Dalbergia brasiliensis Vogel extracts in vitro antioxidant activity and phytotoxic effect in Lactuca sativa and Allium cepa

Based on medicinal use of Dalbergia species, the present study aimed to evaluate D. brasiliensis leaves and barks crude extracts and fractions’ antioxidant and phytotoxic potential, employing allelopathic essays and 3 antioxidant essays: Phosphomolybdenum complex reduction, DPPH free radical scavenging and TBARS (lipid peroxidation). Concerning Phosphomolybdenum complex reduction potential, leaves chloroform fraction (LCF) and barks ethyl acetate fraction (BEAF) overcame rutin’s reduction potential in 90% and 23%. DPPH essay revealed leaves ethyl acetate fraction (LEAF) antioxidant potential (IC50 de 40,629 μg/mL), given standards ascorbic acid (IC50 = 4,503 μg/mL) and rutin (IC50 = 7,330 μg/mL) activities. TBARS essay demonstrated that leaves hexane fraction (LHF) (51%) and barks ethyl acetate fraction (BEAF) (41%) stand out when compared to BHT antioxidant activity (54%). Regarding phytotoxicity, the lowest crude extracts and fractions concentration (250 μg/mL) employed were able to inhibit L. sativa and A. cepa primary root initial growth, specially leaves hexane fraction (LHF) and leaves chloroform fraction (LCF). Dalbergia brasiliensis fractions might function as stress inhibitors in oxidant systems and alter meristem cellular division, due to present substances’ activity.


INTRODUCTION
A wide range of plants produce compounds, which, in certain amounts, may provoke toxic effects to surrounding plant species (Harborne, 1994). Allelophaty defines plant interactions employing phytotoxins, once donor plant negatively affects target plant, providing advantage to donor plant (Dayan & Duke, 2009). Secondary metabolites are able to bring on beneficial and harmful effects, depending on some characteristics such as amount, chemical properties and present functional groups (Barbosa et al., 2005;Goldfarb et al., 2009). Also, are considered as greatly responsible for allelophatic effects observed in nature, since these compounds are important agents against natural predators and ultraviolet radiation, including simple phenolic and heteroside compounds, coumarins, xanthans, tannins, flavonoids, saponins, alkaloids and sulfur compounds (Haida et al., 2010;Jácome et al., 2004).
Dalbergia brasiliensis, popularly known as jacarandá, caroaba-brava, caviúna or marmeleiro, is distributed among Cerrado regions and Brazilian Atlantic Forest (Carvalho, 2004). Dalbergia brasiliensis research scarcity along Dalbergia genus known biological and pharmacological potential, encouraged antioxidant activity evaluation by three methods (phosphomolybdenum complex, TBARS and DPPH) and allelopathic potential evaluation on Lactuca sativa e Allium cepa inicial growth.
Plant material taxonomic identification was determined at Municipal Botanic Museum Herbarium (Curitiba) and kept at room temperature for 10 days, until dehydration. Dryed plant material was stored in plastic bags, protected from light and heat.

Crude Ethanolic Extract and Fractions
Leaves and barks crude ethanolic extracts were obtained in Soxhlet apparatus, employing ethanol 96º GL, approximately at 78ºC. Crude ethanolic extracts were vacuum filtered (Büchner) and concentrated in rotary evaporator (reduced pressure, 78ºC, 150 rpm). Crude ethanolic extracts were fractioned by liquidliquid partition in modified Soxhlet apparatus (Carvalho et al., 2009) employing solvents in increasing polarity scale, obtaining leaves' and barks' hexane, chloroform and ethyl acetate fractions. Crude ethanolic extracts and fractions were evaporated (water bath, 50º C) and stored in cool dry place.

Antioxidant Activity Evaluation
Leaves and barks crude ethanolic extracts and fractions had in vitro antioxidant potential evaluation determined employing three different methods.

 Phosphomolybdenum complex reduction
Samples' ability to reduce phosphomolybdenum complex were evaluated according to Prieto et al. (1999). Samples were diluted in metanol (200 µg/mL). Reagent solution (complex solution) containing sodium phosphate 0,1M solution (28 mL), ammonium molybdate 0.03M solution (12 mL) and sulfuric acid 3M solution (20 mL) had it final volume adjusted to 100 mL. Sample solution was added (0.3 mL) to reagent solution (3 mL) and test tubes were kept sealed in water bath (95º C) for 90 minutes. After cooling, solutions were analyzed in spectrophotometer (Shimadzu, UV 1601) at 695 nm.
Results showed samples' Relative Antioxidant Activity (RAA%) when compared to standards (ascorbic acid and rutin) by following equation: Samples' DPPH scavenging potential were evaluated according to Mensor et al. (2001). Samples and standards (rutin and ascorbic acid) methanolic solutions were obtained (15 to 125 μg/mL, except hexane fractions: 100 e 450 μg/mL). To each solution (2.5 mL), DPPH methanolic solution (0.03 mmol/mL) was added (1 mL). Blank samples (2.5 mL of sample solution and 1 mL of methanol) were also evaluated. After thirty minutes, samples, standards and blank samples were analyzed in spectrophotometer (518 nm, which corresponds to maximum DDPH absorption). Samples' ability to scavenge DPPH was determined as following: Samples IC 50 (50% of inhibitory concentration) were determined by linear regression, establishing an y=ax+b equation, for comparing purposes.

 Thiobarbituric Acid Reactive Substances (TBARS)
Samples' ability to inhibit lipid peroxidation were evaluated according to adapted Moraes et al. (2006). Samples were diluted in methanol (1000 ppm) and butylated hydroxytoluene (BHT) was employed as standard. Filtered egg yolk (5%) in sodium dodecyl sulfate solution (0.55%) was employed as lipid source.
Each Petri dish was randomly sown (30 seedlings) with Lactuca sativa and Allium cepa, separately in quadruplicates, as described in Brasil (2009). Sown Petri dishes were placed in BOD germination chamber (relative humidity ± 80% and constant temperature: 25º C for lettuce and 15º C for onion). Lettuce seedlings were incubated in the dark and onion seedlings in 12 hour photoperiod, as suggested in Brasil (2009).
Germination bioessays followed Macias et al. (2000) methodology. Data collection was performed daily for seven days, evaluating radicle protusion (minimum length 2.0 mm). Essay conclusion was attested when germination became nule for three consecutive days. Speed Germination Index (SGI) was determined according to Maguire (1962), as shown on following equation: Growth bioessays followed Barnes et al. (1987) and Macias et al. (2000) described methods. After seven days of radicle protusion, root and hypocotyl/coleoptile were measured over millimiter paper (10 seedlings from each Petri dish).

RESULTS AND DISCUSSION
Essays performed on D. brasiliensis extracts and fractions, demonstrated fractions' antioxidant activity varied between in vitro methods (Table 1).
Free radicals or reactive oxygen species are generated by exogenous chemical products or endogenous metabolic processes and are able to oxidize biomolecules, such as nucleic acids, proteins, lipid and DNA. These molecules may initiate different degenerative illnesses like neurological disturbs, cancer, emphysema, cirrhosis, arthritis, atherosclerosis, among others (Halliwell, 2007;Sies, 1997).
Mammals natural antioxidant mechanisms are not always efficient, therefore antioxidant compounds intake becomes an alternative supply, pointing lower rates of antioxidant-related diseases (Antolovich et al., 2002). Supplement ou tea intake protects organism against free radical effects (Szent-Giörgyi, 1963). Many medicinal plants showed antioxidant activity throught pharmacological traditional methods for the past two decades (Kasote et al., 2013). Knowing that, the present study aimed evaluating D. brasiliensis antioxidant activity, since other genus species are traditionally known for its antioxidant properties.
Etnophamacologycal and nutraceutical in vitro investigation often employ antioxidant evaluation methods to track and verify plants and its phytochemicals antioxidant potentials. Still, many times, plant extracts oxidant mechanism are difficult to comprehend. Therefore, the present study evaluated antioxidant activity employing methods based on antioxidant mechanism, like DNA damage prevention, lipid peroxidation inhibition or ammonium molibdate reduction (Antolovich et al., 2002). Results on lettuce and onion initial growth show D. brasiliensis allelopathic potential on seedlings, as growth parameters evaluated on laboratory were altered. All samples in every concentration were able to inhibit lettuce germination period. LHF and BHF provided accentuated delay, when compared to other samples. BAEF provided similar effect to BHF, as shown on Table 2. Hormesis effect was observed in onion seedling treated with LCEE and LHF, revealing inhibitory activity in 250 µg.ml -1 concentration (75.91 e 74.43, respectively). LCF and LEAF boosted onion seedlings germination speed in all concentrations in a non dose-dependent mean. All BCEE and BEAF concentrations inhibited onion seedlings germination speed and LHF and LCF (both in 500 and 1000 µg.ml -1 ) provided most damages, revealing selective mechanism to monocotyledons (Table 3). Concerning primary root, samples' 250 µg/ml concentrations were able to affect lettuce root growth, highlighting LCF (24%) and BCEE (17%). Inhibitory effect was also observed in LHF (500 µg/ml: 11.1% and 1000 µg/ml: 11.44%) while LEAF (1000 µg/ml) boosted root growth in 16.15% ( Figure  1A and B). BEAF 1000 µg/ml concentration also showed primary root growth inhibition in L. sativa in 11% ( Figure 1B).
L. sativa and A. cepa root growth were most affected than hypocotyls and coleoptiles. Baziramakenga et al., (1995) emphasizes that, besides root contact with extracts, root tissues are more permeable to allelochemicals which may alter tissues' initial growth (Nishida et al., 2005), damaging root methabolic activities and cellular division on root extremities that might lead to dry weight reduction (Singh et al., 2003).
Over the present investigation, negative impact observed on discussed parameters is possibly due to allelochemicals large amounts, which might, significantly or not, have raised stress levels on L. sativa e A. cepa. Then, in line with plant activity studies strategies' on evaluating antioxidant and phytotoxic potential, it is advisable to combine in vivo antioxidant assays and toxicity evaluation.

CONFLICTS OF INTEREST
The authors declared no conflict of interest.