PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF ACHILLEA MILLEFOLIUM GROUP-RELATED CULTIVAR PROA TO DIFFERENT NITROGEN REGIMES

Transcription

1 Article A&EB PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF ACHILLEA MILLEFOLIUM GROUP-RELATED CULTIVAR PROA TO DIFFERENT NITROGEN REGIMES Özgür Ttr 1, Emine Byrm 1, Emili Geshev, Çiğdem Sönmez 1, Deniz Atsoy 1, Angel Konkchiev 3, Mitht Nuri Gevrek 1 1 Ege University, Fculty of Agriculture, Izmir, Turkey Bulgrin Acdemy of Sciences, Institute of Plnt Physiology nd Genetics, Sofi, Bulgri 3 Bulgrin Acdemy of Sciences, Institute of Orgnic Chemistry with Center of Phytochemistry, Sofi, Bulgri Correspondence to: Özgür Ttr E-mil: ozgur.ttr@ege.edu.tr ABSTRACT In the present study the effect of different nitrogen mounts ( g [N ],.5 g [N 1 ],.9 g [N ] nd 1.35 g N [N 3 ] per pot, corresponding to kg h -1, 5 kg h -1, 1 kg h -1 nd 15 kg h -1 N ppliction in field conditions) nd ppliction times (t sowing, vegettive stge nd pre-loom stge) on the yield, reltive wter content, totl nitrogen content nd nitrogen use efficiency in plnts of the Achille millefolium group-relted cv. Pro ws investigted. Antioxidnt ctivity, flvonoid content, rte nd composition of the essentil oils were lso determined. Appliction of N incresed the dry herge nd flos production, totl nitrogen mount nd nitrogen use efficiency, without ffecting the essentil oil rte while reducing the ntioxidnt ctivity. Higher mounts (N 3 ) of nitrogen mintined the dry herge nd flos yield t levels similr to N ut decresed the ntioxidnt ctivity nd rte of the essentil oil. The mjor component of the essentil oil, chmzulene, ws negtively (N 1, N ) or not influenced (N 3 ) y the nitrogen ppliction, while vrile chnges were registered in the content of the other components. The dt point to the nitrogen regime eing sutle tool for regulting the performnce of cv. Pro plnts. Biotechnol. & Biotechnol. Eq. 13, 7(), Keywords: Achille millefolium group, cv. Pro, nitrogen ppliction, dry mtter, essentil oil, ntioxidnts, chmzulene Arevitions: GC MS: Gs Chromtogrpy Mss Spectrometry; PFD: photon flux density; RWC: reltive wter content Introduction During the lst decdes the production volume of the medicinl plnts hs een constntly incresing s result of the rising demnd for nturl products nd introduction of their new uses in the phrmcy, cosmetics nd food industries (5). Plnts of the genus Achille L. (Astercee fmily), referred to s yrrow, hve een known for over 3 yers (3), nd hve een used in folk medicine ginst severl ilments such s skin inflmmtions, spsmodic nd gstrointestinl disorders, hepto-iliry complints (7). The medicinl properties of Achille re pprecited worldwide nd the plnts re included in the ntionl Phrmcopoeis of some countries like Germny, the Czech Repulic nd Frnce (9). Studies on Achille millefolium s.l. (sensu lto) hve identified different components such s essentil oils, sesquiterpenes nd phenolic derivtives constituting group of secondry metolites with powerful phrmcologicl ctivity (7). One of the min constituents of the essentil oil, chmzulene, contriutes to the nti-inflmmtory efficiency y ntioxidnt-type inhiitory interctions (). On the other hnd, phenolic compounds flvonoid nd phenylcronic Biotechnol. & Biotechnol. Eq. 7/13/ cid complexes re minly recognized s responsile for the nti-inflmmtory (9), nti-microil (1), choleretic () nd cytotoxic (31) ctivities of the plnt. One of the most importnt types of phenolic compounds re flvonoids nd their mount is defined s linked to the ntioxidnt ctivity of plnt extrcts (17). Antioxidnts re ccountle to prevent oxidtion rections which re the mjor cuses for cellulr deteriortion (33). Medicinl plnts re generlly collected from the wild flor, which mkes the qulity of their extrcts unpredictle due to the effect of vrious environmentl fctors (1). Approprite mngement of growth conditions my e effective in incresing the yield nd qulity potentil of the plnts (). One of the most importnt environmentl fctors, nutrient deficiencies, my ffect the plnt growth, increse the production of rective oxygen species (ROS) nd enhnce the ntioxidnt ctivity displyed y phenolic derivtives (). Thus, in nitrogen strvtion conditions ccumultion of phenolic compounds such s flvonols (3), nthocynins (1) nd phenolic cids (19) ws estlished. Accordingly, decrese in the mounts of totl nitrogen, mino cids, chlorophyll nd crotenoids is reported for the nitrogen deficient-plnts of Achille collin Becker ex Rch., wheres the mount of phenolics nd ntioxidnt cpcity re higher in leves nd roots (1, 15). Furthermore, improved nitrogen nutrition leds to higher vlues for lef size, numer of flowering stlks (7), fresh nd dry weight of shoots (15, 5). Therefore, the purpose of this study ws to evlute the effect of different nitrogen mounts nd times of ppliction 39

2 on the yield, reltive wter content, totl nitrogen nd nitrogen use efficiency in plnts of the Achille millefolium grouprelted cv. Pro. Antioxidnt ctivity, flvonoid content, rte nd composition of the essentil oil were lso investigted. Mterils nd Methods Plnts nd growth conditions Plnts of the cv. Pro (relted to Achille millefolium group) were used s experimentl mteril. Seeds of cv. Pro were supplied from Phrmst GmH (Germny). Pot experiments were conducted in 9, in fully controlled climte chmer. The pots were cm in height nd 17 cm in width nd were supplied with net t the ottom to mintin infiltrtion. Ech pot hd 15 kg soil sustrte nd contined one plnt. Sterilized field soil of the cly-lomy type (. % orgnic mtter, ph 7.) ws used s sustrte. Seeds were germinted nd 7-weekold seedlings were trnsferred to pots. Plnts were grown t C to 5 C nd % to 5 % ir humidity during the whole growth period. The verge photon flux density (PFD) ws pproximtely µmol m - s -1 (1 h light/1 h drk). Tretments The study ws crried out in rndomized experimentl design with two fctors nd three replictes. The first fctor ws the nitrogen mount (mmonium nitrte), nmely g (N ),.5 g (N 1 ),.9 g (N ) nd 1.35 g N (N 3 ) per pot, corresponding to kg h -1, 5 kg h -1, 1 kg h -1 nd 15 kg h -1 N ppliction in field conditions. The second fctor ws the time of nitrogen fertiliztion performed t three different points during the growth cycle: t sowing (A 1 ), t the vegettive stge (A ), nd the pre-loom stge (A 3 ). Smpling All nlyses were done t the end of the experiment when plnts were fully loomed. The whole plnts (referred to s her) were divided into flowers (flos) nd stems nd leves (ottom herge), which were seprtely nlyzed. Dry weight nd reltive wter content (RWC) Dry weight ws determined fter drying t 35 ºC. RWC ws clculted sed on the dry nd fresh weight vlues ccording to the following formul: RWC = (FW - DW)/(S - DW), where RWC is reltive wter content, FW is fresh weight, DW is dry weight, nd S is sturted weight of leves. Nitrogen content nd nitrogen use efficiency Finely ground dry plnt smples were used for mesuring the nitrogen content y the stndrd mcro-kjeldhl method (11). Nitrogen use efficiency ws clculted sed on the rtio of totl iomss production of plnts nd totl nitrogen uptke from the soil. Rte nd composition of essentil oil The ir-dried flos smples were sumitted for 3 h to stem distilltion using Likens Nickerson type pprtus to produce the essentil oil. The components of essentil oil were nlyzed y GC MS (Hewlett Pckrd, USA) equipped with n HP 5973 mss selective detector. The cpillry column ws HP-5MS UI, Agilent Technologies, USA ( m.5 mm.5 µm). Antioxidnt ctivity Antioxidnt ctivity (FRAP ssy) ws determined ccording to the method of Benzie nd Strin (). Plnt smple (.5 g) ws ground with ml % (v/v) ethnol nd then centrifuged for min t ºC. The rection ws strted y dding.5 ml extrct to 1.5 ml FRAP regent nd.15 ml distilled wter. The sornce of the rection mixture ws mesured spectrophotometriclly (Cry 5 ) t 593 nm. The results were expressed s μmoles FRAP in 1 g dry mtter. Flvonoid content Totl flvonoid content ws mesured y the luminum chloride colorimetric ssy (31):.3 ml of 5 % NNO ws dded to.3 ml extrct. After 5 min.3 ml of 1 % AlCl 3 solution ws dded. Then, fter min, ml of 1 mol L -1 NOH ws dded nd the totl volume ws mde up to 1 ml with distilled wter. The solution ws mixed well nd kept in drkness for 3 min. The sornce ws mesured spectrophotometriclly (Cry 5 ) t 51 nm. Totl flvonoid content of plnt smples ws expressed s mg rutin in 1 g dry mtter. Sttisticl nlysis Dt were sujected to nlysis of vrince for ech prmeter. All dt were nlyzed y computer softwre (stndrd ANOVA nlysis). The mens were compred y using the LSD test descried y Steel nd Torrie (3). Results nd Discussion Dry mtter yield The effects of different nitrogen mounts nd ppliction times on dry herge, dry flos nd dry ottom herge of cv. Pro re demonstrted in Fig. 1. The highest dry herge yield (15. g/plnt) ws otined from N tretment, wheres the lowest vlues were recorded in N (13. g/plnt) nd N 1 tretments (13. g/plnt). The dry flos weight ws higher in N (3. g/plnt) nd N 3 (3. g/plnt) thn in N 1 (1.7 g/plnt) nd N tretments (. g/plnt). An increse in the dry herge yield s result of incresed nitrogen mounts in the soil ws reported for Orignum vulgre L (3) s well s for Achille collin Becker ex Rch. (15). However, plnts like Artemisi nnul L. showed no etter yield potentil with dditionl rtes of nitrogen (7). The effect of the different times of ppliction of the nitrogen fertilizer on the dry herge yield of cv. Pro ws not sttisticlly significnt (Fig. 1). The effect of higher nitrogen concentrtions on the yield formtion ws etter mrked for dry flos thn for the lower prts of the plnts (dry ottom herge) (Fig. 1). Hence, it could e suggested tht the dditionl nitrogen supply could improve the yield of flowers, the economiclly most importnt prts of the plnt. 35 Biotechnol. & Biotechnol. Eq. 7/13/

3 DrY Herge (g/plnt) Dry Flos (g/plnt) Dry Bottom Herge (g/plnt) LSD: 1.55 Biotechnol. & Biotechnol. Eq. 7/13/ LSD: 1.9 N N1 N N3 A1 A A3 Fig. 1. Effects of different nitrogen mounts ( g [N ],.5 g [N 1 ],.9 g [N ], nd 1.35 g [N 3 ] per pot) nd ppliction times (A 1 : t sowing, A : t the vegettive stge, A 3 : t the pre-loom stge) on dry herge, dry flos nd dry ottom herge weight of cv. Pro plnts. Men vlues mrked y the sme letters re not significntly different t the.5 level, using the LSD test. Totl Nitrogen Content (mg/plnt) - Nitrogen Use Efficiency (mg/g plnt) LSD: 7.5 c LSD: 1. c N N1 N N3 A1 A A3 Fig.. Effects of different nitrogen mounts ( g [N ],.5 g [N 1 ],.9 g [N ], nd 1.35 g [N 3 ] per pot) nd ppliction times (A 1 : t sowing, A : t the vegettive stge, A 3 : t the pre-loom stge) on nitrogen content of dry herge nd nitrogen use efficiency of cv.pro plnts. Men vlues mrked y the sme letters re not significntly different t the.5 level, using the LSD test. RWC RWC of plnts vried etween 35.3 % nd 39.9 % s result of nitrogen levels, the difference etween tretments eing sttisticlly non-significnt. The different times of ppliction lso hd no sttisticlly significnt ffects on RWC (dt not shown). Bhvr et l. () reported tht the wter content of plnt tissues rose with n increse in the nitrogen mount in the soil. However, there re lso findings tht RWC is not ffected y dditionl nitrogen mounts (9), which is comptile with our results on cv. Pro. Totl nitrogen content nd nitrogen use efficiency The vilility of nitrogen is the primry, considerle fctor limiting plnt growth in mny environments; therefore rections of plnts to different nitrogen mounts re suject of significnt interest. The responses of vrious medicinl plnts, such s Meliss officinlis L. (), Thymus vulgris L. (13), nd Lvndul ngustifoli Mill. (), to nitrogen fertiliztion hve een reported. In the present study the highest totl nitrogen content per plnt (35. mg/plnt) ws found in the N tretment, wheres the lowest one (5. mg/plnt), in the N tretment (Fig. ). Although n increse in the totl nitrogen content of plnts ws oserved with incresing the level of nitrogen ppliction, there ws no discernile difference fter N tretment. Correspondingly, the highest dry herge nd flos yield ws lso otined from the sme tretment (N ) (Fig. 1). The nitrogen use efficiency (NUE) incresed with ll nitrogen pplictions (N 1, N nd N 3 ) reltive to N tretment (Fig. ). However, there were no significnt differences etween N 1 (. mg g -1 ), N (.1 mg g -1 ), nd N 3 (3. mg g -1 ) tretments. An increse in nitrogen use efficiency with incresing the nitrogen pplictions is reported for Achille collin Becker ex Rch. (15). The dt of these uthors show tht the nitrogen uptke of the plnt ws. mg under nitrogen-deficient conditions nd 3. mg under nitrogensufficient conditions to produce 1 g of dry mtter. Our results clerly gree with this study. On the other hnd, the different times of nitrogen ppliction did not significntly ffect the totl nitrogen content nd nitrogen use efficiency (Fig. ). Antioxidnt ctivity nd flvonoid content The chemicl nd iologicl properties of medicinl plnts mostly chnge depending on the growing conditions, environmentl effects nd geneticl chrcteristics of the species (). Although severl studies hve een performed out the environmentl impct on the iochemicl properties of some medicinl plnts, there is limited informtion out the effect of nitrogen fertiliztion on the ntioxidnt ctivity of Achille millefolium L. In the present study the highest ntioxidnt ctivity (51.9 µmol FRAP/g DM) ws otined in the N tretment, wheres the lowest vlues were recorded for N (13.1 µmol FRAP/g DM) nd N 3 (395.7 µmol FRAP/g DM). Nitrogen ppliction t sowing (A 1 ) nd t the vegettive stge (A ) led to significnt increse in the ntioxidnt ctivity in comprison to tht oserved fter ppliction t the preloom stge (A 3 ) (Fig. 3). The flvonoid content of the plnts vried etween 7. mg rutin/g DM nd.7 mg rutin/g DM. However, oth fctors, nitrogen mount nd ppliction time, did not hve significnt effects on the flvonoid content of the plnts (dt not shown). A previous study on Achille collin 351

4 Becker ex Rch. (15) showed tht nitrogen strvtion my increse the ntioxidnt ctivity compred to nitrogen-sufficient conditions. Studies on Mtricri chmomill L. plnts lso reported decrese in ntioxidnt ctivity with incresing the soil nitrogen content (19). Accordingly, the highest ntioxidnt ctivity in the present study ws oserved in nitrogen-deficient conditions. The lck of significnt ltertions in the flvonoid content suggests tht the chnge of the ntioxidnt ctivity is result of other enzymtic or non-enzymtic fctors such s the phenyllnine mmoni lyse (PAL) nd hydroxycinnmic cid derivtives, s shown y Giorgi et l. (15). The uthors oserved significnt ccumultion of 3-O-cffeoyl quinic cid (chlorogenic cid), nd 3,5- nd,5-di-o-cffeoyl quinic cids in nitrogen-deficient Achille collin Becker ex Rch. plnts ccompnied y n incresed ctivity of PAL, key enzyme in the phenylpropnoid iosynthesis. Similr results were reported out nitrogen-deficient Mtricri chmomill L. plnts (19). Essentil Oil Rte (%) Antioxidnt Activity (µmol FRAP/ g DM),7,,5,,3,,1, LSD:.95 N N1 N N3 LSD: 3.3 LSD: 31. A1 A A3 Fig. 3. Effects of different nitrogen mounts ( [N ],.5 [N 1 ],.9 [N ] nd 1.35 [N 3 ] g N/pot) nd ppliction times (A 1 : t sowing, A : t the vegettive stge, A 3 : t the pre-loom stge) on essentil oil rte nd ntioxidnt ctivity in dry flos of cv. Pro plnts. Men vlues mrked y the sme letters re not significntly different t the.5 level, using the LSD test. Rte nd composition of the essentil oil The effect of nitrogen ppliction on the rte of essentil oil hs een investigted for different medicinl plnts. Higher mounts of nitrogen led to decrese of the essentil oil rte of Orignum vulgre L. (3), wheres no vrition ws detected in Artemisi nnul L. (7) nd Artemisi sinthium L. (). In the present study the rte of the essentil oil rnged etween. % nd.9 %. It ws not influenced y N 1 nd N supply ut significntly dropped to its lowest vlue (. %) in the N 3 tretment (Fig. 3). It ws reported tht the essentil oil of Achille millefolium L. contins minly sinene, β-pinene, 1. cineole, rtemisi ketone, linlool, α-thujone, β-thujone, cmphor, orneol, fenchyl cette, ornyl cette, (E)-β-cryophyllene, germcrene D, cryophyllene oxide, β-isolol, δ-cdinol, nd chmzulene (). In our work the presence of most of these components ws detected (Tle 1). According to Mrqurd nd Kroth (1), chmzulene is the min constituent of the essentil oil in the Achille millefolium group, reching % in some species. Our dt showed tht chmzulene is the mjor component in ll tretments, vrying etween.1 % nd 37. % of the essentil oil (Tle 1). TABLE 1 Effects of different nitrogen mounts ( g [N ],.5 g [N 1 ],.9 g [N ], nd 1.35 g [N 3 ] per pot) nd ppliction times (A 1 : t sowing, A : t the vegettive stge, A 3 : t the pre-loom stge) on the content of essentil oil components (pek re, % of totl) in dry flos of cv. Pro plnts. Components of Nitrogen mounts Appliction times the essentil oil N N 1 N N 3 A 1 A A 3 Sinene + β-pinene Cineole Artemisi keton Terpinen--ol α-terpineol β-cryophyllene α-humulene Germcrene D Cryophyllene oxide Chmzulene There is limited informtion on the effect of different nitrogen mounts on the essentil oil composition of Achille millefolium. Mnsour nd Ali () reveled tht vrious nitrogen tretments my not ffect the chmzulene content of Artemisi sinthium L. In the present work chmzulene ws either negtively (N 1, N ) or not influenced (N 3 ) y the nitrogen supply. However, little increse could e noted with the ppliction time of nitrogen closer to the looming (Tle 1).Vrile tendencies were oserved with regrd to the other essentil oil components, s ffected y the nitrogen regime (Tle 1). Conclusions As medicinl plnts re generlly collected from the wild flor, it is of fundmentl importnce to identify the optimum growth conditions for incresing their yield nd qulity potentil. Our reserch suggests generl trend of inverse reltion etween the romtic type components ntioxidnts (most proly hydroxycinnmic cids), essentil oil, chmzulene, nd the degree of nitrogen supply. This could e ccounted for y the competition of romtic nd nitrogen metolism for common cron precursors. Nevertheless, the dt point to the nitrogen regime eing sutle tool to regulte plnt performnce y improving dry mtter production without loss of essentil oil 35 Biotechnol. & Biotechnol. Eq. 7/13/

5 rte nd with no significnt dmge to chmzulene content nd ntioxidnt ctivity. Our results re generlly in greement with reserch on other medicinl plnts; the diversity of dt could e due to specificities of plnt species, orgn, tissue, developmentl stge, s well s experimentl conditions such s mount, time nd durtion of nitrogen ppliction. Acknowledgements The uthors thnk the Reserch Fund of TUBITAK (The Scientific nd Technologicl Reserch Council of Turkey) nd BAS (Bulgrin Acdemy of Sciences) for their finncil support (Project No. O9). REFERENCES 1. Aljncic I., Vjs V., Menkovic N., Krdzic I., et l. (1999) J. Nt. Prod.,, Arcı O., Byrm E. (5) Journl of Agriculturl Fculty ADU,, Azizi A., Yn F., Honermeier B. (9) Ind. Crop. Prod., 9, Bhvr N., Edi A., Toeh A., Jmti-E-Somrin S. (9) Reserch Journl of Environmentl Science, 3, Byrm E., Kırıcı S., Tnsı S., Yılmz G., et l. (1) In: VII Turkey Technicl Congress of Agriculturl Engineering, Jnury, Ankr, Benedek B., Geisz N., Jger W., Thlhmmer T., Kopp B. () Phytomedicine, 13, Benedek B., Koop B. (7) Wien. Med. Wochenschr., 157, Benzie I.F.F., Strin J.J. (9) Method. Enzymol., 99, Blumenthl M., Golderg A., Brinckmnn J. () Herl Medicine. Expnded commission E monogrphs, Newton: Integrtive Medicine Communictions (Yrrow). 1. Bongue-Brtelsmn M., Phillips D.A. (1995) Plnt Physiol Biochem., 33, Bremneri J.M., Mulvney C.S. (19) In: Methods of Soil Anlysis, Prt II, Chemicl nd Microiologicl Properties (A.I. Pge, R.H. Millernd, D.R. Keeney, Eds.), Americn Society of Agronomy, Mdison, WI, Cnter P.H., Thoms H., Ernst E. (5) Trends Biotechnol., 3, Ceyln A., Byrm E., Özy N. (199) Turk. J. Agric. For.,, Giorgi A., Licheri G.L., Mingozzi M., Cocucci M. (7) Plnt Med., 73, Giorgi A., Mingozzi M., Mdeo M., Spernz G., Cocucci M. (9) Food Chem., 11, Ktr D., Gürüz B. () J. Agric. Sci., 1, Konylioglu S., Krmenderes C. (5) J. Ethnophrmcol., 1, Kovcik J., Bckor M. (7) Plnt Soil, 97, Kovcik J., Klejdus B., Bckor M., Repck M. (7) Plnt Sci., 17, Mnsour G., Ali A. () Agr. Res.,, Mrqurd R., Kroth E. (1) Anu und Qulittsnforderungen Ausgewhlter Arzneipflnzen, 3 syf, ISBN X.. Miliusks G., Venskutonis P.R., Vn Beek T. () Food Chem., 5, Mitich L.W. (199) Weed Technol.,, Montnri M., Degl Innocenti E., Mggini R., Pcifici S., et l. () Food Chem., 17, Nms V.O., Folkrd N.F.G., Smith J.N.M. (1993) Cn. J. Botny, 71, Orv A., Ark E., Rl A. () Nt. Prod. Res.,, Ozgüven M., Şener B., Orhn I., Şekeroğlu N., et l. () Ind. Crop. Prod., 7, -.. Sfyhi H., Sierj J., Siler E.R., Ammon H.P. (199) Plnt Med.,, Schlemmer M.R., Frncis D.D., Shnhn J.F., Schepers J.S. (5) Agron. J., 97, Steel R.G.D., Torrie J.H. (19) Principles nd procedures of sttistics: A Biometric Approch, nd Ed., Mc Grw-Hill, NY, USA. 31. Stewrt A.J., Chpmn W., Jenkins G.I., Grhm I., et l. (1) Plnt Cell Environ.,, Trifunovic S., Vjs V., Jurnic Z., Zizk Z., et l. () Phytochemistry, 7, Trumeckite S., Benetis R., Bumluskiene L., Burdulis D., et l. (11) Food Chem., 17, Zhishen J., Mengcheng T., Jinming W. (1999) Food Chem.,, Biotechnol. & Biotechnol. Eq. 7/13/ 353