Byrsonima intermedia preparations inhibits trypsin and chymotrypsin activities from Aedes aegypti larval gut

This work describes the larvicidal activity of the ethanolic crude extract (EEB) as well as the hexanic (FH), chloroform (FCL), ethyl acetate (FAE) and remaining (FR) fractions obtained from Byrsonima intermedia leaves and bark against Aedes aegypti. Hexanic and remaining fractions from the leaves demonstrated larvicide activity against A. aegypti larvae on the 3rd stage, with LC50 values of 250 and 125 μg/ml -1 , respectively, with less mortality as compared to the other fractions. Hexanic and remaining fraction from the leaves demonstrated larvicide acitivity against A. aegypti larvae on the 3rd stage, with LC50 values of 250 and 125 μg/ml -1 , respectively, with less mortality as compared to the other fractions. Trypsin activities from gut of larvae treated with the extract or the fractions were significant (p < 0.05), and the major effects occurred with hexanic fraction (0.06 μmol min -1 ) and remaining fraction (0.09 μmol min -1 ) in the 1000 μg/ml -1 concentration, respectively. The same fractions inhibited chymotrypsin activity in 0.06 μmol/ml -1 (hexanic fraction) and 0.13 μmol/ml -1 (remaining fraction) at 1000 μg/ml -1 . The inhibition was dose-dependent. The results show the inhibitory effects of the hexanic and remaining fractions from the leaves of B. intermedia on trypsin and chymotrypsin activity, which may be responsible for the deleterious effects on larvae mortality. The crude ethanolic extract and fractions of the bark does not inhibit the synthesis of trypsin compared to control. The results show the inhibitory effects of the hexanic and remaining fraction from the leaves of B. intermedia on trypsin and chymotrypsin activity, which may be responsible for the deleterious effects on larvae mortality.


INTRODUCTION
Classical and hemorrhagic dengue epidemics have reappeared in the past 25 years in tropical regions which present a warm and humid weather and propitious socioenvironmental conditions for the proliferation of the mosquito vector Aedes aegypti (Silva et al., 2008).A. aegypti control has been performed using synthetic chemicals with larvicidal effect (Luna et al., 2004;Lima et al., 2006).However, its frequent use is costly and can lead to environmental contamination, public health damage and the arising of resistant insects (Oga, 2003;Braga and Valle, 2007).In this sense, the use of natural bioactive products with insecticide potential emerges as aviable alternative.Malpighiaceae is one of the major families of angiosperms, comprising about 66 genus and 1,200 species, spread in tropical and subtropical regions (Anderson, 1990).In Brazil, 55 genus occured, with emphasis on Byrsonima.
Although not very studied in scientific works, Byrsonima intermedia is known as murici anão-do-campo and murici-anão.It occurs with predominance in the Cerrado region of Mato Grosso do Sul, Brazil and it shows adstringent activity against diarrheas and dysenteries when used as an infusion from the bark stark (Rodrigues and Carvalho, 2001).In the literature, anti-inflammatory and anti-nociceptive activities are reported for the bark extract (Orlandi et al., 2011), and the leaves show gastroprotective and antidiarrheal activities (Santos et al., 2012).The floral oil of B. intermedia is rich in free fatty acids and the main constituent is byrsonic acid (Reis et al., 2007).
Current strategies based on the elimination of breeding sites and applications of chemical insecticides for larval and adult mosquito control have resulted in development of resistance without eliminating the constant risk of dengue epidemics (Lima et al., 2011).Thus new approaches are urgently needed.Interest on possible use of environment friendly natural products such as oils of plants or plant parts increased for vector control.Plant derived products have received increased attention from scientists and more than 2000 plant species are already known to have insecticide properties (Pankaj and Anita, 2010;Kamaraj et al., 2011).Considering the advancement of researches on the botanical larvicies and their potential for larvae control, the objective of this work was to evaluate, under laboratory conditions, the bioactivity of crude ethanolic extract (CEE), hexane (FH), chloroform (FCL), ethyl acetate (FAE) and remaining fractions (FR) from the leaves and barks of Byrsonima intermedia, against A. aegypti, through assays of mortality, trypsin and chymotrypsin activities.
da Silva et al. 417

Vegetal material
B. intermedia was collected in the Cerradinho of the Universidade Federal do Mato Grosso do Sul, from July to August, 2011, and a species sample was identified by comparison with material deposited in the Campo Grande, Mato Grosso do Sul herbarium, under number 21176.

Crude extract and fractions obtainment
The collected material was dried at 25°C, milled and submitted to exhaustive extraction with ethanol in a Sohxlet apparatus, consisting of a vertical glass cylindrical extraction tube that has both a siphon tube and a vapor tube, that is fitted at its upper end to a reflux condenser and at its lower end to a flask so that the solvent may be distilled from the flask into the condenser whence it flows back into the cylindrical tube and siphons into the flask to be distilled again.After extraction, the ethanolic crude extract was dried and submitted to fractioning with solvents of different polarity degrees, obtaining the hexanic (FH), chloroform (FCL), ethyl acetate (FAE) and remaining (FR) fractions.

Aedes aegypti mortality
The larvicide activity under A. aegypti was assessed following the World Health Organization (1981) methods, with some modifications.Eggs from A. aegypti, Rockefeller strain, were provided by the Fundação Oswaldo Cruz -Rio de Janeiro.For egg eclosion, they were put in a plastic tray and 500 ml of chlorinelacking water was added, and then taken to a BOD incubator in 27 ± 2°C temperature and 80 ± 5% relative humidity.Regarding the assays, we chose to evaluate the extract activity using 3rd stage larvae, as they are considered more resistant (Silva and Silva, 1999).Larvae feeding was prepared with Fish's diet (Aldon Basic™, MEP 200 Complex) from the eclosion period to the 3rd larvae stage, and solutions of the extract and its fractions in the concentration of 1000 µg ml -1 were prepared by solubilizing the samples with 0.5% dimetilsulphoxide (DMSO) and diluted in water in the concentrations of 1000, 500, 250 and 125 µg ml -1 for the assays.The stearic acid activity was evaluated as well.The same method was used to prepare a 50% concentration.Samples containing 15 larvae at the 3rd stage were put in plastic cups and the volume was completed to 5 ml.The assays were performed in triplicates, totaling 45 larvae.A 0.5% DMSO aqueous solution was used in triplicate, as a negative control.Temephos™ was used as a positive control, technical grade of 90%, lot #005/2011, manufactured by Fersol Mairinque Laboratories, São Paulo.The calibration was made following the protocol recommended by the World Health Organization described in World Health Organization (2009), Lima et al. (2003) and Braga et al. (2004) using 0.060 mg ml -1 as a diagnostic concentration (DC), which is twice the lethal concentration that causes 99% mortality in a susceptible strain, as defined by World Health Organization (2009).The larvicide activity was evaluated after 24 h, by counting the number of dead larvae in each sample.Moribund larvae, unable to reach the water surface when touched, were considered dead (World Health Organization, 2009).

Larvae homogenate preparation
A. aegypti larvae homogenates, submitted to the extract and fractions during 6 h were prepared according to Macedo et al. (1993), with modifications.Live larvae were homogenized by hand Table 1.Effect of different concentrations of the crude ethanolic extract (CEE) and hexanic (HF) chloroform, ethyl acetate (EAF) and remaining (RF) fractions of leaves and barks of the B. intermedia in the mortality and LD50 against A. aegypti larvae.

Determination of protein content
The protein content in the homogenate preparations from larvae was determined according to Lowry et al. (1951), using serum bovine albumin (25 to 500 µg/ml) as standard.

Protease activity assay
The total trypsin activity was determined using the N-benzoyl-D, Larginine-ρ-nitroanilide (BAPNA) reagent as substrate.About 500 μl of the larvae homogenate (0.8 μg of protein) were added to BAPNA and left to rest for 20 min at 37°C; after this, the reaction was stopped by adding 30% acetic acid (v/v).The trypsin activity was read in a microplate reader at 410 nm (ε, 10.0 mM -1 cm -1 ) in accordance with Silva et al. (2009).The chymotrypsin activity was determined using N-succinyl-Ala-Ala-Pro-Phe-p Nitranilide (SAAP) as substrate.About 50 μl (0.08 μg of protein) of the homogenates were incubated in 450 µl of Tris-HCl Buffer 0.1 M (pH 8.0) for 10 min.Afterwards, 1.0 ml of SAAP 2 Mm dissolved in pure DMSO was added.The reaction mixture was incubated at 37°C for 20 min and then the reaction was ended by the addition of 30% acetic acid (v/v).The absorbance was read at 405 nm (ε, 8.8 mM -1 cm -1 ) (Silva et al., 2009).

Statistical analysis
Values of lethal concentration (LD50) in µg/ml were determined using the Probit analysis method (Finney, 1974).For each evaluated sample, triplicates were used and data was submitted to analysis of variance, and when a difference was detected, the averages were compared by the Dunnet test, with 5% of probability.

RESULTS
The mortality of Aedes aegypti larvae varied according to extract and concentration tested.The hexanic (LD 50 < 250 μg ml -1 ) and remaining fractions (LD 50 < 125 μg ml -1 ) from the leaves demonstrated more activity, showing a mortality of 11.9 ± 1.5 and 11.7 ± 0.7 individuals (mean ± SD), respectively for the 1000 μg ml -1 concentration.Chloroform and remaining fractions showed lower activity with a LD 50 < 500, with mortality of 7.7 ± 1.6 (mean ± SD) and 6.0 ± 1.1 (mean ± SD), respectively.Larvae treated with fractions from the barks showed less mortality, with a LD 50 > 1000 μg ml -1 .The crude ethanolic extract demonstrated larvicide activity with a LD 50 > 500 (Table 1).No concentration had an effect comparable to that of the diagnostic concentration (DC) of 0.060 mg mL -1 of the pesticide Temephos.The inhibition in trypsin activity was also observed and a dose-dependent relationship was verified, with larger inhibitory effects for the 1000 μg ml -1 concentration.The hexanic and remaining fractions from the leaves inhibited the trypsin synthesis in 0.06 and 0.09 μmol min -1 , respectively, demonstrating more activity of the fractions when compared to crude ethanolic extract (0.13 μmol min -1 ), chloroform (0.20 μmol min -1 ) and ethyl acetate fractions (0.21 μmol min -1 ).Only the ethanolic crude extract (EEB) from the barks caused an accentuated inhibition in trypsin synthesis (0.21 μmol min - 1 ).The fractions did not lead to alterations in trypsin synthesis (Figure 1).
The EEB and all the fractions from the leaves inhibited the chymotrypsin synthesis.A higher inhibitory effect was verified for the hexanic (0.06 μmol min -1 ) and remaining fractions (0.13 μmol min -1 ) in the 1000 μg ml -1 concentration.The crude ethanolic extract, chlorophorm and ethyl acetate fractions affected the chymotrypsin activity as well, albeit in a less proportion.The crude ethanolic extract and fractions of the barks did not alter the activity of chymotrypsin when compared to control (Figure 2).

DISCUSSION
The results of the larvicidal assay demonstrated that the crude ethanolic extract and fractions of B. intermedia was effective against the last A. aegypti larval stage.Secondary metabolites have been reported as active principles from insecticidal plant extracts.
Compounds such as quercetin may be involved in larvicidal activity since they are able to kill mosquito larvae (Rahuman et al., 2008;Ochieng et al., 2010).Quercetin can bind to trypsin S1 region through hydrogen bonds and electrostatic interactions and promote enzyme inhibition (Maliar et al., 2004).Secondary metabolites have been reported as active components of vegetal extracts which exhibit insecticide activity.Detected substances such as quercetin may be involved in the larvicide activity because they are able to kill the mosquito larvae in all its development stages (Rahuman et al., 2008;Nikkon et al., 2010;Ochieng et al., 2010).Toxic substances interact specifically with apical membrane receptors of the median gut, causing serious damage to the epithelium that culminate with larvae death (Charles, 1981;Gill et al., 1992).The hexanic and remaining fractions from the leaves were more active when compared to the other fractions.These results demonstrate that it is possible that one of the action mechanisms occurs by ingestion of the extract and fractions which are solubilized in the intestinal lumen and cleaved by trypsin and chymotrypsin, then becoming toxic.
Trypsin and chymotrypsin may play various important roles in food digestion, immune defense and zymogen activation in insects (Ge et al., 2012).Despite several studies concerning adult A. aegypti digestive biochemistry and molecular biology, very few studies have been performed to elucidate the digestion in A. aegypti larvae.Trypsin-like and chymotrypsin-like activities are known in A. aegypti larvae (Mesquita- Rodrigues et al., 2011).Enzymatic inhibitors, when added to the diet of insects, interfere with their digestive process by decreasing the assimilation of nutrients, leading to delayed development and mortality (Napoleão et al., 2012).Zhang et al. (2010) reported what enzymes involved in the food digestion; it has to be secreted into the interspaces between the epithelium and peritrophic membrane or the lumen of the gut, where it digests the ingested food proteins, and the results showed that digestive enzymes was present not only in the epithelium of the anterior, middle and posterior midgut, but also in the lumen food residues of the anterior, middle and posterior midgut, as well as the feces of the larvae, suggesting that the protein was secreted into the lumen of the gut.
Current strategies based on the elimination of breeding sites and applications of chemical insecticides for larval and adult mosquito control have resulted in development of resistance without eliminating the constant risk of dengue epidemics (Lima et al., 2011).Thus new approaches are urgently needed.Interest on possible use of environment friendly natural products such as oils of plants or plant parts increased for vector control.Plant derived products have received increased attention from scientists and more than 2000 plant species are already known to have insecticide properties (Pankaj and Anita, 2010;Kamaraj et al., 2011).The protease inhibitors show insecticide activity because of the damage in processes related to digestion and absorption of nutrients (Carlini and Grossi-De-Sá, 2002).The effects of trypsin inhibitors in insect larvae include reduction of body weight, decrease of survival rate, as well as delay or blockade of larvae development (Macedo et al., 2002(Macedo et al., , 2003;;Bhattacharyya et al., 2007;Ramos et al., 2009).
It is plausible to infer that hexanic and remaining fractions obtained from the leaves of B. intermedia cause the same effects on larvae of A. aegypti, and these effects are associated with inhibition of gut enzymes.Thus, the use of hexanic and remaining fractions for the control of insects is an alternative way to minimize the harmful effects of pesticides used to control mosquitoes.Further, samples of locals where there has already been intensive use of pesticides by the control programs demonstrate that the larvae and mosquitoes became resistant by mechanisms such as increase in the synthesis of acetylcholinesterase (Pinheiro and Tadei, 2002).Extracts of plants could be used in stagnant water bodies which are known to be the breeding grounds for mosquitoes.However, further studies on the active principals involved and their mode of action and field trials are usually needed to recommend any of these plant materials as an anti-larvicidal product used to combat and protect people from mosquitoes in a control program.Plant could be an alternative source for mosquito larvicides because they constitute a potential da Silva et al. 421 source of bioactive chemicals and generally free from harmful effects.Use of these botanical derivatives in mosquito control instead of synthetic insecticides could reduce the cost and environmental pollution.Taking this into consideration, the hexanic and remaining fractions of leaves represent an alternative to the larvae control, as they acted as inhibitors of digestive enzymes such as trypsin and chymotrypsin, demonstrating its potential as physiologic pesticide.The isolation of substances present in hexanic and remaining fractions, and new research on the isolated activity of these substances could improve our understanding on the popular indication of this plant as pesticide by the Cerrado population, and evolve to the developing of formulations that could be used for the control of larvae and mosquitoes.

Conflict of Interests
The author(s) have not declared any conflict of interests.

Conclusion
The hexanic and remaining fraction of B. intermedia leaves can be considered as a larvicidal agent against A. aegypti on the last developmental 3rd stage, and this activity is related to trypsin and chymotrypsin inhibition.New insecticides of herbal origin discovered through ethnopharmacological studies have shown interesting results.Our laboratory has initiated and developed original investigations, and we have evaluated the larvicidal effect from natural extracts of plants.Purification of the bioactive component(s) from B. intermedia leaves is underway, and further investigations may improve our understanding of possible developmental changes from hexanic and remaining fraction of this plant used in folk medicine.

Figure 1 .
Figure 1.Effects of different concentrations of the crude ethanolic extract (CEE) and hexanic (HF) chloroform, ethyl acetate (EAF) and remaining (RF) fractions of leaves and barks of the B. intermedia on trypsin synthesis in A. aegypti larvae.*Means followed by the same letter from the control have no significant difference among them, according to Dunnet test (p < 0.05).

Figure 2 .
Figure 2. Effects of different concentrations of the crude ethanolic extract (CEE) and hexanic (HF) chloroform (CLF), ethyl acetate (EAF) and remaining (RF) fractions of leaves and barks of the B. intermedia on chymotrypsin synthesis in A. aegypti larvae.*Means followed by the same letter from the control have no significant difference among them, according to Dunnet test (p < 0.05).
Means followed by the same letter from the control have no significant diff erence among them, according to Dunnet test (p < 0.05).