Quantitative Fluorimetric Analysis of Plant Nicotinamide

ISINGHUA SCIENCE AND TECHNOLOCYISSN 1007-0214 05/21 pp417 - 420Volume 6. Number 5. Deember 2001Quantitative Fluorimetric Analysis of Plant NicotinamideWANG Zebin (王泽斌), FENG Wenzheng (冯闻铮),CAO Zhu'an (曾竹安), LIU Jinyien (刘进元)“Q94ADepartment of Biologlcal Seiences and Biotebnology, Tsinghun University, Beijing 10084, ChinaAbstract:Fluorlmetry was used to measure the amount of nicotinamide in plant samples. The nicotinamidewas extracted and puritfied from plant tsses with thy| acetate; converted to the fluorescent derivative,N-methylnicotnamide, by reacting with methy! idide; and quantitied according to its ftuorescent strength.The nicotinamide in the leaf tssue of ten kinds of plants was measured, and the results showed that thenicotinamide content for dfferent plants varied from 0.1 to 3.0 g/g of fresh leaf weight. In addition, thecrossing value of the fluorescent strength and the nicotinamide amount demonstrated that the linear correlationcefficient generally reached 0. 997, with a detectable limit of 0. 02 mg/L and the relative standard deviation ofless than 9%. The results suggested that this method of quantlfying niootinamide in plants is useful andbeneficial tor functional research.Key words : plant nicotinamide; quantiative detection; fluorimetric methodmutagens or UV light, could activate in virvo polyIntroduction( ADP-ribose ) polymerase( PARP )， whichNicotinamide (NIC), also called vitamnin B: ordegraded nicotinamide adenine dinucleotide (NAD)vitamin PP，functions as a plant growth factor 10to produce polymers of ADP-ribose by releasingincrease the dry weight of plants and fresh fruitNIC. When NIC exceeded certain thresholdthrough improving photosynthesis, stimulatingamounts，it then reduced the activity of PARPCO2 absorption， and restraining respiration[-s.stabilizing NIC content in plants(s.s. However ，Furthermore , recent research reported that NIC inlittle is known about the action mechanism of NICplants might be related to their defensein plants.mechanisms. There was an increase in the activityTo investigate NIC's action mechanism, itof phenylaniline ammonialyase (PAL) and chalcorewas first necessary to find a suitable assay systemsynthase (CHS) in plants treated with NIC, whileto quantify the plant NIC. NIC quantifications inPAL was one of the critical enzymes inanimal tissues have been done-1o], but almost allphenylalanine and flavonoid pathways whichof these used methods such as polarography?,included many key substances involved in defensemechanisms]. On the other hand， strandchromatograph (HPLC)9, could not be applied tobreakage in DNA, caused by various stresses suchquantification of plant NIC due to the lowas oxidative stress, treatments with varioussensitivity and selectivity of the methods or due tothe low NIC content and the complicatedcomponents in plant tissues.Although highlyReceived: 2000 12-27* Supported by the National Natural Science Foundationsensitive and selective, radioassayloJ requiredof China (No.39780030) , the International Foundationconsiderable time and effort, and had the safetyfor Science (No. C/1992-1). and Fund of the Ministryand economic shortcomings making it difficult toof Education.be used for plant NIC quantification. The# *To whom correspondence should be addressedfluorin中国煤化士serum NICTHCNMHGrsinghua Seience and Technology, December 2001, 6(5): 417-420418analysis by Clark[1] was fast, accurate and safe fortwice with 10 mL water -saturated ethyl acetate. .quantifying plant NIC, and therefore, was used forThe combined water phase was added by 0. 25 mL.this work.o[ NaCl-saturated 0. 75 mol/L NazHPO、 anNIC, as well as its derivative nicotinic acid,extracted with 10 mI dry ethyl acetate. The 1 Idid not fluoresce under UV iradiation. Since thereresultant ethyl acetate phase containing NIC waswere a pyridyl and amido groups in NIC molecules,transferred to an Eppendorf tube, and the ethylhe NI_ alkyl-pyridinium derivative, N-methyl-acetate was evaporated overnight in a water-nicotinamide fluoresced under the ultraviolet toaspirated vacuum desiccator until it was dry. Theallow the quantification of plant NIC by adried NIC samples were analyzed fluorimetricallyspectrophoto-fluorometer. In this paper,for N'-methylnicotinamide as presented below.fluorescent quantitative assay system01 was1.4 Fluorimetric assayapplied and the NIC of leaf tissues in ten kinds ofplants was determined. This appears to be the firstThe dried NIC sample was dissolved in 0. 5 mLapplication of fluorescent quantification of plantmethyl iodide and stirred until it was completelyNIC.dissolved. Then， the solution was placed in thedark for 24- 30 hours.Afterevaporation of the1Methods and Materialsmethyl iodide in the chemical hood, 0. 1 mL of10% acetophenone in 80% ethanol was added.1.1 PlantsAfter being completely mixed， 0.1 mL ofThe plants used in this study were eggplant1.0 mol/L KOH in 80% ethanol was added andSolanummelongena )chili( Capsicumstirred immediately. Exactly 8 min after thefrutescans), kangkong (Ipomea aquatica), lettuceaddition of alcoholic KOH, 1. 0 mL of 99% formic( Lactuca sativa )，three-coloured amaranthacid was added and mixed immediately to terminate( Amaranthus tricolor )，rape ( Brassica napus ),he reaction. The order of reagent addition wascabbage (Brassica oleracea var capitatas), tomatoreversed for the tube serving as an extract blank.( Lycopersicon esculentum )，spinach ( SpinaciaThus, to each blank, 1.0 mL of 99% formic acidoleracea)，and cotton (Gossypium herbaceum ).was added first， followed by the addition ofYoung leaves from mature plants grown in fields0.1 mL of 1.0mol/L KOH in 80% ethanol ancwere used for the NIC extraction.then, after mixing, 0. 1 mL of 10% acetophenonein 80% ethanol. After standing for 4 - 6 hours,1.2 Chemicalsthe resulting solution was measured at an emissionPurified NIC was purchased from Sigma, all otherexcitationchemicals were domestic of analytical grade.wavelength of 370 nm on a spectrophoto-fluorometer (Hitachi- 850).1.3 Preparatlon of NICA series of standard solutions containingAccording to Berflund's methodl2], 5.0g freshnicotinamide was prepared in 0. 1 mmol/L aqueousleaves were completely homogenized with siliconHCl and processed as desctibed above. A pre-dioxide using a mortar and pestle, and extracteddetermined amount of the solution's ethyl acetatewith 5 mL of boiling H2O. After being centrifugedphase was added to the same phase of the extractat 7000 r/min for 5 min, the residue was re-sample as an internal standard.extracted twice with the same volume of boiling2 Results and Discussionwater and the combined water solution wascentrifuged at 7000 r/min for 10 min. The2.1 Application of the fluorimetric method toresulting supernatants were adjusted to the volumeplant NIC quantificationof 20 mL with H2O and used in the next step toisolate the plant NIC.The method for quantifying NIC from serumIn order to isolate the NIC from the extracteddescribed by Clarl] was partially modified anwater solution, the method described by Clark[1]used to isolate NIC from plant leaves. After beingwas modified. 300 mg NaCl and 0. 25 ml of 0. 3converted to its fluorescent derivative, the plantmol/I. HCl were added to 0. 5 mL aliquot of theNIC from fresh tissue was quantified using aaqueous supernatant, which were stirred on aspectrophotofluorometer. Using Clark's method ，Vortex mixer until the NaCl dissolved. Thewater-saturated ethyl acetate was used to extractresultant saturated NaCl solution was then washed中国煤化工:etophenone. In thisYHCNMHGWANG Zebin (王泽斌) et al; Quantitative Flworimeric Analysis of Plant .....419step, some of the NIC in the water phase was lost,the volume of the extract sample (Lfollowed by discard of the organic layer，increasingThe NICcontents for three -colouredsomeunavoidable error. The accuracy of theamaranth, cotton and chili were 2. 12, 1. 09 andresults would have been better if this step had been0.36 rg/g, respectively.ormitted. After extracting NIC from the aqueous50(A)(BC)(D)solution with ethyl acetate, only 1 mL of the40methyl acetate phase was desiccated in the next30step. If the NIC content was high enough, a smallvolume was used to decrease the evaporation time.出20When the NIC content was low, the volume of the10solution needed to be greater for satisfactoryresults.0 42044040 40440 420 40 460、420 440 460Since the sample impurities might haveWavelcngth (am)diminished the fluorescent strength and theFig.1 Fluorescence spectra of the solutions fromH2O (A), three-coloured amaranth (B), cotton (C)compositions of extracts from different plants wereand chl]i (D). ( 一: the extract sample containing thedifferent，relative fluorescent strength ( RFS )interna] standard, RFS,+1;he extract sampleproduced by the same amount of NIC in differentalone. RFS,; .... : the extract blank, RFSb)samples varied. The RFS for the same amounts ofhe internal standard in H2O， three colouredAfter each standard solution of 0-0.1 mg/Lamaranth, cotton and chili were 16.5, 8.5， 12. 0,was obtained as the procedures described in theand 14. 6, respectively, with a standard deviationmethods section, a standard curve was made. Theof less than 5% (Fig. 1). The different RFSlinear relative coefficient was 0. 997, the relativevalues for the extract sample containing thestandard deviation was less than 9% and theinternal standard and for the extract sample weredetectable limit was. 0. 02 mg/L. The resultsconsidered as resulting from the internal standarddemonstrated that the amounts of NIC in plantin that sample. Similarly ，the different RFS valuestissues could be quantified using this fluorimetricfor the extract sample and the extract blankmethod.tesulted from the authentic NIC of that extract2.2 NIC contents in 10 kinds of plantssample. The ratio of the internal standard to theundetermined NIC should have been equal to thatNIC contents in 10 kinds of plant leaves wasof the RFS produced by both. Thus, the NIC wasquantified using this method. As shown in Fig.2,reversely quantified according to its fluorescentthe NIC content in different plants varied fromstrength.approximately 0. 1 to 3, 0 ug/g of fresh leafThat is,weight. The NIC content in eggplant leaves wasRFS,+i- RFS._ VCi2.9 1ng/g, while that in spinach leaves was onlyRFS,- REFS。= v,C:0. 19 pg/g. The NIC content in eggplant leavesTherefore,was approximately 15 times greater than that inVIC(RFS, - RFS%)V,(RFSs+i- RFS,)Here RFSo+iis the RFS of the extract samplecontaining the internal standard; RFS, is the RFSof the extract sample; RFSb is the RFS of theblank; C, is the NIC concentration of the extractsample (mg/L); Ci is the NIC concentration of theinternal standard added (mg/L); V, is the volumeof the extract sample treated (L); V; is the volumeof the internal standard added (L) .5678910PlantsWhile NIC content in a leaf was calculatedFlg. 2 NIC content in 10 kind o{ plants. Results areusing the following equation:presented as the mean (士SD) from at least threeindependentexperiments.1- 10representsMsuccessively rape, tomato, cabbage, three-colouredHere C is the NIC content of the leaf,amaranth. epenlant. sninarh. chiti. lettutuce, kangkong(ug/g); M is the weight of the leaf (g); and V.isand a中国煤化工FYRCNMHG420Tsinghua Science and Technoiogy, December 2001, 6(5): 417 -420special reference to oxidative stress in plants. FEBSspinach leaves.The results showed that plant NIC could beLetters, 1994, 351; 145- 149.quantified quickly, accurately and safely using the5] Yang Qiwei, Chen Defeng. Structure, expressionregulaion of gene of poly ( ADP-ribose )fluorimetric method. Compared to other methods,polymerase. Chinese Bulletin of Life Science, 1996,this method had the following advantages. First, it8 (3): 13- 17. (in Chinese).was convenient， fast andcould simultaneouslytreat many samples. Secondly, it was economical,6] Suginura T， Miwa M. Poly ( ADP -ribose ):Historical perspecive. Mol Cell Biochem， 1994,costing only 1% of the cost of radioassay.138: 5-12. .Thirdly, it produced no radioactive pollution.[7] Xin Hong, Yuan Zuobin, Kuang Pingxian, et al.Although the sensitivity may have been lower thanSimultaneous determination of mixture of vitamins bythat of the radioactive method, it still satisfactorilyusing single sweep oscillopolarography and targetquantified plant NIC. In this study, the NICfactor analysis. Analyical laboratory, 1997, 16(4);content in only young leaves of ten kinds of mature21 - 23. (in Chinese)plants were studied. The NIC content in different[8] Dang Quanxun. Sun Zengpei, Ling Dakui. Analysistissues, at different stages of growth and under>f nicotinamide and vitamin B。 by micellardifferent growth conditions also needs to beelectrokinetic capillary chromatography. Chinesemeasured. 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