The   tomato   (Solanum  Licopersycum  L.,  Solanaceae) adapts well to subtropical and temperate climate. Tomato crop is infested by various pests that can substantially alter their productivity and quality. Of all the insect pests, the tomato moth Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) has been infesting the tomato crop in Brazil since 1980 (Carvalho and Borgoni, 2006; Araújo et al., 2013).

Farmers have attempted to control this insect pest with insecticides. Though these are effective management options, but insecticides carry the risk of environmental degradation. In addition, as use of these chemicals became routine, their efficacy against T. absoluta decreased (Silvério et al., 2009; Lebdi-Grissa et al., 2010). Several researchers have reported alternatives to the use of conventional pesticides, including plant extracts as bioinsecticides or mass capture with sexual pheromones that attract males to traps where they are killed (Hassan and Alzaidi, 2009; Moreno et al., 2011, Tomé et al., 2013; Cocco et al., 2013). The bioinseticidal botanical families include Asteraceae, Meliaceae, Rutaceae, Annonaceae, Lamiaceae and Canellaceae (Zabel et al., 2002, Pereira et al., 2002, Tamm, 2004). Morinda citrofolia L., from the Rubiaceae family, popularly known as noni, has been used since antiquity for the treatment and prevention of various diseases in humans and animals. This species is well adapted to the various regions of cultivation in Brazil (Navarro-Silva et al., 2009). Among the chemicals found in M. citrifolia fruits, phenolic compounds such as anthraquinones (Deng et al., 2007) are found in trace quantities (Lin et al., 2007). Reyes et al. (2011) reported that ethanolic and aqueous extracts of the fruit contained free quinones, steroids and flavonoids. Studies have tested the bioactivity of extracts of M. citrifolia against Diptera such as Drosophila sechellia (Tsacas and Baechli, 1981) (Diptera: Drosophilidae). The plant's fruit contains secondary defense compounds, primarily octanoic acid that are lethal to most other flies of the same genus (Morales et al.; 2010; Kovedan et al., 2012; Silva et al., 2015; López et al., 2017). The bioactivity of noni extracts (M. citrifolia) has chemical similarities with other natural products in terms of bioinsecticidal activity. The hypothesis raised is that the phenolic compounds of this plant have bioinsecticidal properties. Therefore, the objective of this work was to evaluate the leaf and fruit ethanolic extracts in the control of the tomato moth, in addition to identifying and quantifying its phenolic compounds, to assist in the integrated pest management.

Quantification of total phenols

The method for the determination of total phenols consisted of the reaction of the constituent acids of the Folin-Ciocalteau reagent and phenolic or non-phenolic compounds. The Folin-Ciocalteau reagent is composed of phosphomolybdic and phosphotungstic acids, in this solution, molybdenum is in the oxidation state +6 with a yellowish coloration; in the presence of phenolic compounds, it undergoes a reduction reaction and passes to a +5 oxidation state (Rezende, 2010). With the reduction reaction there is the formation of blue-colored molybdenum-tungsten complexes. This reaction occurs in alkaline medium, specifically in the presence of Na₂CO₃ (Rezende, 2010). The total phenol content was quantified by the method described by Freitas et al. (2014) with some adaptations. To create the gallic acid calibration curve, 0.04 g of gallic acid was weighed into 8 mL of MeOH (stock solution). Dilutions (Gallic acid test solutions) were then prepared at concentrations of 0.15, 0.1, 0.05, 0.025, 0.01 and 0.005 mg/mL.

The following procedures were performed from the dilutions of gallic acid (in triplicate for each concentration): into an amber glass we added 100 μL of gallic acid test solution, 500 μL of Folin-Ciocalteau reagent and 1 μL of distilled H₂O and then vortexed for one minute. Subsequently, 2 mL of 15% sodium carbonate was added and stirred for another 30 s in the vortex. Subsequently, the solution was filled into a 10 mL volumetric flask. The solution was incubated in the dark for two hours. Absorbance readings were obtained using a UV-VIS spectrophotometer with a wavelength of 750 nm. To obtain the test solution of the vegetable sample, 0.005 g of each vegetable ethanolic extract was diluted in 5 mL of MeOH. Then, a 0.075 mL aliquot of this solution was added to 0.425 mL MeOH. To perform the test, the same procedure was performed as for gallic acid, replacing the test solution of gallic acid with the test solution of the vegetable ethanolic extract. To obtain the blank control solution, a solution of 100 μL of MeOH, 500 μL of the Folin-Ciocalteau reagent and 1 mL of distilled H₂O was prepared. The procedure described for gallic acid was repeated, replacing the gallic acid test solution with the blank control solution. The absorbances were read in a UV-VIS spectrophotometer with a wavelength of 750 nm. Before any reading, the blank was used to reset the spectrophotometer.

Quantification of flavonoids

The method for the quantification of flavonoids consisted of preparing the calibration curve of quercetin, where 1 mg of quercetin was weighed and diluted in 1 ml of MeOH. Dilutions were then carried out at concentrations of 0.03, 0.025, 0.020, 0.015, 0.01, 0.005, 0.0025 and 0.00125 mg/mL. Subsequently, solutions were made of the extract, where 1 mg of the extract was weighed and diluted in 1 mL of MeOH. After preparation of the test solution, the solutions (in wells in triplicate) were prepared for reading with 200 μl of the test solution of the plant sample and 100 μl of 2% aluminum chloride methanolic solution. The solution was prepared for the blank (in triplicate) with 200 μl of MeOH and 100 μl of 2% methanolic chloride of aluminum chloride. Thereafter, the well plate was placed in the dark for 30 min. Then, the reading was performed in UV-VIS spectrophotometer at 420 nm. The flavonoid content was determined by interpolating the mean absorbance of the samples against the quercetin calibration curve and expressed in mg QE (quercetin equivalent) per gram of ethanolic extract.

Separation, identification and quantification of phenolic compounds

The determination of the phenolic compounds was performed using high-performance liquid  chromatography  (HPLC).  The  equipment used was a Shimadzu HPLC, equipped with four high pressure pumps, model LC-20AT, degasser model DGU-20A 5R, interface model CBM-20A, automatic injector model SIL-20A HT and detector model SPD-20A. The chromatographic column employed in both analyses was an Agilent-Zorbax Eclipse XDB-C18 column (4.6 × 250 mm, 5 µm).

The standards used for chromatographic analysis of the phenolic compounds were gallic acid, catechol, vanillic acid, salicylic acid, vanillin, syringaldehyde, coumaric acid, chlorogenic acid, coumarin, rutin, quercetin, kaempferol and caffeic acid. All standards were purchased from Sigma-Aldrich or AcrosOrganics. All solvents used for chromatography were of analytical grade; methane was from Panreac, formic acid was from Dynamic and ultrapure water was obtained from a Milli-Q system. For the standards, stock solutions with a concentration of 40 mg/L in water/alcohol at 30%/70% were prepared. The method used for quantification was that of external standardization. For the construction of analytical curves, dilutions of an intermediate solution were carried out containing a mixture of all the standards. This was obtained by diluting the stock solutions previously prepared. In this intermediate solution, all standards were at a concentration of 10 mg/L. The 1% formic acid solution in milli-Q water (Solvent A) and methanol (Solvent B) were used as the mobile phases for the elution of the tested compounds.

Plant samples and standards were eluted according to the gradient (Table 1) with a total run of 66 min. The wavelength used was 290 nm at 33°C, flow rate of 0.6 mL/min and injection volume  of 20 μL. Samples and standards were filtered on 0.45 μm polyethylene membranes (Milipore) and were injected directly into the chromatographic system. Each injection was performed three times in the HPLC system, in order to obtain the mean concentrations and retention times. Thus, the identity of the analytes was confirmed by the retention time, and the profile of the peaks of the sample was compared to the patterns.

Bioassay for evaluation of insecticidal activity

The data in Tables 2 and 3 refer to the average obtained by the Dunnett test at 5% probability, as an alternative bilateral hypothesis shown to be effective in relation to the control. The hypothesis was that both the ethanolic extract of the leaf and the fruit in the concentrations 0.01 to 0.05 mg/L would provoke a mortality effect in T. absoluta in a similar way. There was a higher mortality of T. absoluta with the leaf extracts at the concentration of 0.02  mg/L   than   in  the  control,  with  88.04%  mortality (Table 2). In the fruit extracts, the highest mortality was observed at the concentration of 0.03 mg/L, and the difference from the control was 34.62% (Table 3). To the best of our knowledge, this study was the first to show M. citrifolia bioinsecticidal activity against insects of agricultural interest, microlepidoptera such as T. absoluta. To date, there have been no reports on the bioactivity of this plant in relation to caterpillars of this order of insects. In the difference of the means of the treatments with the control, we observed that the results were better than the control, both for the leaf extracts and for the fruit extracts in terms of insect mortality.

According to Chan-Blanco et al. (2006), the most abundant bioactive compounds in M. citrifolia were phenolics, including damnacanthal, scopoletin, morindone and rubiadin, with rutin and scopoletin as major components and damanacantal with demonstrated anti-carcinogenic properties. In reports of phenolic compounds with insecticidal bioactivity of M. citrifolia on dipterans, nanoparticles synthesized from root extracts were used, with significant results against Aedes aegypti larvae (Suman et al., 2015).  Many  phenolic  compounds have antioxidant, anticarcinogenic, antimutagenic and anti-inflammatory activities. However, most interest is focused on antioxidant activity, and the attributed physiological and pharmacological functions originate in this activity (Thani et al., 2010).

Zin et al. (2002) demonstrated high levels of antioxidant activity in M. citrifolia extracts when using methanolic extracts of roots, fruits and leaves. Faria et al. (2014) measured the presence of various phytochemical compounds, including tannins, flavonoids, conjugated anthraquinones, saponins, coumarins and alkaloids and suggested pharmacological and functional functions. Studies demonstrate current interest in the study of phenolic compounds, primarily due to the antioxidant potential of these substances in terms of sequestering free radicals that are harmful to human health (Alves et al., 2007; Neves et al., 2008).

Costa et al. (2013) carried out studies with M. citrifolia using the seed, peel and pulp, and demonstrated the antioxidant capabilities of this plant. The bioactive compounds found in plants are phenolic substances with diverse functions including plant growth, sensory properties, seed germination processes, and defense against pests and oxidative damage (Liu, 2007).

Phenolic compounds identified and quantified

In this study, 11 phenolic compounds were detected in the fruit and leaf extracts of M. citrifolia. Figure 1 shows the chromatogram with spectrophotometric detection at 290 nm. The  phenolic profile of the plant samples is very similar except for the detection of chlorogenic acid and vanillin patterns found only in the extract of the fruits of M. citrifolia. The other compounds were detected in both the fruit and leaf extracts.  The concentrations of the identified products were calculated from standard curves generated with commercial products chromatographed under identical conditions (Table 4). The bioactivity of M. citrifolia on the mortality of caterpillars can be attributed to phenolic compounds that were found in greater quantity in leaf extracts than in fruit extracts. Some of these phenolic compounds are distinguished terms of insect control in bioassays.

Bendassolli et al. (2010) reported mortality of ants of the genus Atta, with extracts of Anarcadiaceae. The results obtained with methyl gallate and quercetin were significant, with quercetin resulting in mortality of 50% and Gallic acid resulting in 50% mortality only at the end of the experiment. In the present study was this effect was found at concentrations 24.767 mg/L in the leaf and in the fruit 16.515 mg/L. Rutin had a bioinseticidal effect on caterpillars (Anticarsia gemmatalisas) that was potentiated when the caterpillars fed on mixed diets with rutin. In that study, the rutin was present in the leaf at 89.1063 mg/L and in the fruit at 21.1252 mg/L (Wang et al., 2002; Céspedes et al., 2004; Scott et al. 2010). Villaño et al. (2006) found that phenolic acids (syringic acid, vanillic acid and p-coumaric acid) protected plants against insects. Saponins have been shown to be substances in plant secondary metabolism that protect from insects as well. Peyser et al. (2017) suggested that octanoic acid and hexanoic acid were the toxins that mediated the effects of M. citrifolia, and that octanoic acid was a neurotoxin as well.

The  bioinseticidal   action   of   M.  citrifolia  was  efficient against the tomato moth (T. absoluta), especially the ethanolic extract of its leaves. The effect of M. citrifolia leaves in terms of bioinseticidal activity may be related to their chemical composition, evidenced here by the quantification of phenolic compounds and flavonoids.         The mortality data from the tomato moth resulting from the bioactivity of M. citrifolia extracts and their chemical compositions suggest that they can be used to control the tomato moth.

The authors have not declared any conflict of interests.