<sup>-8&nbsp;</sup>mol/L (0.05-0.01 mg/L). The tests for six narrow fractions obtained&nbsp;from the overall mixture of acids through fractioning on the basis of different&nbsp;solubility at different pH values show the same activity as 3-indolacetic acid&nbsp;(0.5 mg/L), with the fraction isolated from the aqueous solution at pH 7 also&nbsp;active but in comparison to 3-indolacetic acid this activity was 10 times&nbsp;lower. Hormone activity of gibberellinic type of the potassium salts of&nbsp;naphthenic acids was determined using &ldquo;Endosperm Test&rdquo; where amylase&nbsp;<br />activity was determined by spectrophotometric measuring of the increase of&nbsp;the concentration of reducing sugars in endosperm in the treated barley&nbsp;seed which was then compared to the activity of the gibberellic acid solution&nbsp;(GA3).&nbsp; The results of these tests indicate that the activity of aqueous&nbsp;solutions of narrow fractions of naphthenic acids in the concentration of&nbsp;1.0 mg/L (3.5 x 10<sup>-6&nbsp;</sup>mol/L)the fraction isolated &nbsp;at pH 8 has the activity&nbsp;which is approximately within the range of activities of gibberellin of the&nbsp;concentration of 10<sup>-2</sup>-10<sup>-3&nbsp;</sup>mg/L.&nbsp;</p><p>Sodium salts of naphthenic acids in concentrations of 10<sup>-6&nbsp;</sup>do 10<sup>-8&nbsp;</sup>mol/L&nbsp;stimulated formation of adventitious roots in&nbsp;<span style="font-size: 12px;">sunflower cuttings even by a&nbsp;</span><span style="font-size: 12px;">factor of 40 compared with control, the effect being also observed lateral&nbsp;</span><span style="font-size: 12px;">branches of interspecies sunflower hybrids. The obtained results suggest&nbsp;</span><span style="font-size: 12px;">the possibility of using naphthenic acids as a means for rooting of plant&nbsp;</span><span style="font-size: 12px;">cuttings.</span></p><p>Rooting of hardwood plants was investigated on the cuttings of white poplar&nbsp;(Populus alba) and black poplar (Populus deltoides). The distinct inhibitory&nbsp;effect on the root and shoot growth in water culture was detected in the&nbsp;concentration of naphtenic acids 10<sup>-4&nbsp;</sup>mol/L, but 24-hour treatment raised&nbsp;the number roots on undermost 5 cm of the cutting, as well as the total&nbsp;number of roots, comparing to the control.</p><p>In the test of rooting microshoots ofchrysanthemum the highest number of&nbsp;<span style="font-size: 12px;">roots was achieved after the treatment with solution containing 10 &micro;mol/L of&nbsp;</span><span style="font-size: 12px;">total napthenates, as &nbsp;well as after the treatment with fraction of naphthenic&nbsp;</span><span style="font-size: 12px;">acids which was isolated at pH &nbsp;7 in concentration of 50 &micro;mol/L. Both results&nbsp;</span><span style="font-size: 12px;">are on the level of activity of 3-ndolbutyric acid in concentration of 10 &micro;mol/L&nbsp;</span><span style="font-size: 12px;">which suggests that in this case ractionation is not necessary.</span></p><p>The effects of sodium naphthenates on rooting were investigated by&nbsp;measuring the level of total peroxidases and amylase, along with the&nbsp;contents of reducing sugars and total proteins in basal parts of cuttings of&nbsp;black locust (Rozaszin-AC). After 1; 3 and 6 days cuttings were taken for&nbsp;biochemical analysis. In all cases, the activities of IAA-oxidase and amylase&nbsp;increased to the third day and showed a decrease afterwards. The effect&nbsp;was more pronounced after the three-hour treatment with sodium&nbsp;naphthenates, compared to the six-hour treatment and control. The content&nbsp;of soluble proteins increased one dayafter the treatment and decreased to&nbsp;the third and again increased to the sixth day, an exception being the&nbsp;six-hour treatment with sodium naphthenate, when the effect was&nbsp;completely opposite.</p><p>The test of naphthenic acids influence on the level of cadmium in plants&nbsp;showed physiological activity of naphthenic acids on the decrease of the&nbsp;level of heavy metals, &nbsp;in this case cadmium, in the plant. The effect of low&nbsp;concentrations (10<sup>-7&nbsp;</sup>mol/L) of sodium naphthenate on total content of Cd in&nbsp;the intercellular space and inside cells, as &nbsp;well as on some physiological&nbsp;and biochemical parameters of young soybean plants grown in the&nbsp;presence of 1 mmol/L solution of cadmium chloride was investigated.&nbsp;Presence of naphthenate reduced in average by 40 % content of total and&nbsp;intracellular Cd in root, stem and leaves and alleviated the harmful effect of&nbsp;<br />Cd on activity of nitrate reductase and content of photosynthetic pigments.</p><p>Treatment of soybean plants with low concentrations of sodium&nbsp;naphthenate influenced the accumulation ofsome essential elements by the&nbsp;young plants. The presence of naphthenates (10<sup>-7&nbsp;</sup>mol/L) significantly&nbsp;increased content of Mn, Fe, Zn and Ni in root, but in stem and leaves&nbsp;increased only contents of Fe and Mn. Inroot, the content of Mn increased&nbsp;four times while contents of Fe, Zn and Ni increased by 17%, &nbsp;60% and 68%&nbsp;respectively compared to the control.</p><p>Investigations on celery and carrot&nbsp; confirmed that the influence of&nbsp;naphthenates on mineral nutrition depends on the applied element and that&nbsp;the way of uptake of certain ions is different, which results in either increase&nbsp;or decrease of the contents of some ions in certain parts of plants. Young&nbsp;plants of celery and carrot, were grown in the nutrient medium and treated&nbsp;with sodium salts of naphthenic acids (10<sup>-7&nbsp;</sup>mol/L) over the leaves or with its&nbsp;presence in the nutrient medium. Only the treatment based on the &nbsp;presence&nbsp;of naphthenates in the nutrient medium resulted in the decrease of the fresh&nbsp;mass of root and aboveground part by about 20% for both plants. Both&nbsp;treatments had influence on the content of Fe, Cu, Mn, Mg and Ca in the&nbsp;plants of celery and Fe, Mn, Zn and Na in the plants of carrots. In the plants&nbsp;of celery the content of the mentioned nutrients was decreased, while in the&nbsp;root of plants of carrot &nbsp;the content was increased, Fe by 45%, Mn by 70%&nbsp;and Zn by 37%. With these plants &nbsp;the presence of naphthenates in the&nbsp;nutrient medium caused the reducing roots and aboveground parts, which is&nbsp;completely opposite from the results obtained with soybean plants. The&nbsp;treatment by leaves significantly increased the mass of aboveground part&nbsp;only with celery.</p>

Identiferoai:union.ndltd.org:uns.ac.rs/oai:CRISUNS:(BISIS)23726
Date04 December 2009
CreatorsGrbović Ljubica
ContributorsĆirin-Novta Vera, Kuhajda Ksenija, Penov-Gaši Katarina, Kevrešan Slavko
PublisherUniverzitet u Novom Sadu, Prirodno-matematički fakultet u Novom Sadu, University of Novi Sad, Faculty of Sciences at Novi Sad
Source SetsUniversity of Novi Sad
LanguageSerbian
Detected LanguageUnknown
TypePhD thesis

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