Natural Products Chemistry & Research

ISSN - 2329-6836

Research Article - (2014) Volume 2, Issue 6

Acetone Fraction of Annona squamosa Seed Extract Inhibits Mitochondrial Complex II of Musca domestica

Vivek Kempraj1,2* and Sumangala K Bhat3
1Biowave Resources, 519, 33rd Cross, 9th Main, IV Block, Bangalore-560011, India
2Division of Entomology and Nematology, Indian Institute of Horticultural Research, Hesserghatta Lake Post, Bangalore -560011, India
3Department of Biotechnology, Acharya Institute of Technology, Bangalore-560011, India
*Corresponding Author: Vivek Kempraj, Division of Entomology and Nematology, Indian Institute of Horticultural Research, Hesserghatta Lake Post,Bangalore-560011, India, Tel: +91-9731372149, Fax: +91-08-26541973 Email:

Abstract

Mitochondrial respiratory complexes in insects are involved in diverse physiological processes including energy production. Specific and potent inhibitors of complex I, III and IV are described and has allowed in determining the importance of these enzymes in the physiological processes. However, there seems a paucity of information regarding potential inhibitors of complex II of insect mitochondria. In this study we demonstrate the toxicity of acetone fraction (ASF) of Annona squamosa seed extract to Musca domestica and its mitochondrial complexes. Effectiveness of ASF as oral bait and contact poison was also evaluated. Inhibition of Complex II in treated flies has been related to its lethargic nature. Energy inhibition study of ASF further confirmed reduction in ATP.

Keywords: Housefly; Complex II; Insecticides; Annona; ATP, Mitochondrial

Introduction

Synthetic insecticides (SI) are widely used for the control of M. domestica. Prolonged and continuous use of SI causes occupational hazard, damage to environment and development of resistance in insects. Therefore, there is a need for an alternative and effective insecticide. Several phytochemicals including acetogenins have been proved effective against houseflies [1-4]. Several compounds and extracts derived from plants of the family Annonaceae are known to exhibit toxic effect on a plethora of organisms [1,5-12] however, studies regarding ASF of seeds of A. squamosa seem meagre [13]. M. domestica is an insect closely associated with the lifestyle of human beings [14]. They are prominent vectors of many diseases [15,16] and affect performance of industries leading to heavy financial loss [17,18]. Hence, maintaining the premises free from houseflies is critically important. This study has evaluated the chemical composition and toxic effects of the ASF of A. squamosa seeds on M. domestica and the mode of action of the fraction.

Materials and Methods

Plant material and extraction

Seeds of A. squamosa (234.8 g) were collected from ripe fruits between September to January during 2008-09, washed under tap water to remove the pulp and were shade dried at 27 ± 1°C for 15 days. The seeds were then pulverized in an electric blender (powdered seed size range from 0.1-2 mm). The dried powder was defatted by extracting in petroleum ether for 48 h. The defatted dried seed powder was then extracted with ethanol (1:2.5 w/v) at 27 ± 1°C for 48 h. The crude extract was filtered and concentrated on a water bath at 40°C to obtain a brown thick paste (~5 g). The concentrated crude extract was dissolved in acetone to yield a soluble and an insoluble fraction. Acetone soluble fraction (hereafter referred as ASF), measuring 150 ml was passed through a membrane filter (Millipore, 0.45 μm) and concentrated to yield a light brown waxy paste (~2 g) and refrigerated until further use.

Chemical analysis and identification of the components of the active fraction

Chemical composition of the active fraction (ASF) was analysed using GC-MS (Perkin Elmer Clarus Gold 500) apparatus equipped with a capillary column (SGE-BPX-5) of 30 m length and 0.25 mm ID and 0.25 mm film thickness. Oven temperature was programmed at 40-270°C. Helium was used as carrier gas at a flow rate of 1 ml/min. Compounds were identified by GC retention time and mass spectrum using NIST library as reference.

Housefly culture

A wild strain of housefly (M. domestica) was used in the assay. Houseflies were collected from poultry near the premises of the research centre. They were transferred to culturing cages of dimension, 30 × 60 × 30 cm3 and allowed to acclimatize to laboratory conditions (27 ± 1°C, 75 ± 2% RH and 15 h light and 9 h dark photoperiod). Autoclaved concoction of wheat flour, yeast pellets and meat (2:1:2 ratio) served as oviposition medium and as food source for larval stages. Adults were fed on sterile sucrose solution (10% w/v) and peptone solution (10% w/v). The pupae formed were collected from culturing cages and transferred to new cages for emergence of adults. The newly emerged (2-day-old) adults were used throughout the study.

Bioassay

Oral toxicity:

Test concentrations ranging from 0.1-5 mg ml-1 were prepared by incorporating appropriate amount of ASF in sucrose solution (10% w/v) with the help of an emulsifier (Triton X-100; 0.01% v/v). Test emulsions (5 ml) were socked on sterile filter paper discs (diameter 10 cm; Whattman) using a micropipette and dried at 27 ± 1°C. This was repeated several times until 5 ml of the emulsion was completely absorbed by the filter paper. The emulsion impregnated filter paper was cut into square pieces of 1 × 1 cm dimension (hereafter referred to as “Bait”). Ten baits were distributed throughout the rearing cages containing 30 numbers of 2-day-old houseflies. This was done to negate the possibility of contact toxicity. Cotton pad socked in water was provided to the houseflies. Bait containing all ingredients excluding ASF served as control. The mortality data was collected after 24 h exposure period. The tests were conducted in triplicates and the results analysed statistically. The relation between concentration and mortality was analysed using log-concentration probit analysis (LC50) software SYSTAT. If mortality exceeds 10% in the control batch, the whole bioassay was discarded or if below 10% the results of the treated sample were corrected using Abbott’s formula [19,20].

Contact toxicity: Contact toxicity of ASF to 2-day-old adult houseflies was tested with concentration ranging from 0.1–5 mg ml- 1. Houseflies were anaesthetised with 1 ml of diethyl ether on cotton pads for 20s and placed on plates cooled to 4°C to maintain anaesthesia during the application. Thirty adults were used for each concentration tested. Test concentrations of ASF were prepared by adding appropriate amounts into acetone. A micro-syringe was used to apply 0.1 μl of the test concentrations onto the thorax of anesthetized houseflies with the help of stereomicroscope (20x). A separate batch (n=30) treated with 0.1 μl of acetone served as control. The control and treated flies were transferred into separate rearing cages for acclimatization. The bioassay was conducted in triplicates. The mortality data collected after 24 h exposure period was subjected to log-concentration probit analysis software SYSTAT.

Insect mitochondrial preparation

Four hundred 2-day-old houseflies were refrigerated for 15 min to stop their activity. The thoraxes were separated from the head and abdomen using a scalpel with the help of a stereomicroscope (20x) under cold conditions. The mitochondrial preparation method by Knecht et al. [21] was followed with slight modifications. One gram of thorax was homogenized with 10 ml of mitochondrial inhibition buffer (MIB) in a pre-cooled homogenizer. The homogenate was centrifuged at 500 g for 10 min at 4°C. The supernatant was taken in sterile tubes and centrifuged at 30,000 g for 10 min at 4°C. The pellets formed were washed thrice, re-centrifuged, weighed and used immediately.

Determination of protein content

Protein concentration of the mitochondrial suspensions was determined by the method of Lowry, BSA was used as standard [22].

Mitochondrial enzyme inhibition studies

Succinate dehydrogenase (SDH) (EC number: 1.3.5.1) activity was measured according to Singer [23]. Mitochondria isolated from thoraxes (0.26 mg) were added to a reaction medium containing 20 mM Sodium succinate, 40 mM HEPES (pH 7.5) and 1% (v/v) Triton X-100. After 1 min, the reaction was initiated by the addition of 10 μL of 0.5% (w/v) DCIP and ASF at varying concentration of 0.0-1.0 μg ml- 1. The reaction was followed at 25°C in a spectrophotometer at 600 nm. Activity was calculated from the absorbance decrease, using an extinction coefficient for DCIP of 19.1 mM- 1cm- 1. Similarly, toxicity of ASF to mitochondrial malate dehydrogenase (MDH) (EC number: 1.1.1.37) was studied. The reaction medium contained 100 mM Tris– HCl (pH 7.8), 20 mM MgCl2, 1 mM EDTA, 0.1 mM NADH and 0.5 mM oxaloacetate. The reaction was initiated by the addition of the enzyme source and ASF at varying concentration of 0.0–1.0 μg - 1. The oxidation of NADH was monitored spectrophotometrically at 340 nm. Activity was calculated from the absorbance decrease, using the extinction coefficient of NADH (6.22 mM- 1 cm- 1). The IC50 values for each enzyme were calculated using the data obtained.

Estimation of total ATP production

ATP extraction method described by Yang et al. [24] was followed with slight modifications to determine the total ATP production of treated flies. Oral and contact treated houseflies (n=60) were collected at different time intervals (1,6,8,12 and 24 h) and frozen for 20 min. Whole flies were homogenized in distilled water (5 ml) and boiled for 15 min. The boiled sample was centrifuged at 5000 g for 10 min. The supernatant was collected and ATP content determined using the method described by Lamprecht and Trautschold [25]. Flies without any treatment were used as control. The experiments were conducted in triplicates.

Statistical analysis of data

Data in the graphs are result obtained with independent mitochondrial preparations and are presented as means ± standard errors (S.E.M). The data were subjected to either Student’s t-test or analysis of variance (ANOVA) with significant differences among means being identified by Duncan’s multiple range test. P<0.0001 was adopted as a criterion of significance. The 95% confidence intervals (CI) of the lethal concentration (LC) at 50% and inhibitory concentration (IC) at 50% were analysed.

Results and Discussion

Results of the toxicity studies using oral and contact formulations of ASF on adult houseflies are shown in Figure 1 and are clear that the toxicity is dose-dependent. The LC50 for oral and contact formulations of ASF were 2.3 ± 0.8 mgml- 1 and 1.9 ± 0.6 mgml- 1, respectively. The treated houseflies frequently rubbed their body and became inactive (unable to fly) 1 h after treatment in both mode of application. Following a 2h exposure, the treated flies completely stopped feeding. From the results, it can be perceived that irrespective of the mode of application, ASF influences the physiology of the flies equally. From the behavioural changes observed in treated flies, it was presumed that ASF targets on sites of energy production.

natural-products-chemistry-research-Effect-ASF

Figure 1: Effect of ASF to M. domastica treated with two different kind of formulation (as Bait and Topical treatment).

Major mitochondrial enzymes regulating energy production were subjected to inhibition activity assay. A dose-dependent inhibition of SDH and MDH by ASF is shown in Figures 2 and 3 respectively. The inhibitory effect of ASF was found to be less on malate dehydrogenase (MDH) (IC50=0.72 μg ml- 1, R2=0.9924; P<0.0001) than that on succinate dehydrogenase (SDH) (IC50=0.65 μg ml- 1, R2=0.9817; P<0.0001).

natural-products-chemistry-research-succinate

Figure 2: Effect of ASF on the succinate dehydrogenase of M. domastica mitochondria.

natural-products-chemistry-research-dehydrogenase

Figure 3: Effect of ASF on the malate dehydrogenase of M. domastica mitochondria.

Figure 4 shows the amount of ATP present in the whole fly following 24 h exposure to ASF by oral and contact applications. Total ATP content decreased to 0.075 μmols in flies treated with oral formulation (R2=0.6497; P<0.0001) and 0.020 μmols in flies treated with contact formulation (R2=0.7513; P<0.0001) from the initial concentration of 0.45 μmols for topical treated and 0.47 μmols in the case of bait treated flies, respectively. However, the ATP concentration remained same as initial level (i.e. 0.45-0.50 μmols) in control flies. From the above data it is evident that ASF interrupts ATP production by inhibition of mitochondrial enzymes. The behavioural observation correlated with these findings. GC-MS analysis of ASF (Table 1) revealed two major compounds, ethyl ester of oleic acid (ethyl oleate) and Iso-octyl phthalate, but previous studies on A. squamosa seeds have reported acetogenins as the active compound [11,15,26-30]. The current study shows the inhibitory activity of ASF on MDH and SDH (Complex II) in housefly, M. domestica. The GC-MS analysis proved that ASF contained no trace of acetogenins. Studies regarding the activity of the major compounds are planned to be carried out in our laboratory.

natural-products-chemistry-research-ATP-production

Figure 4: Effect of two different treatments on ATP production by M. domastica.

Sl.No. CAS.No. Compounds          mg g-1
1 6871-44-9 Diatine 3.82
2 465-11-2 Gamabufagin 2.68
3 547-98-8 Cholestanoic acid 2.06
4 35536-76-6 Corchoroside B 1.67
5 6625-20-3 Sanchycline hydrochloride 1.73
6 76-25-5 Coupe-A 1.72
7 509-60-4 Paramorfan 1.99
8 87-44-5 Beta-caryophyllene 25.22
9 1080-26-0 Ethyl arachidonate 3.43
10 96-76-4 2,4-di-t-Butylphenol 5.04
11 112-39-0 Methyl palmitate 31.41
12 507-79-9 Ungernin 19.82
13 628-97-7 Ethyl palmitate 68.46
14 20987-26-2 Resibufaginol       1.55
15 2777-58-4 Methyl petroselinate 54.15
16 112-61-8 Methyl stearate 16.02
17 111-62-6 Oleic acid, ethyl ester 197.59
18 111-61-5 Ethyl stearate 27.96
19 128-13-2 Ursodiol 1.52
20 27554-26-3 Iso-octyl-phthalate 532.09

Table 1: GC-MS analysis of the acetone-soluble fraction (ASF) of ethanol extract
of A. squamosa.

References

  1. Dos Santos AF, Sant’ana AE (2001) Molluscidal properties of some species of Annona. Phytomedicine 8: 115-120.
  2. Pavela R (2007) Lethal and sublethal effects of thyme oil (Thymus vulgaris L.) on the House fly (Musca domestica Lin.). Journal of Essential Oil Bearing Plants 10: 346-356.
  3. Tarelli G, Zerba EN, Alzogaray RA (2009) Toxicity to vapor exposure and topical application of essential oils and monoterpenes on Musca domestica (Diptera: Muscidae). J Econ Entomol 102: 1383-1388.
  4. Pohlit AM, Rezende AR, Lopes Baldin EL, Lopes NP, Neto VF (2011) Plant extracts, isolated phytochemicals and plant-derived agents which are lethal to arthropod vectors of human tropical diseases – a review. Planta Medica 77: 618-630.
  5. Moeschler HF, Pfluger W, Wendisch D (1987) Pure annonin and a process for the preparation thereof, US Patent 4689232.
  6. Mikolajczak KJ, McLaughlin JL, Rupprecht JK (1988) Control of pests with annonaceous acetogenins, US Patent 4721727.
  7. Mikolajczak KJ, McLaughlin JL, Rupprecht JK (1989) Control of pests with annonaceous acetogenins, US Patent 4721727.
  8. Teresa G, Raul A, Jose RT, Amparo MB, Carmen ZMP (1998) Acetogenins from Annona glabra seeds, Phytochemistry 47: 811-816.
  9. Mehra BK, Hiradhar PK (2000) Effect of crude acetone extract of seeds of Annona squamosa Linn (Family: Annonaceae) on possible control potential against larvae of Culex quinquefasciatus say. J Entomol Res 24: 141-146.
  10. George S, Vincent S (2005) Comparative efficacy of Annona squamosa Linn. and Pongamia glabra Vent. to Azadirachta indica A. juss against mosquitoes. J Vector Borne Dis 42: 159-163.
  11. González-Coloma A, Guadaño A, de Inés C, Martínez-Díaz R, Cortes D (2002) Selective action of acetogenin mitochondrial complex I inhibitors. Z Naturforsch C 57: 1028-1034.
  12. Coelho MB, Marangoni S, Macedo ML (2007) Insecticidal action of Annona coriacea lectin against the flour moth Anagasta kuehniella and the rice moth Corcyra cephalonica (Lepidoptera: Pyralidae). Comp Biochem Physiol C Toxicol Pharmacol 146: 406-414.
  13. Kempraj V, Bhat KS (2011) Acute and reproductive toxicity of Annona squamosa to Aedes albopictus. Pesticide Biochemistry and Physiology 100: 82-86.
  14. Anderson JR, Poorbaugh JH (1964) Observations on the ethology and ecology of various Diptera associated with northern California poultry ranches. Journal of Medical Entomology 1: 131-147.
  15. Guadaño A, Gutiérrez C, de La Peña E, Cortes D, González-Coloma A (2000) Insecticidal and mutagenic evaluation of two annonaceous acetogenins. J Nat Prod 63: 773-776.
  16. Tan SW, Yap KL, Lee HL (1997) Mechanical transport of rotavirus by the legs and wings of Musca domestica (Diptera: Muscidae). J Med Entomol 34: 527-531.
  17. Crosskey RW, Lane RP (1993) Medical Insects and Arachnids. Medical and Veterinary Entomology 8: 178.
  18. Howard J (2001) Nuisance flies around a landfill: patterns of abundance and distribution. Waste Manag Res 19: 308-313.
  19. Abbott WS (1925) A method for computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267.
  20. Finney DJ (1971) Probit Analysis. Cambridge, UK: Cambridge University Press.
  21. Knecht W, Altekruse D, Rotgeri A, Gonski S, Löffler M (1997) Rat Dihydroorotate Dehydrogenase: Isolation of the recombinant enzyme from mitochondria of insect cells. Protein Exp Purif 10: 89-99.
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275.
  23. Singer TP (1974) Determination of the activity of succinate, NADH, choline, and alpha-glycerophosphate dehydrogenases. Methods Biochem Anal 22: 123-175.
  24. Yang NC, Ho WM, Chen YH, Hu ML (2002) A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP. Anal Biochem 306: 323-327.
  25. Lamprecht W, Trautschold I (1974) Methods of Enzymatic Analysis. Weinheim: Germany, Verlag Chemie.
  26. Zafra-Polo MC, González MC, Estornell E, Sahpaz S, Cortes D (1996) Acetogenins from Annonaceae, inhibitors of mitochondrial complex I. Phytochemistry 42: 253-271.
  27. Byun HO, Kim HY, Lim JJ, Seo YH, Yoon G (2008) Mitochondrial dysfunction by complex II inhibition delays overall cell cycle progression via reactive oxygen species production. J Cell Biochem 104: 1747-1759.
  28. Greenberg B (1973) Flics and disease. Vol. II. II. Biology and disease transmission. Flics and disease. Vol. II. II. Biology and disease transmission 1-447.
  29. Kazuyoshi K, Jocelyn PA, Kobayashi A (1989) Isolation, Structure of Neoannonin, A novel insecticidal compound from the seeds of Annona squamosa, Agric Biol Chem 53: 2719-2722.
  30. Richter C, Park JW, Ames BN (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci U S A 85: 6465-6467.
Citation: Kempraj V, Bhat SK (2014) Acetone Fraction of Annona squamosa Seed Extract Inhibits Mitochondrial Complex II of Musca domestica. Nat Prod Chem Res 2:155.

Copyright: © 2014 Kempraj V, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.