Turmeric powder and its derivatives from Curcuma longa rhizomes: Insecticidal effects on cabbage looper and the role of synergists

Curcuma longa has well-known insecticidal and repellent effects on insect pests, but its impact on Trichoplusia ni is unknown. In this study, the compound ar-turmerone, extracted and purified from C. longa rhizomes, was identified, and its insecticidal effects, along with turmeric powder, curcuminoid pigments and crude essential oil were evaluated against this important agricultural pest. The role of natural (sesamol and piperonal) and synthetic [piperonyl butoxide (PBO)] synergists under laboratory and greenhouse conditions were also evaluated. The concentration of ar-turmerone in C. longa rhizomes harvested was 0.32% (dwt). Turmeric powder and its derivatives caused 10–20% mortality in third instar T. ni at a very low dose (10 μg/larva). Addition of PBO increased toxicity of turmeric powder and its derivatives (90–97% mortality) in most binary combinations (5 μg of turmeric powder or its derivatives +5 μg of PBO), but neither piperonal nor sesamol were active as synergists. The compound ar-turmerone alone and the combination with PBO reduced larval weight on treated Brassica oleracea in the laboratory and in greenhouse experiments, compared with the negative control. The compound ar-turmerone could be used as a low cost botanical insecticide for integrated management of cabbage looper in vegetable production.


Results and Discussion
Yield of ar-turmerone. The yield of C. longa essential oil with hexane was 0.39% (dwt) (1.93 g) and that of ar-turmerone from this essential oil was 82% (dwt) after the chromatographic separations from the initial material (1.58 g). A total of 3.2 g of ar-turmerone was present per kg of rhizomes of this plant grown in Catalão, Goiás, Brazil. Consistent with our results, high concentrations of ar-turmerone in non-polar extracts and essential oils of C. longa have been reported from China (Asia), India (Asia), Nigeria (Africa), Pakistan (Asia), and the islands of Sao Tome and Principe (Africa) 29,[45][46][47] . The quantitative and qualitative compositions of plant extracts and essential oils depend on genetic factors and on the environmental conditions of the area where the plant is grown, with variations in the essential oils of C. longa occurring at different localities [30][31][32] . Curcuma longa can be cultivated at low cost and sustainably in Brazil using minimal labour, a range of growing seasons and spacing, and organic fertilization with 50 tons of cattle manure per ha 48 . Contact toxicity (topical application) of turmeric powder and derivatives on cabbage loopers in the laboratory. The initial screening dose (10 μ g/larva) demonstrated low toxicity through topical application to third instars, ranging from 10-20% at 24 h (Table 1). Binary mixtures with piperonal as a synergist slightly improved toxicity of treatments in some cases (turmeric powder + piperonal and ar-turmerone + piperonal). Sesamol did not enhance activity in any treatment. However, addition of PBO as a synergist increased toxicity of turmeric powder and all of its derivatives.
Dose response effects of turmeric powder and derivatives on cabbage loopers in the laboratory through contact toxicity (topical application). Based on the LD 50 values (Table 2), binary mixtures of turmeric powder + PBO (LD 50 = 0.03 μ g) and turmeric crude essential oil + PBO (LD 50 = 0.05 μ g) were both significantly more toxic than ar-turmerone + PBO (LD 50 = 0.26 μ g) which itself was more toxic than curcuminoid pigments + PBO (LD 50 = 0.61 μ g) against third instar cabbage loopers (based on non-overlapping 95% confidence intervals) ( Table 2). PBO synergized toxicity of turmeric compounds to the cabbage looper, as previously reported for alpha-cypermethrin (a synthetic pyrethroid insecticide) and xanthotoxin (a furanocoumarin-type plant natural product) 72 h after exposure to the navel orangeworm, Amyelois transitella (Walker, 1863) (Lepidoptera: Pyralidae) 39 . PBO is well recognized to enhance the action of pyrethroids and organophosphates in the malaria mosquito, Anopheles gambiae Giles, 1902 (Diptera: Culicidae) 40 , and deltamethrin in a pyrethroid-resistant strain of A. aegypti 41 .  Table 1. Toxicity of turmeric powder and its derivatives from Curcuma longa (Zingiberaceae) rhizomes with or without synergists against third instar cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae) at an initial screening concentration in the laboratory. n = 3 × 10 insects per treatment. Turmeric crude essential oil, turmeric powder, ar-turmerone, and curcuminoid pigments were tested at 10 μ g/larva. Mixtures of turmeric powder or its derivatives + synergists were tested in a 1:1 ratio of each constituent (dose = 5 μ g + 5 μ g/larva). PBO = Piperonyl butoxide. Shultz Insect Spray ® was tested at 10 μ g/larva. 1 Standard error. Synergists alone did not cause any mortality at the dose tested (5 or 10 μ g/larva) and are therefore not included in the analysis. Mortality of cabbage looper larvae was < 10% for individual treatments of turmeric crude essential oil, turmeric powder, ar-turmerone, and curcuminoid pigments at a dose of 5 μ g/larva (data not reported). We decided to use 1:1 ratio in binary mixtures as this is a common practice in our laboratory. PBO is usually added in a ratio of 10:1 in many commercial insecticides; we have made a simple combination consisting of equal parts of each constituent.
Scientific RepoRts | 6:34093 | DOI: 10.1038/srep34093 Growth inhibitory effects of turmeric powder and its derivatives on cabbage loopers in the laboratory through feeding. Weight of cabbage loopers reared on artificial diets incorporating turmeric powder or its derivatives and their binary mixtures with PBO after seven days in the laboratory showed a significant effect (one-way ANOVA; F 13,105 = 5.5; p < 0.05). Weights of larvae were significantly lower than negative controls for all treatments (Tukeys' test; p < 0.05). Larval weight was significantly lower on ar-turmerone (119.1 mg), ar-turmerone + PBO (81.8 mg) and ar-turmerone + sesamol (116.7 mg) treatments, compared to all other treatments including the negative control (297.8 mg) and the positive control (200.0 mg). Weight reduction for these treatments varied from 60 to 72% compared with the negative control ( Table 3). The positive control reduced growth by 35.2% at 1,000 ppm (Table 3). Red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae) adults, fed on wheat flour (Triticum aestivum L., Poales: Poaceae), which had been treated with turmeric oil at 200 ppm produced fewer and underweight larvae, pupae, and adults compared with those fed on untreated flour 51 . Curcuminoids, comprising three closely related curcumins (I, II, and III) of turmeric rhizome powder, were screened for their growth inhibitory activity against the desert locust, Schistocerca gregaria (Forsskål, 1775) (Orthoptera: Acrididae) and the red cotton bug, Dysdercus koenigii (Fabricius, 1775) (Hemiptera: Pyrrhocoridae) nymphs. At a dosage of 20 μ g per S. gregaria fifth instar nymph, curcumins injected into the hemolymph, produced 40-50% growth inhibition and 10-15% mortality. Turmeric oil produced 10% growth inhibition and 60% nymphal mortality at the same dosage. Topical application of a dosage of 50 μ g of curcuminoids (I, II, and III) produced 45% growth inhibition of D. koenigii nymphs 52 .

ar-Turmerone
Oil Pigments Powder LD 50 (μ g/larva) 1 (95% CI) 2 0.26 (0.14-0.38) 0.05 (0.03-0.08) 0.61 (0.447-1.00) 0.03 (0.00-0.11)  Table 2. Dose response effect of binary mixtures of turmeric powder ('Powder') and derivatives [ar-turmerone, turmeric crude essential oil ('Oil') and curcuminoid pigments ('Pigments')] from Curcuma longa (Zingiberaceae) rhizomes with the synergist piperonyl butoxide (PBO) at a 1:1 ratio against third instar cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae) via topical application. n = 3 × 10 insects per treatment. 1 Concentration causing 50% mortality. LD 50 values were based on 4-5 concentrations (0.01-10 μ g/larva). 2 Confidence interval. 3 Standard error. Mixtures (turmeric powder and its derivatives + PBO) were tested in a 1:1 ratio. Chi-squared test is measuring the null hypothesis that the slope is zero.  Protection of cabbage leaves treated with turmeric powder and derivatives with or without synergists in the laboratory. Weight of cabbage loopers reared on cabbage leaves treated with turmeric powder or its derivatives and their binary mixtures with PBO for four days in the laboratory showed a significant effect (one-way ANOVA; F 7,122 = 4.9; p < 0.05). Larval weight was significantly lower on ar-turmerone (24.3 mg) and ar-turmerone + PBO (14.5 mg) treatments compared with the negative control (46.1 mg) (Tukeys' test; p < 0.05). Larval weights were reduced by 47 and 69% respectively in the ar-turmerone and ar-turmerone + PBO treatments, compared with the control. Similarly, the number of larvae recovered from the leaves was also the lowest on these two treatments (Table 5). Larval recovery from the leaves treated with ar-turmerone + PBO was only 36% relative to the control. Reductions in weight and number of cabbage loopers recovered from cabbage leaves treated with ar-turmerone or ar-turmerone + PBO are similar to those seen with the cotton bollworm, Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae) larvae. First instar H. armigera were reared on a semi-synthetic diet treated with 5% of C. longa rhizome powder for 7-10 days. Larval and pupal weights, survival, development time, and adult emergence rate were adversely affected by C. longa treatments. There was 69% growth inhibition in larvae and the adult emergence period was prolonged by 8 days compared with the control 54 . Turmeric extracts have been shown to protect stored wheat, and increase egg mortality in the Angoumois grain moth, Sitotroga cerealella (Olivier, 1789) (Lepidoptera: Gelechiidae) when treated with 1,000 ppm of turmeric extracts prepared in acetone, ethanol or petroleum ether 23 .

Treatments Mean (mg) ± SE 1 Losses (%) Reduction (%)
Protection of intact cabbage plants treated with turmeric powder and derivatives in the greenhouse. Greenhouse results (   test; p < 0.05). Although larval weights were lowest on ar-turmerone and ar-turmerone + PBO, they did not differ significantly from turmeric crude essential oil + PBO (22.6 mg). Larval weight was significantly lower on turmeric crude essential oil + PBO (22.6 mg) compared with the crude essential oil (36.9 mg) alone. Numbers of larvae recovered from these treatments were also the lowest (Table 6). Larval recovery from cabbage plants treated with ar-turmerone + PBO was only 38% relative to the control. Results in the greenhouse confirm the toxicity of ar-turmerone (+ /− PBO) and although not insecticidal in some cases, it can suppress larval growth and reduce feeding damage caused by this pest. Larvae from the ar-turmerone treatment (+ /− PBO) were significantly lighter (~70% reduction in weight) and weaker than the control, increasing their probability of being preyed upon by natural enemies, as suggested for S. frugiperda larvae fed on an artificial diet treated with an acetonic solution of ar-turmerone 13 .

Conclusion
An insecticide based on turmeric powder or some of its derivatives, especially the sesquiterpene ar-turmerone, could potentially control the cabbage looper larvae. In contrast, curcuminoid pigments were not active. Addition  of PBO increased efficacy of turmeric solutions in most combinations, whereas the natural products, piperonal and sesamol, were not synergistic. The ar-turmerone could be a low-cost and sustainable alternative for IPM of cabbage looper larvae and the addition of PBO can improve its efficacy. Since the treatments exhibit more than one mode of action, it is believed that this will delay resistance development in cabbage looper and other insects. Plant defense chemicals that attack pests at multiple levels are especially suitable for crop protection.

Chemicals. Schultz Insecticide, Houseplant & Indoor Garden Insect Spray ® (Premier Tech Home & Garden
Inc., Brantford, Canada) was used as the positive control. It contains 0.02% pyrethrins as the active ingredient and 0.20% PBO as a synergist ( Fig. 2A) 55 . Piperonal (C 8 H 6 O 3 ) (aka heliotropin) and sesamol (C 7 H 6 O 3 ) (aka 3,4methylenedioxyphenol or 1,3-benzodioxol-5-ol) were used as natural synergists. Piperonal is a compound commonly found in fragrances and flavors. It is structurally related to other aromatic aldehydes such as benzaldehyde and vanillin. Piperonal naturally occurs in various plants, including dill (Anethum graveolens L., Apiales: Apiaceae), violet flowers (Viola odorata L., Malpighiales: Violaceae) and black pepper (Piper nigrum L., Piperales: Piperaceae) (Fig. 2B). Sesamol is a natural component of sesame oil, which is an edible oil derived from sesame seeds (Sesamum spp., Lamiales: Pedaliaceae). It can also be produced via synthesis from heliotropine ( Fig. 2C) 56,57 . PBO was used as a synthetic synergist. All synergists were purchased from Sigma-Aldrich (Canada) and their purity varied from 98 to 100%.  Extraction and structural characterization of ar-turmerone. Rhizomes of C. longa were air-dried at 40 °C for three days and ground into a fine reddish-yellow powder (turmeric powder). The major chemical constituents of turmeric powder are curcuminoids, including bisdemethoxycurcumin (Fig. 2D), curcumin (3.14%) (Fig. 2E) and demethoxycurcumin (Fig. 2F) 58 . Other general constituents include proteins, resins and sugars 59 . Some volatile components could be lost by drying the rhizomes at 40 °C. However, our objective was to test the non-volatile components in the present study. Moreover, ar-turmerone has a high molecular mass and is not volatile at 40 °C.
An aliquot of the turmeric powder was reserved for bioassays and the part of the remainder extracted by steeping in hexane freshly distilled at 25 ± 3 °C with occasional stirring for a period of six hours. Five hundred grams of rhizome powder was extracted with 1 L hexane. The solution obtained was filtered and the solvent removed in a rotary evaporator under low pressure, yielding a light-yellow oil (= crude essential oil). Some volatile compounds in turmeric crude essential oil include atlantone (Fig. 2G), turmerone (Fig. 2H) and zingiberene (Fig. 2I).
An aliquot of the crude essential oil was reserved for bioassays and the remainder was separated by column chromatography on silica gel (Vetec, 60-270 mesh), eluted with hexane:ethyl acetate (9:1). The fractions of interest, containing ar-turmerone (Fig. 3A,B), were analyzed by thin-layer chromatography (0.20 mm thickness, 60-mesh silica gel; Macherey-Nagel) visualized with iodine vapor (sublimation) and compared with a previously isolated and identified standard.
Contact toxicity (topical application) of turmeric powder and derivatives on cabbage loopers in the laboratory. Contact toxicity (measured as 24-48 h mortality) of turmeric powder and its derivatives with or without synergists was determined by topical application to early third instar T. ni following previous methodology with slight modifications 8,61 . Each larva received 1 μ L of acetonic solution of turmeric powder or derivatives (dose = 10 μ g per 3 rd instar cabbage looper) or a 1:1 binary mixture with one of the synergists (5 μ g of turmeric powder or its derivatives + 5 μ g of PBO or other synergists), on the dorsum with a repeating dispenser attached to a 50 μ L syringe. Acetone and Shultz Insect Spray ® , alone, were used as negative and positive controls, respectively. After the compounds were applied, the larvae were transferred to Petri dishes (90 mm diameter × 15 mm height) in groups of 10 along with a small piece of artificial diet (1.12 g). There were three replicates of 10 larvae each per treatment. Treatment groups were placed in sealed plastic boxes lined with moistened paper towels and held for 48 h in a growth chamber (22 ± 3 °C, 16:8-h L:D). The mortality of cabbage loopers was determined after 24 and 48 h. Larvae were considered dead if they did not respond to prodding with forceps according to methodology described for velvetbean caterpillar, Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Noctuidae) larvae treated with neem oil 14 . The LD 50 (lethal dose causing 50% mortality) value was determined for treatments demonstrating > 50% mortality at the initial screening concentration of 10 μ g/larva. Mixtures were tested in a 1:1 ratio (dose = 5 μ g of turmeric powder or its derivatives + 5 μ g of PBO or other synergists). LD 50 values were calculated using the software EPA Probit Analysis Program, version 1.5 62 .
Growth inhibitory effects of turmeric powder and derivatives on cabbage loopers in the laboratory. The effect of turmeric powder and its derivatives was assessed following modified methodology 63 .
Acetonic solutions of the samples (20 mg/mL) were admixed with 3.5 g of dry artificial diet and allowed to dry in a fume hood for approximately 30 min. Following evaporation of the solvent, the artificial diet was mixed with an agar solution (0.5 g agar + 16 mL water were boiled and cooled before mixing to prevent the loss of compounds) to produce 20 g of treated artificial diet (1,000 ppm fwt). The negative control was an artificial diet prepared with  After seven days, all larvae were removed from the trays and individually weighed. The mean larval weight from each treatment was expressed as a percentage of the controls. The EC 50 (effective concentration reducing larval growth by 50%) was determined using four concentrations of each sample (250, 500, 750 and 1,000 ppm fwt). Synergists were added at 1:1,000 part of the mixture (v/v).
Our contact toxicity experiments demonstrated high mortality in binary mixtures (turmeric powder or its derivatives + synergist in a 1:1 ratio) and, therefore, we could not use this ratio for growth inhibition experiments. Effect of an insecticide on the growth of larvae is normally assessed at 1,000 ppm. Synergists tested alone at this concentration did not cause any growth inhibition or mortality of the larvae and were therefore not included in statistical analysis.
Protection of cabbage leaves treated with turmeric powder or its derivatives with or without synergists in the laboratory. Five cabbage leaves were treated with 160 μ L of acetonic solution of turmeric powder or one of its derivatives with or without synergists (PBO, piperonal or sesamol). Solutions were applied and carefully dispersed over the entire area of the leaves using a pipette. Larvae (third-instar) were introduced onto each leaf and allowed to feed for four days. There were five replicates per treatment with six insects per replicate. Larvae collected from each leaf were counted and weighed on day four. Individual treatments were tested at 1% and mixtures in a 1:1 ratio of each constituent. Shultz Insect Spray ® (diluted to 1%) and acetone were used as positive and negative controls, respectively. Shultz Insect Spray ® was used as a positive control because it is based on pyrethrins that are both toxic and inhibit growth of cabbage looper larvae. It was tested at 1,000 ppm in the artificial diet and 10 μ g/larva in contact toxicity bioassays.
Protection of intact cabbage plants treated with turmeric powder and derivatives in the greenhouse. We modified previous methodology 8,61 to demonstrate the protection of intact cabbage plants in the greenhouse. Cabbage plants were grown individually in plastic pots over five to six weeks in the greenhouse until the six to eight leaf stages. Plants were then removed from their trays and arranged into three groups of five plants. All groups were sprayed with treatments until runoff with a hand-held sprayer. Treatments consisted of 1% acetonic solutions of turmeric powder or its derivatives with or without PBO (1:1 ratio). Acetone was used as a negative control and Shultz Insect Spray ® (1%) as a positive control.
The plants were air-dried, and five third instar cabbage loopers were introduced onto each plant (n = 75 larvae per treatment). The plants were randomly placed on a table under grow lights (400-Watt light bulbs per square meter) in the greenhouse (28 ± 3 °C, 16:8-h L:D photoperiod). On day four, larvae were removed from the plants, placed in separate polystyrene cups, and weighed in the laboratory. Experiments were repeated twice.
Statistical Analysis. Mortality data were subjected to Probit analysis to determine LD 50 values (lethal dose causing 50% mortality) and their corresponding 95% confidence intervals using the EPA Probit Analysis Program version 1.5. LD 50 's are considered significantly different from one another if their 95% confidence intervals (C.I.) do not overlap. EC 50 (effective concentration reducing larval growth by 50%) values were calculated by using linear regression analyses in Microsoft Excel. Growth inhibition data were analyzed using the Statistics 7 program for analysis of variance (ANOVA). When significant F values were found, Tukey's HSD multiple-comparison tests were used to test for significant differences between individual treatments. Experiments were repeated at least twice.