Artigo Científico/Cientific Article Competitive enzyme ... ?· Competitive enzyme immunoassay for antibodies…

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  • 45Rev. Bras. Farm., 88(1), 2007

    Competitive enzyme immunoassay for antibodies to3-Acetyllupeol in brazilian Vernonia scorpioides

    grown under domesticationMrcia de Ftima Incio Freirea & Ronald Bastos Freireb

    SUMMARY A solid phase indirect competitive enzyme immunoassay using polyclonal IgY antibodies raised to 3-Acetyllupeol (3-AL) was applied to quantify the content of 3-AL in crude extracts obtained from stalks, leaves and rootsof Vernonia scorpioides domesticated in individual vessels containing brazilian yellow-red podsolic soil added of diffe-rent combinations Ca (NO 3)2 and of CaHPO 4. Test sensitivity for 3-AL decreased more than five orders of magnitude asthe number of 3-AL ligands on the processed plant material reduced antibody recognition of the 3-AL conjugated to acarrier protein. Soil CaHPO4 concentration was directly related to crescent root production of lupeol-acetate, sugges-ting a phosphoenzyme metabolic activation, warranting further studies on this region of synthesis. The technique isdesigned for qualitative and quantitative studies and allows ready identification of 3-AL. It may also be further appliedin the study of other plant bioactive substances lending support to certify raw products from natural sources.

    GRAPHICAL ABSTRACT A competitive ELISA was applied to quantify the triterpene named 3-Acetyllupeol (1), an

    Artigo Cientfico/Cientific Article

    Ensaio imunoenzimtico competitivo para anticorposanti-3-Acetil-lupeol Vernonia scorpioides brasileira

    cultivada sob condies de domesticaoMrcia de Ftima Incio Freirea & Ronald Bastos Freireb

    RESUMO Aplicou-se um ensaio imunoenzimtico competitivo indireto em base slida, utilizando-se anticor-pos IgY policlonais anti-3-Acetil-lupeol (3-AL), para a quantificao de 3-AL endgeno em extratos vegetaisdiferenciados (razes, caules e folhas) a partir de Vernonia scorpioides domesticada em vasos individuaiscontendo lato-solo brasileiro adicionado de diferentes concentraes de Ca (NO3)2 e CaHPO4. A magnitude dasensibilidade do ensaio decresceu ao quntuplo quando os anticorpos foram previamente neutralizados pelohapteno livre contido nos extratos e deixaram de reconhecer o 3-AL conjugado protena carreadora presos base slida. A quantificao dos extratos das plantas crescidas sob os diferentes regimes de tratamento indicouque a maior fertilidade do solo foi proporcional recuperao do 3-AL. A concentrao de CaHPO4 mostrou-sediretamente proporcional maior produo de 3-AL nas razes, sugerindo uma ativao metablica de fosfoen-zimas, indicando a possibilidade de futuros estudos nessa regio de sntese. A tcnica mostrou-se indicadapara ensaios qualitativos e quantitativos e permite uma identificao bastante precisa de 3-AL endgeno. Estametodologia pode, tambm, ser aplicada para estudos futuros com outras substncias bioativas vegetais, ofere-cendo suporte para a certificao de matrias-primas oriundas de fontes naturais.

    Rev. Bras. Farm., 88(1): 45-49, 2007

    important secondary metabolite with medicinal activity, in crude extracts of cultured stalks fromVernonia scorpioides added of different macroonutrient concentration. The technique allows rea-dy identification of 3-Acetyllupeol and may be further applied in the study of other bioactivesubstances giving support to certify raw products from natural sources.

    KEYWORDS Vernonia scorpioides; 3-Acetyllupeol; Hapten, competitive ELISA.

    Recebido em 03/11/2006 aPrograma de Diversidade Taxonmica/Instituto de Pesquisas Jardim Botnico do Rio de Janeiro/JBRJ, Rio de Janeiro, RJ, Brasil.

    bDepartamento de Biologia Animal/Instituto de Biologia/Universidade Federal Rural do Rio de Janeiro, Seropdica, RJ, Brasil

    INTRODUCTION

    T rends have been developped in many countries as good alternative to preserve medicinal plants(Freire et al, 1996, Lans et al, 2001, Calixto, 2000).

    During the two past decades, biotechnological andgenetic studies of medicinal plants were made to im-prove methods to the analysis of medicinal constitu-ents. GC-MS when used with stable isotopically labe-led carriers (Morris et al., 1991) is specific, precise and

    SUMRIO GRFICO Um ensaio de ELISA competitivo foi aplicado para a quantificaodo triterpeno 3-Acetil-lupeol (1), importante metabolito secundrio com atividade medi-cinal, em extratos brutos de Vernonia scorpioides crescidas por estaquia e adicionadade diferentes concentraes de macro-nutrientes. A tcnica permite a identificao preci-sa de 3-Acetil-lupeol e poder ser aplicada no estudo de outras substncias bioativas,como suporte para a certificao de matrias-primas obtidas a partir de fontes naturais.

    PALAVRAS-CHAVE Vernonia scorpioides; 3-Acetil-lupeol; hapteno, ELISA competitivo.

    Recebido em 03/11/2006 aPrograma de Diversidade Taxonmica/Instituto de Pesquisas Jardim Botnico do Rio de Janeiro/JBRJ, Rio de Janeiro, RJ, Brasil.

    bDepartamento de Biologia Animal/Instituto de Biologia/Universidade Federal Rural do Rio de Janeiro, Seropdica, RJ, Brasil

  • 46 Rev. Bras. Farm., 88(1), 2007

    sensitive. On the otherhand, capture ELISA is fast,very sensitive, and multiplesamples can be processed.While each technique offersadvantages over the other,the speed, sensitivity, andsample handling capabilityof the immunological metho-ds are attractive, and theyhave been widely applied tothe analysis of bioactivesubstances with variable

    method for domesticated plants certification (Freire etal., 2006).

    The time required for plant has been a problem.Many variations for rapid assays have been proposed,but most require extensive preparation and solventextraction. The sample size also has been a substantialproblem overcome by the immunological methods, allo-wing many more samples to be handled at one time.This in effect provides for multiple parallel analysis ofdifferent (or replicate) samples. The anti-3-AL IgY atthe 1: 2000 work dilution were 100% specific to the hap-ten conjugated to chicken egg albumin (OVA), a carri-er protein different from bovine serum albumin (BSA),the carrier protein used to elicite the anti-hapten res-ponse in the immunized animals (Fig. 2).

    The 3-AL competitive ELISA showed a strict com-patibility with the therpene presence in each extractassayed. The signal inhibition, not detectable when 3-AL were absent, was ever inversely proportional to theamount of antigen in solution and correlated with theamount of 3-AL present in the sample. It was not asurprise, once the prediction values for the competiti-ve ELISA were settled every time as high as 80%. Be-cause of that, it could be determined high lupeol-ace-tate concentrations even when the extract yield was notthat big, and vice versa.

    Thus a substantial time element is reduced by ena-bling direct measurement of 3-AL in its biologic sour-ces. For pure grade 3-AL, the LOD was found to be100pg/mL (5pg/well). The 50% intercept of the calibra-tion curve was 1.5ng/mL (75pg/well). The working rangeof the assay for pure grade 3-AL, as with all other ma-trices (roots, stalks and leaves), was 0.124ng/mL (51,200pg/well), corresponding to 0.01-4.8ng 3-AL/500mgV. scorpioides stalking in soil added of different macro-nutrient concentration (Figure 3).

    The intra and interassay variation-coefficients es-tablished through V. scorpioides extracts and puregrade 3-AL spiked fractions did show 100% of concor-dance, with no significant difference to the estimationof 3-AL concentration obtained for both, the pure gra-de 3-AL and the 3-AL content in all the extract sam-ples. In all cases, 3-AL alone or in mixture producedthe same inhibition curves (Figure 4).

    In all plants, there exists a complex conjunction ofmetabolites, which may change according to their en-vironmental and genetic profiles in order to guaran-tee their best survival (Emerenciano et al., 1998, Got-tlieb & Kaplan, 1993).

    The plant response to the environmental stress con-

    FIG. 1 - Structural formula of 3-acetyllupeol (1)

    degrees of success (Honda et al., 1981, Morris et al.,1991, Avrameas et al., 1992, Abramson et al., 1995, Ri-beiro-Neto et al., 1997, Strachan et al., 2002, Almquistet al., 2004, Lapc c k et al., 2004).

    Competitive enzyme immunoassays (competitiveELISA) are widely used because they are rapid, sim-ple, accurate and specific. They are also a cost-effecti-ve way to quantify many biologically important mole-cules such as steroids (Lap c c k et al., 2003), peptidesand nucleic acids (Brown-Augsburger et al ., 2004).

    Recently we reported an accurate detection methodfor a rapid recognition to 3-Acetyllupeol (3-AL), a bio-logically active triterpene (1) (Fig. 1) naturally occur-ring in brazilian V. scorpioides, using IgY (egg antibo-dies) raised in hiperimunized hens (Freire et al., 2004).We now report a competitive ELISA to determine therelative quantitation of LAc produced by greenhousepropagated Vernonia scorpioides stalks in brazilianyellow-red podsolic soil added of phosphorus (CaHPO4)and nitrogen (Ca (NO3)2). The specificity of IgY selec-tion and resolution adds a biosynthetic origin informa-tion.

    2. RESULTS AND DISCUSSION

    It was the first time a coompetitive ELISA was carri-ed out to detect the relative concentration of 3-AL con-tent in domesticated V. scorpioides. The success of thiskind of assay to quantify different haptens is well do-cumented (Suzuki et al, 2000) and is accomplishedby the sample-antibody preincubation before additionto microtiter plates.

    The competitive ELISA can be used to measurebioactive chemicals in this study quickly and accura-tely, without sample pretreatment or dilution. Beingsensitive, rapid and accurate with a simple plant frag-ment sample homogenate; this in vitro assay appearsto be a rapid medicinal source monitoring and a good

    FIG. 2 - Influence ofspecificity in indirect ELISA.Phytohapten-conjugated tochicken egg albumin (OVA-3LAc, 250 ng/mL in PBS) orcontrol chicken egg albumin(OVA, 250 ng/mL PBS) wereadsorbed to plastic wells ofimmunoplates beforeblocking. Diluted anti-3-ALIgY antibodies wereincubated on the plate andpresence of immobilizedantibodies was revealed by athree-step amplificationprocedure. The serumdilution factor (1,000)produced a non detectablebackground signal to OVAand a sensitive specific OVA-3LAc signal (arrow). n = 4,error bars indicating SD.

    FIG 3 - Competitive inhibitionof IgY antibody binding toOVA-3-AL-coated plates byfree 3-AL. Phytohapten-protein conjugates (OVA-3-AL, 250ng/mL in PBS) wereadsorbed to immunoplatesbefore blocking. Antibodies(1/1,000 in PGT) wereincubated on the plate andafter washing steps,immobilized antibodies wererevealed using the two-stepprocedure. Results areexpressed as inhibitionpercent from control. Linesare indicative of theconcentration of 3-AL thatcaused 50% of signalinhibition. Arrows indicatethe lowest (0.1ng/mL) andthe highest (24ng/mL)detection considered asdetection limits for the test(n = 4, in all cases SD was

  • 47Rev. Bras. Farm., 88(1), 2007

    ditions should be unpredictable in many aspects (Wa-termam & Mole, 1989). As the metabolic condition togenerate bioactive terpenes seems to be a result of theindividual constitution (Dovjak & Andrascik, 1987; Cor-ra-Jnior, 1998), the environmental nutrients dispo-sition may be strategically modified to benefit the desi-red metabolism.

    Unfortunately, the knowledge of how to cultivate and,at the same time, maintain the bioactive delivering wi-thout depriving their natural behaviour continues tobe a problem to be solved, due to the few accessibleinformation about the best irrigation condition, the soilpH correction needed and the fertilization of the groundnecessary for a successful cultivation.

    We found in these samples differences correlatedthe soil treatment with macronutrients but neither re-lated to the intrinsic qualities of the soil or the matri-ces processing. As the yellow-red podsolic soil, the

    natural soil from which samples were taken from, hasa poor nutritional value there was no over loading ofnutrients to the plants, approaching the stalking con-dition very closely to the natural behavior V. scorpioi-des presented at the time it was collected.

    Stalkings propagated in soil added 200kg.ha-1 Ca(NO3)2 presented a great aerial part development,while those added 80 kg.ha-1CaHPO4, great rooting.Nevertheless, the plant relative biomass productionseems to exert no interference on the 3-L.Ac yiel-ding in the processed aerial part of the plant. Basedon the high accuracy of the test assay we observedinorganic nitrogen added to the soil, despite of in-fluencing on the total weigh of fresh collected stalksand leaves did not interfere augmenting the relative3-AL concentration of in any sample (Fig. 5).

    Similarly, the yielding of the triterpene for eachgram of dry root was constant without a significantcorrelation to the overall biomass production, inde-pendently of the time plant samples were haversted.

    The inorganic phosphorus disposition is an es-sential feature of Vernonia scorpioides artificial gro-wth on driving the secondary metabolism to genera-te a cumulative yield of 3-AL which decreased withthe nitrogen augmentation, probably favoring 3-ALmigration to the leaves under nitrogen feeding cir-cumstances.

    In fact, ATP is definitive for phosphatase to ge-nerate therpenoids from isoprene unities (Bramley,1997) and iso-penthenyl-pyrophosphate formation towhich the high-energy inorganic phosphorus is in-trinsically necessary. The clear needs of ATP seemedto be obvious since, as it was already demonstrated,deficiency of this constituent leads to sugar accumu-lation in all parts of the plant (Maia, 1998; Shuman,1994).

    Terpenoids have a preferential radicular origin inseveral natural sources of terpenes as observed un-der natural conditions (McCaskill & Croteau, 1998;Flores & Curtis, 1992; Flores et al. 1999). Being anindependent variable, this macronutrient might exerta down regulation on the genetic expressions of ter-penes in cultured plants, during the developmentalstages of the plant organism or tissue leading theyoungest stalkings to concentrate more 3-AL in theroots and the eldest to acumulate higher 3-AL con-tent in their leaves and stalks.

    Unfortunately we could not establish a long termand a short period exposure, warranting a further time-course experiment to determine the 3-AL productionin the different parts of the plant. Previous experi-ments, however, demonstrates there is a seasonal pat-tern that directs the terpene content and emission inplants naturally grown under field conditions (Llusiand Pauelas, 2000). Our observations corroboratewith these findings and use that plants domesticationto give good results if stalks cultured in the presenceof ideal nutrients concentration. Finally, it must be em-phasized that anti-hapten antibodies, constitute the-mselves a very sensitive tool for tracking bioactive mi-cro molecules. Being reproducible, it certainly will behelpful on generating, further comprehensive and lessexpensive methodology for accessing the expressionof important medicinal drugs as well as from substan-ces, warranting further studies of...