Journal of Scientific & Industrial Research Vol. 73, July 2014, pp. 465-468

The chemical exploration of () in Brazil, with emphasis on insecticidal response: A review

Wagner de Souza Tavares1, Alexandre Igor de Azevedo Pereira2, Silvia de Sousa Freitas3, José Eduardo Serrão4 and José Cola Zanuncio5* 1Departamento de Fitotecnia, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais State, Brazil 2Instituto Federal Goiano, Campus Urutaí, 75790-000, Urutaí, Goiás State, Brazil 3Departamento de Química, Campus Catalão, Universidade Federal de Goiás, 75704-020, Catalão, Goiás State, Brazil 4Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais State, Brazil 5Departamento de Biologia Animal, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais State, Brazil

Received 20 July 2012; revised 20 October 2013; accepted 16 March 2014

This review links two apparently unconnected subjects supporting strategies for sustainable biodiversity use and conservation of the Cerrado (Savanna-type) biome in Brazil with the flavonoid astilbin from Dimorphandra mollis Benth. (Fabaceae) flowers. This is abundant in the Cerrado flowering from October to January. Larvae and hives of the honey bee Apis mellifera L., 1758 (Hymenoptera: Apidae) died after bee workers have pollinated crops of orange Citrus spp. (Rutaceae) near D. mollis in São Paulo State, Brazil, suggesting that bee workers might have collected pollen from D. mollis intoxicating themselves, other adults and the offspring. Pollen from D. mollis was collected and dried out of direct sunlight. Dichloromethane and methanol solutions were obtained from extracts of this pollen and the second was toxic to honey bees in the laboratory. Methanol solutions of astilbin from D. mollis were tested against insect-pests, with better results to Atta sexdens rubropilosa Forel, 1908 (Hymenoptera: Formicidae) [pest of eucalyptus Eucalyptus spp. (Myrtaceae)] and the defoliator caterpillars Anticarsia gemmatalis Hübner, 1818 [pest of soybean Glycine max L. Merrill (Fabaceae)] and Spodoptera frugiperda J.E. Smith, 1797 (Lepidoptera: Noctuidae) [pest of corn Zea mays L. (Poaceae)]. The abundance in the Cerrado and the toxic activity of astilbin from D. mollis flowers against forest and agriculture insect-pests show the potential to develop a new insecticide.

Keywords: Anticarsia gemmatalis, Apis melífera, astilbin, Atta sexdens rubropilosa, Botanical extract, Flavonoid, Insecticidal , Neoisoastilbin, Rutin, Spodoptera frugiperda

Natural botanic insecticides and astilbin Cerrado; laboratory synthesis of active ingredients The choice of Dimorphandra mollis Benth. from material collected in situ and chemical (Fabaceae) for studying insecticide activity was efficiency of its flavonoids against insect-pests and based on ethnobotanical information. Beehives of selectivity to natural enemies, places D. mollis as one Apis mellifera L., 1758 (Hymenoptera: Apidae) in of the most promising plant for natural botanic orange Citrus spp. (Rutaceae) plantations in São insecticides prospection4,5. Paulo State, Brazil showed high mortality during In a previous study, methanol solutions from D. mollis flowering, which indicates the use of this plant. extracts of anthers with pollen grains, floral peduncles Brazilian laws impair the use of natural botanic and stems of D. mollis, from which the astilbin and insecticides and the exploitation of plant resources neoisoastilbin were isolated, were toxic to the honey (prospecting of insecticidal plants) is not bee A. mellifera. The astilbin is a major component of sustainable1,2. Dimorphandra mollis, native to pollen grains and may be one of those responsible for Cerrado (Savannah-type) biome of Brazil, has ideal bee mortality3. This compound was toxic to larvae characteristics for providing insecticide molecules3. and hives of A. mellifera6, workers of Atta sexdens Knowledge on aspects such as germination, rubropilosa Forel, 1908 (Hymenoptera: Formicidae)7, seasonality and responses to fertilization; not being at and to defoliator caterpillars Anticarsia gemmatalis extinction risks; relative wide distribution in the Hübner, 1818 (velvetbean caterpillar) and Spodoptera frugiperda J.E. Smith, 1797 (fall armyworm) —————— 8 *Author for correspondence (Lepidoptera: Noctuidae) . These last three are Email: [email protected] insect-pests in Brazil damaging on eucalyptus 466 J SCI IND RES VOL 73 JULY 2014

Eucalyptus spp. (Myrtaceae), soybean Glycine max L. controls (only water or artificial diet). Treatment with Merrill (Fabaceae) and corn Zea mays L. (Poaceae) astilbin at 0.1 mg.ml–1 had a significant effect on the plants, respectively showing the potential to develop a leafcutter ant survival rate, with death occurring new insecticide molecule. mostly between the 3rd and 8th days of the treatment. Extracts of cork, flower peduncles, flowers, leaves, The leafcutter ant workers treated with ingestion petioles and stems bark of D. mollis were obtained in experiments of astilbin showed symptoms of studies in Corumbataí and Rio Claro, São Paulo State, intoxication, such as lower movements, leg tremors 7 Brazil. Dichloromethane and methanol solutions from and prostration with all concentrations tested . these extracts at 0.2, 0.5 or 1.0% or pure astilbin were Dichloromethane and methanol solutions were incorporated to the A. mellifera artificial diet obtained from extracts of flower peduncles and consisting of sucrose and honey (5:1) and offered flowers of D. mollis in Rio Claro, São Paulo State, (30 mg per bee) to newly emerged honey bees. The Brazil. A solution of astilbin in water + dimethyl daily survival rates were evaluated during 25 days3,6. sulfoxide (99.9 + 0.1 by volume; 3 ml) was added to Astilbin was isolated as a major component from the ascorbic acid (1.8 g; an ingredient of the artificial methanol extracts of flower peduncles and flowers diet), its solvent evaporated under vacuum at 40 ºC in (concentration higher than 99%), while the methanol a rotary evaporator and incorporated to the artificial extracts of the cork yielded a mixture of astilbin, as diet at final concentrations of 0.1, 1.0 and 10.0 mg.kg–1. the major constituent (60.6%), neoisoastilbin (16.6%) Neonate caterpillars of A. gemmatalis and and catechin (9.0%). The methanol solutions of S. frugiperda fed on this artificial diet. Duration of flower peduncles, flowers and stems bark proved to larva and pupa stages; duration of the life cycle (larva be toxic to honey bees6. The highest concentration of to adult emergence); weight of pupa and percentage flower peduncles and flowers solutions killed all of living insects (viability) at the end of each stage honey bees after 20 days. The methanol solution from were daily evaluated. The experiment with the stems bark showed to be active, killing all the S. frugiperda larva and pupa did not show differences honey bees within eight days3. The methanol cork between the treatments and the control (artificial diet solution was toxic to honey bees and was partitioned was prepared without the astilbin). However, the into fractions 1, 2, 3, 4 and 5. Only fraction 1 did not increase of astilbin concentration in the artificial diet show toxic effects against this insect. Fractions 2, 3 increase in 1.2 days the duration of larva stage and 4 showed toxic effects against honey bees and compared to the control. Anticarsia gemmatalis had a were mixtures of tannins and astilbin. Fraction 5 shorter larva (0.5 days) and pupa (1.3 days) period consisted of tannins and was also toxic to honey bees6. without differences to the control. A small increase on Astilbin was obtained in Corumbataí, São Paulo duration of the larva and pupa stages of this insect State, Brazil, from D. mollis flowers, dissolved in produced increases the total cycle in 1.53 days for methanol and incorporated to an artificial diet S. frugiperda and 2.27 days for A. gemmatalis [glucose (50 g.l–1), bacto-peptone (10 g.l–1), yeast compared to the controls. A small but significant extract (1.0 g.l–1) and agar (15 g.l–1) in distilled water reduction (264-240 mg) in pupa weight of (100 ml)] heated, immediately after the sterilization S. frugiperda treated with astilbin was observed. process with an autoclave or in the moment the However, no differences were found for the pupa ingredient mixture dried, without being dissolved with weight of A. gemmatalis. The viability of larva, pupa methanol beforehand. The final concentrations of and total cycle for S. frugiperda and A. gemmatalis astilbin incorporated or added into the diets were 1.0, were observed with increasing concentration of 5.0, 10.0, 20.0 and 30.0 mg.ml–1 or 0.1, 0.5, 1.0 and astilbin in the artificial diet. This viability varied from 2.0 mg.ml–1, respectively. Mean survival (50%) of 100.0% (control) to 93.3% (0.1, 1.0 and 10.0 mg.kg–1) adult workers of A. sexdens rubropilosa maintained for S. frugiperda larva stage and A. gemmatalis on two artificial diets was calculated. The showed a higher reduction, from 90.0% (control) to survivorship rate of leafcutter ant treated with 76.7% (1.0 and 10.0 mg.kg–1). The viability of the artificial diet containing a solution of astilbin caused pupa stage decreased, 80.0% in the control to 33.3% mortality to the leafcutter ant with all concentrations. (10.0 mg.kg–1) for S. frugiperda and from 96.7% Survivorship rates for the leafcutter ant treated with in the control to 53.36% (10.0 mg.kg–1) for artificial diet containing astilbin dry-mix added at low A. gemmatalis. These variations affected the concentrations had a higher mortality rate than the total cycle from 80.0% in the control to 26.2% TAVARES et al: THE CHEMICAL EXPLORATION OF DIMORPHANDRA MOLLIS (FABACEAE) IN BRAZIL 467

(10.0 mg.kg–1) for S. frugiperda and from 86.7% The structural identification of astilbin and in the control to 33.3% (10.0 mg.kg–1) for neoisoastilbin was performed by analyzing the spectra A. gemmatalis8. of NMR 1H, 13C and PENDANT. 1 Flowers of D. mollis are collected with great waste The NMR H spectra of the main solid of rutin from their shells and pods. About 600 tons of showed hydrogen’s signals connected to seeds are discarded per year during extraction of this aromatic rings between δH 6.85 and 5.78 flavonoid, which could provide up to 160 t per year of (5H), characteristics of flavonoid compounds 9 galactomannan . This biomolecule is used in and sugar signals between δH 4.5 and 3 (5H) and in 13 pharmacology, oil, cosmetics and food industries to δ 0.83 (3H). The NMR C-PENDANT spectra viscous solutions at low concentrations, with potential shows signal of ketone carbonyl α,β-unsaturated in to replace imported polysaccharides. Its thickener, δC 195.9; five signals of oxygenated aromatic stabilizing and gelling (galactomannan content) effects carbon between δC 169 and 146; seven aromatic show potential for gums for the food industry10,11. carbon signals between non-oxygenated between The material provided by D. mollis is extracted δC 131.8 and 102 and signals in δC 78.6 and 84 from its natural habitat without care about its completed the typical skeleton of dihydroflavonoids. reproduction. Natural populations of this plant are The signal in δC 17.8 and those set in δ 102.1; under local risk of extinction, due to poor 71.7; 70.4; 70.1 and 69 confirmed the presence of management reducing the number of individuals the monosidic rhamnose unit. These data are without criteria by cropping beans and other consistent with the structure of dihydroflavonol structures to obtain active ingredients. Fruit collectors glycosylated astilbin, which was confirmed by comparison of the NMR 13C signals of this cut D. mollis trees to increase their efficiency because 14 12 solid with of astilbin . they are paid by the numbers collected . Dimorphandra 1 13 genus has nine species with The NMR H and C data of the astilbin and neoisoastilbin are similar. The main difference in the Rizzini (Fabaceae) having similar potential and rare 1 since its description in 196913. NMR H spectra is in constant coupling between the hydrogens H-2 and H-3, being δ 5.2 (d, J= 10 Hz, The methodology to isolate and to characterize the H H-2) and 4.6 (d, J= 10 Hz, H-3) in astilbin and δ 5.5 flavonoid with insecticide activity astilbin from H (d, J= 2.5 Hz, H-2) and 4.2 (d, J= 2.5 Hz, H-3) in flowers of D. mollis in methanol is described. The neoisoastilbin. The comparison of the NMR 13C flowers of D. mollis were collected and dried out of data of neoisoastilbin from D. mollis and of the direct sunlight. The extracts were obtained from 450 g literature14 confirms that its structure is as of D. mollis flowers in dichloromethane and diastereoisomer of astilbin. methanol, with three extractions of the first solvent and two of the second. Both were evaporated under Conclusion reduced pressure. The methanol extract showed A model for plant resources exploration based round-shaped crystals. Dichloromethane and on activities that promote the sustainability, methanol solutions from these extracts were subjected generating socio-economic development of areas to biological testing toxicity on honey bees and only with biodiversity and encouraging the use of natural 3 the methanol one showed toxicity . resources with aggregate value was presented. The methanol extract of D. mollis was fractionated This type of strategy must be expanded to other by high-performance liquid chromatography (HPLC). plant species due to the poor knowledge of the The analytical conditions were: column polymer potential of Cerrado flora of Brazil. Astilbin is ASAHIPAC 310 s (2.15 cm ID × 50 cm length); flow abundant on parts (cork, flower peduncles, flowers, 7 ml.min–1; = 290 nm UV detector; injection volume leaves, petioles and stems bark) of D. mollis 2 ml; eluent methanol 100% and sample concentration plants, which is native at the Cerrado biome of of 50 mg.ml–1. Two yellow solids were isolated, Brazil. Methanol solutions of this flavonoid are highly the first with 95% and the second with 5% of the toxic at low doses to leafcutter ant A. sexdens extract concentration, respectively, identified as the rubropilosa workers and defoliator caterpillars, flavonoids astilbin (3-ß-O-rhamnosideo of 5,7,3’,4’– A. gemmatalis (velvetbean caterpillar) and tetrahidroxi-2,3-diidroflavonol) and its diastereoisomer S. frugiperda (fall armyworm), forest and agriculture neoisoastilbin. insects-pest of eucalyptus, soybean and corn, 468 J SCI IND RES VOL 73 JULY 2014

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