Potentialization of antioxidant activity of Piper umbellata ( Piperaceae ) leaves after their metabolism in Heraclides brasiliensis larvae ( Lepidoptera : Papilionidae )

Piper umbellata leaves accumulate the compound, 4-nerolidylcatechol, which is responsible for the antioxidant power of the plant. Surprisingly, in this study we observed a significant increase in the antioxidant activity of P. umbellata leaves after their metabolism on Heraclides brasiliensis larvae. The total phenolic concentrations of the extracts from the leaves and fecal matter (metabolized leaves) measured using the Folin-Ciocalteu reagent, were 14.78 and 27.13 mg GAE/g, respectively. The antioxidant activity, based on 2,2-azinobis 3-ethylbenzothiozoline-6-sulfonic acid (ABTS), was EC50 of 120.10 and 62.50 ug/ml for the leaves and fecal material, respectively. These values were correlated with the total phenolic content.


INTRODUCTION
Piper umbellata (L.) Miq.(Piperaceae) [syn.Heckeria umbellata (L.) Kunth., Pothomorphe umbellata L., Piper hilarianum Stend.],known popularly as "caapeba" in Brazil and "cowfoot" in English speaking countries, has been reported to be a potent antioxidant (Kijjoa et al., 1980).This antioxidant activity of P. umbellata is attributed to its major constituent, 4-nerolidylcatechol (1), detected in its root and leaf tissues (Pinto et al., 2010;Ropke et al., 2003;Noriega et al., 2008a;b).Others minor compounds (N-p-coumaroyl tyramine, piperumbellactam A, B and C) isolated of P. umbellata branches showed antioxidant activity (Tabopda et al., 2008).The compound (2) during metabolism of the leaves by Heraclides brasiliensis, a host insect (Figure 1) (Ramos et al., 2012).Whereas alterations in the chemical profile of plant tissues can  Dried leaves of P. umbellata (10 g, 40°C for 48 h) were milled and subjected to ethanol extraction (120 m) twice, and then concentrated in a vacuum to yield 0.91 g of crude extract.Five individuals of H. brasiliensis were collected on P. umbellata leaves and were maintained in cages for a month under a diet consisting of P. umbellata leaves, with feces collected daily.Dried feces (2 g, 40°C for 48 h) were milled and subjected to two methanol extractions (100 ml), which after concentration in a vacuum yielded 0.25 g of crude extract.

LC-MS Analysis
HPLC analysis of extracts and pure compounds was performed with a Shimadzu chromatograph model SCL-10A with UV-VIS detector, using a Supelco reversed phase column.Elution was carried out in a gradient mode starting with acetonitrile:water (4:6) for 10 min, rising to (9:1) in 30 min and maintained for to 35 min.The flow rate was 1 mL/min; injection volume 20 μL; UV scan, 200-400 nm, and all chromatograms were obtained at λ max = 214 nm.

Antioxidant activity
Significant levels of antioxidant activity of the leaf and fecal extracts were found using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS •+ ) radical scavenging assays (ABTS •+ scavenging method).The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging activity of the isolated compounds was measured as reported in the literature (Slinkard and Singleton, 1977) with some modifications.Briefly, ABTS •+ was generated by reacting ABTS solution (7 mM) with potassium persulphate (2.45 mM, final concentration) for 12 -16 h in the dark at room temperature.Then, the ABTS •+ solution was diluted with ethanol to obtain absorbance of 0.70 (± 0.02) at 734 nm and further equilibrated at 30°C.The fecal and leaf extracts were diluted in ethanol at a concentration of 1 mg/ml, and the ABTS •+ solution was added to both extracts to obtain concentrations of 1.0 to 100.0 μg/ml.The absorbance of the reaction mixture was measured at 734 nm after reaction at room temperature for 10 min.Trolox was used as positive control.The capability of scavenging the ABTS+• radical was calculated using the following equation: where A 0 is the initial concentration of ABTS •+ and A 1 is the absorbance of the remaining concentration of ABTS •+ in the presence of the sample.

Total phenolic content
The total phenolic content of the fecal and leaf extracts was determined using the Folin-Ciocalteu reagent according to the method of Slinkard and Singleton (Duh et al., 1999), modified by the use of gallic acid as a standard phenolic compound.Appropriate amounts of extracts (500 μL; 50 μg/mL) were diluted in a volumetric flask with distilled water (3 ml).The Folin-Ciocalteu reagent (100 μL) was added and the contents of the flask were thoroughly mixed.After 3 min, Na 2 CO 3 (15%, 300 μL) was added and the mixture was completed with distilled water (5 ml) and allowed to stand for 2 h in an ultrasonic bath.The absorbance was measured at 760 nm in a spectrophotometer.The total amount of phenolic compounds was determined in micrograms of gallic acid equivalents, using the equation obtained from the standard gallic acid graph.

RESULTS AND DISCUSSION
The chemical profiles of the leaf and fecal extracts obtained obtained by LC-MS indicated that compounds 1 and 2 are major constituents, respectively, as previously reported.The evaluation of the biological activity of both extracts showed that the antioxidant activity of the metabolized P. umbellata leaves (fecal extract), as determined by the total phenol and ABTS •+ methods, was higher than that of the leaves (Table 1).The results indicated that the leaf and fecal extracts of P. umbellata both possess potent antioxidant activity.However, the fecal extract was twice as active as the leaf extract.There was a direct correlation between the phenolic content and the antioxidant activity of the fecal and leaf extracts because phenolic compounds contribute directly to antioxidant activity (Re et al., 1999;Chowdhury et al., 2012).The higher total phenolic content observed for the fecal extract can be associated with the release of phenolic compounds linked to the leaf cell walls during the insect's digestive process.
In summary, the potentialization of the antioxidant activity of leaves of P. umbellata after metabolism in H. brasiliensis can be associated with the biotransformation of compound 1 into 2 as well as the increase of the total phenolic content of the fecal extract.This study type contributes to the discovery of new natural bioactive compounds because it describes, for the first time as a novelty, the modification of a natural source (P.umbellata leaves) with potent antioxidant activity by insect.

Table 1 .
Results of antioxidant activity of leaves and fecal extracts.