A simple procedure for the purifi cation of active fractions in aqueous extracts of plants with allelopathic properties

Most studies conducted to test the allelopathic activity of plant parts have made use of water as solvent. However, the presence of polar, water-soluble substances, such as proteins and carbohydrates, tends to hamper the purifi cation of active compounds. In this study, we present a simple purifi cation procedure that separates the active fraction of the extract from the undesirable substances, thus facilitating the search for active molecules through standard chromatographic methods. Aqueous leaf extracts of three Cerrado species (Caryocar brasiliense, Qualea parvifl ora and Eugenia dysenterica) were prepared at 5% concentration (w/v) and stored at 4°C (crude extracts). Aft er 24 h, these solutions were fi ltered and freeze-dried. Th e powder obtained was dissolved in methanol, fi ltered again, evaporated and dissolved in water for bioassays (purifi ed extracts). For the bioassays, seedlings of Sesamum indicum were grown for fi ve days in aqueous solutions prepared from crude and purifi ed extracts at concentrations ranging from 0.1% to 1.0% (w/v). Seedling growth in distilled water was set as a control. In comparison with the control, we found that test solutions prepared from both crude and purifi ed extracts signifi cantly inhibited sesame seedling growth. However, solutions prepared from purifi ed extracts were two to ten times more inhibitory to seedling growth than were those prepared from crude extracts. Th e inhibition of root growth ranged from 35% to 77%, depending on the plant species, at a concentration as low as 0.1%. Roots were more aff ected than were shoots. Th e eff ects of purifi ed extracts on seedling morphology were similar to those observed when crude extracts were employed, indicating that the procedure of purifi cation of crude extracts did not interfere with the mode of action of the active substances


Introduction
Th e number of studies evaluating the allelopathic potential of the Brazilian fl ora has increased considerably in recent decades, and many native species have been recognized as presenting some type of allelopathic activity.Allelopathy is defi ned as the eff ect that a plant has on the development of another organism by means of the production and liberation into the environment of chemical substances that in turn aff ect the growth of receptive species (Rice 1984).Th ese compounds, known as allelochemicals, can be released by the plant into the environment through lixiviation, wash-off of leaves by rain, root exudation, volatilization and even by decomposition of the organic matter.
Aqueous extracts prepared from plant parts of many Brazilian species, including Andira humilis Mart.ex Benth.(Periotto et al. 2004), Aristolochia esperanzae O. Kuntze (Gatti et al. 2004), Campomanesia adamantium Cambess., Qualea parvifl ora Mart., Trembleya parvifl ora (D.Don) Cogn.(Borghetti et al. 2005), Solanum lycocarpum St. Hil.(Oliveira et al. 2004), Eugenia dysenterica DC. (Pina et al. 2009) and Sapindus saponaria (Grisi et al. 2012) have been shown to have a strong inhibitory eff ect on the growth of target species such as Lactuca sativa (lettuce), Raphanus sativus (radish) and Sesamum indicum L. (sesame) (Periotto et al. 2004;Pina et al. 2009).However, none of those studies, which were designed to evaluate the allelopathic potential of native plant species, have actually identifi ed the active molecules responsible for such eff ects.Th e purifi cation and identifi cation of the active substances involved in the observed interaction has been considered an essential step if one intends to a study to be recognized as a true allelopathy study (Blair et al. 2009).
Th e preparation of aqueous extracts of plant parts and the subsequent test for their eff ects on the germination and growth of the target species have been recognized as a classic procedure in the fi eld of allelopathy (Inderjit & Dakshini 1995;Blum 2011) been seen as a model used to simulate, under laboratory conditions, the eff ect that wash-off would have on plant tissues, which would lead to the liberation of soluble substances into the environment, thereby infl uencing the growth of neighbor plants, (Ferreira 2004).In brief, the simplest laboratory procedure to test for allelopathic activity has been to grind plant tissues and solubilize them in water; to separate water-soluble from water-insoluble substances by fi ltering; to prepare solutions that are more diluted (w/v); and to test for their eff ects on the germination and growth of target species (Jacobi & Ferreira, 1991;Gatti et al. 2004;Periotto et al. 2004;Borghetti et al. 2005;Carmo et al. 2007;Pina et al. 2009;Souza Filho et al. 2010;Grisi et al. 2012).Some authors avoid grinding plant parts, because this process can dissolve cell contents that would not be released under a natural wash-off or even during tissue decomposition in soil (see Ferreira 2004).In many cases, solvents such as methanol, ethyl acetate and dichloromethane are used in the preparation of plant extracts.Th is procedure, although eff ective for the solubilization of active substances (Souza Filho et al. 2005;Grassi et al. 2005;Cunico et al. 2006;Lôbo et al. 2008;Souza Filho et al. 2010), has been viewed with caution in the description of allelopathic eff ects, given that the use of organic solvents does not refl ect what would actually occur under natural conditions (Inderjit & Weston 2000;Ferreira 2004;Blum 2011).
From an analytical perspective, the use of water as a solvent for the solubilization of active substances can hamper subsequent stages in the purifi cation process of allelochemicals, because water also solubilizes molecules that are polar but undesirable in this type of study, such as carbohydrates, amino acids and nucleic acids.Once they are in the solution, these substances make it more diffi cult to search for and purify the active molecules by conventional methods, such as column chromatography and high-performance liquid chromatography.However, the removal of water by freeze-drying and the resuspension of the powder in organic solvents (such as chloroform and ethyl acetate) can render solutions that are easier to handle in the subsequent procedures of purifi cation of the active fractions (Macias et al. 2004).Although this procedure should be avoided in an allelopathic study (Inderjit & Weston 2000;Ferreira 2004), the use of methanol rather than water for the solubilization of plant extracts facilitates subsequent chromatographic purifi cation procedures (Souza Filho et al. 2005;2010) in the search for the allelochemicals that are more polar.As an alternative, extracts to be prepared from plant tissues should fi rst be solubilized in water.If allelopathic activity is detected, the aqueous extracts could then be dried to a powder and resuspended in solvents of lower polarity to facilitate subsequent analyses.Th erefore, it becomes necessary to evaluate whether such sample preparation procedures alter the allelopathic activity of the crude aqueous extracts.In vi ew of this, the objective of this paper was to present a laboratory procedure, starting from an aqueous crude extract, that separates the active fractions from the inactive ones through simple purifi cation techniques such as freeze-drying, powder resuspension and fi lter-paper fi ltration.Our hypothesis was that this procedure would not interfere with the mode of action of the plant extracts under investigation.

Preparation of the extract solutions
Caryocar brasiliense A. St.-Hil.(Caryocaraceae), Qualea parviflora Mart.(Vochysiaceae) and Eugenia dysenterica DC. (Myrtaceae) were the Cerrado species selected for this study.Previous studies have described the allelopathic eff ects that leaf extracts of these species have on the growth of sesame (Borghetti et al. 2005;Pina et al. 2009).Mature leaves were collected from at least fi ve individuals of each species standing on the campus of the University of Brasilia (15°46'13" S and 46°52'07" W), in Brasília, Brazil.In the laboratory, the leaves were ovendried (NT-516; Nova Técnica, Piracicaba, Brazil) at 50°C for 24 h.Th ey were then lightly triturated in an electric blender (Skymsen; Siemsen, Brusque, Brazil) and immersed in distilled water in order to obtain stock solutions in a concentration of 5% (w/v).Th e stock solutions were stored at 4°C for 24 h in darkness for the solubilization of active compounds (Borghetti et al. 2005;Pina et al. 2009).Subsequently, they were vacuum-fi ltered through a qualitative paper fi lter (no.1; Whatman, Clift on, NJ, USA) to obtain a crude leaf solution of each species (crude extract).Part of each crude solution was diluted to 1.0%, 0.5% and 0.1% with distilled water to test for its phytotoxic activity on the growth of sesame.Th e remaining crude solutions were stored at −18°C and subsequently freeze-dried in a rotary evaporator (MA-120; Marconi, Piracicaba, Brazil) until a soft white powder was obtained.Th e powder was weighed and dissolved in methanol (Merck P.A.), aft er which the solution was fi ltered through a Whatman no. 1 qualitative paper fi lter.Each solution was evaporated (MA-206; Marconi), and the powder thus obtained was weighed again.Th e powder was then dissolved in distilled water to obtain a 5% concentration solution (w/v), the purifi ed extract, which was diluted to 1.0%, 0.5% and 0.1% with distilled water in order to test for its phytotoxic activity on the growth of sesame.

Bioassays
Th e bioactivity of the crude and purifi ed extracts was tested on the growth of sesame seedlings.For this purpose, seeds of sesame (Sesamum indicum L. -Pedaliaceae) were fi rstly germinated in distilled water at 30°C in a growth chamber (MA-403; Marconi), and seedlings with a radicle size of approximately 2 mm were selected for bioassays.Th e selected seedlings were immediately placed for growth on 150 × 10 mm Petri dishes (ten per plate) lined with a Whatman no. 1 qualitative paper fi lter moistened with 10 ml of each solution to be tested.Th e bioassays were conducted in the same growth chambers at 30°C, on a 12/12-h light/dark cycle (white light) for fi ve days.Th e concentrations ranged from 0.1% to 1.0% (w/v), depending on the extract (crude or purifi ed).Th e control of the experiments consisted of seedlings placed in distilled water to grow.Aft er fi ve days of incubation, the roots and shoots of the seedlings were measured with a caliper (Mitutoyo, Aurora, IL, USA) for comparisons among treatments.To calculate the percentage of growth inhibition of the treated seedling in relation to the control, the root and shoot growth of the seedlings incubated in distilled water (controls) was taken as 100%.

Experimental design and statistical analysis
At least three replicates of 10 seedlings each were employed for each treatment.Th e experiment was conducted in a completely randomized design (with the deviance information criterion), consisting of 4 treatments (extract concentration) for each extract (crude or purifi ed) and for each species.Th e average length of roots and shoots of the seedlings was compared among the treatments through ANOVA followed by a posteriori Student's t-test (Statistical package BIOSTAT 2.0, Ayres et al. 1998).Th e signifi cance level was 0.05.

Results and discussion
In this study, we have demonstrated that freeze-drying of the aqueous leaf extracts followed by resuspension in methanol does not aff ect their allelopathic potential (Tab.1).Root growth was signifi cantly inhibited by these extracts, in a dose-dependent manner, and the roots were more aff ected than were the shoots (Tab.1).Th is greater inhibition of the root in respect to the shoot growth has been observed in many allelopathic and phytotoxic studies employing aqueous extracts obtained directly from plant tissues (Jacobi & Ferreira 1991;Oliveira et al. 2004;Borghetti et al. 2005;Maraschin-Silva & Áquila 2006;Carmo et al. 2007;Pina et al. 2009;Grisi et al. 2012).
In comparison with their respective crude extracts, the inhibition of sesame growth was greater when purifi ed extracts were utilized; at concentrations as low as 0.1%, the root growth of seedlings incubated in purifi ed extracts was inhibited by approximately 34% for Qualea parvifl ora and by approximately 77% for Eugenia dysenterica (Tab.2).For the three Cerrado species, the morphological eff ects of the crude and purifi ed extracts on the seedlings were similar; there was a reduction in the number of lateral roots and in the number of root hairs, as well as reversion of root gravitropism at higher concentrations.Th e major diff erence between the seedlings treated with the crude extract and those treated with the purifi ed extracts was the length of the roots and shoots, indicating that the mode of action of the bioactive substances is not aff ected by the procedure described here.Th is method also contributes to increasing the biological activity of the extracts, probably because fi ltration of the methanol-dissolved solution removes insoluble and inactive substances.In fact, the inhibitory eff ect that the purifi ed extracts had on seedling growth was found to be 2-10 times greater than was that achieved with the original, crude solutions (Tab.2).When the proposal of a study is to reveal some kind of allelopathic interaction taking place in the fi eld, the use of organic solvents in the preparation of extracts should be avoided (Blum 2011).Instead, the use of water has been recommended in allelopathic studies because it refl ects more closely what would happen under natural conditions (Ferreira 2004).However, once the allelopathic activity of a species has been demonstrated, the solubilization of the active fractions in organic solvents may be desirable because it facilitates the subsequent steps of separation and identification of the active molecules (Lôbo et al. 2006;Macias et al. 2004).Th e procedure described here allows the purifi cation of allelochemicals that are more polar, because it helps to remove from the aqueous solutions insoluble and undesirable substances that could hamper further chromatographic analysis, a useful procedure if one intends to delve further into the study of allelopathy.

Table 1 .
Average growth of Sesamum indicum L. seedlings incubated in aqueous leaf extracts of Caryocar brasiliense A. St.-Hil., Qualea parvifl ora Mart.andEugenia dysenterica DC.Th ree replicates of ten seedlings were used per treatment.Values in the same column followed by the same letter do not diff er signifi cantly, according to the ANOVA and a posteriori Student's t-test (p=0.05).

Table 2 .
Proportional growth inhibition of Sesamum indicum L. seedlings incubated in aqueous leaf extracts of Caryocar brasiliense A. St. Hil., Qualea parvifl ora Mart.and Eugenia dysenterica DC.Number of replicates (of ten seedlings) per treatment is in parentheses.For comparisons among treatments, the root and shoot size of the seedlings incubated in water was taken as 100%.