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  1. 1. SAIFUL IRWAN ZUBAIRI Supervisor: Prof. Dr. Mohammad Roji Sarmidi The Yield and Biological Activity of Rotenone Extracted From Derris elliptica
  2. 2. Presentation Outline <ul><li>Introduction </li></ul><ul><li>Derris elliptica Background </li></ul><ul><li>Methodology </li></ul><ul><li>Results and Discussion </li></ul><ul><li>Conclusion </li></ul> 
  3. 3. Introduction <ul><li>Despite decades of warnings, the inappropriate use of chemical pesticides continues to pose threats to the environment and human health, especially in developing countries. </li></ul><ul><li>There have been massive upsurges in pesticide use in recent years (Harris, 1999), increasing use and often misuse has lead to increased problems of insecticide resistance. </li></ul>
  4. 4. Introduction <ul><li>In this research, the roots of Derris elliptica which is better known locally as Tuba plant is utilized as a raw material. </li></ul><ul><li>It is believed that the tuba plant contains an active ingredient known as Rotenone , which can be used to manufacture bio-pesticides. </li></ul><ul><li>Rotenone is a naturally occurring chemical with insecticidal and piscicidal properties extracted from the roots of several plant species belonging to genus Derris spp grown in Malaysia, East Indies and South America or Lonchocarpus spp grown in Central and South America . </li></ul>
  5. 5. Introduction <ul><li>This non-polar molecule, acts toxic towards cold-blooded animals but harmless to the man and environment. When exposed to sunlight, it is easily biodegrades to form Carbon Dioxide (CO 2 ) & Water (H 2 O). </li></ul><ul><li>Low concentration in commercial products, degradability , and poor absorption across gut and skin in humans; are factors accounting for the good safety record of rotenone . </li></ul><ul><li>Rotenone has been approved by EPA as organic pesticide for organic farming due to low toxicity and low detectable residues in foods (Baker et. al., 2002). </li></ul> 
  6. 6. Engineering Questions <ul><li>What are critical process parameters? </li></ul><ul><li>How do we maximise yield? </li></ul><ul><li>What are the economically optimal operating conditions? </li></ul><ul><li>What are the processing parameters that reduce dissipation of the active ingredient throughout the whole extraction process? </li></ul><ul><li>How do we ensure active ingredient is present and in the correct amounts i.e. Standardisation </li></ul>
  7. 7. Objective & Scopes <ul><li>The main objective of the study is to study the effect of processing parameters and the biological activity of rotenone extracted from Derris elliptica using a batch solid-liquid extraction process. </li></ul><ul><li>In order to achieve the objective, four scopes have been identified in this research. The scopes are: </li></ul><ul><li>To study the effect of processing parameters on the yield of rotenoids resin. </li></ul><ul><li>To study the effect of processing parameters on the rotenone content. </li></ul><ul><li>To study the effect of processing parameters on the biological activity. </li></ul><ul><li>To study the interactions between the biological activity with the yield of rotenoids resin and the rotenone content . </li></ul>
  8. 8. Derris elliptica Background <ul><li>Biology </li></ul><ul><li>Morphology </li></ul><ul><li>Phytochemistry </li></ul><ul><li>Analysis </li></ul><ul><li>Uses </li></ul>
  9. 9. Biology & Morphology <ul><li>Biology </li></ul><ul><li>Member of the Leguminosae </li></ul><ul><li>Part of Fabaceae family </li></ul><ul><li>Composed of 200 genera and 68 species including 21 species of Tephrosia,12 of Derris, 12 of Lonchocarpus, 10 of Millettia and several of Mundula (John, 1944). </li></ul><ul><li>Three species are found in Malaysia, which is Derris elliptica, Derris malaccensis and Derris uliginosa (Gaby Stroll, 1986). </li></ul><ul><li>Morphology </li></ul><ul><li>Small shrub originating in the tropical rainforests </li></ul><ul><li>Grows in lowland areas and does not thrive at higher altitudes </li></ul><ul><li>Required 26 months for the maximum development of the rotenone. </li></ul><ul><li>Derris can propagate vegetatively and fully developed after six weeks. </li></ul><ul><li>Derris thrives on many soils but particularly on loams and clays (Gaby Stroll, 1986). </li></ul>
  10. 10. Phytochemistry <ul><li>Active chemical constituents </li></ul><ul><ul><ul><li>Isoflavonoids - Major component is rotenone. </li></ul></ul></ul><ul><ul><ul><li>Rotenoids - consisted with all isoflavonoids. </li></ul></ul></ul><ul><ul><ul><li>Other Isoflavonoids - deguelin, elliptone, tephrosin, toxicarol, </li></ul></ul></ul><ul><ul><ul><li>malaccol & sumatrol (15-hydroxyrotenone). </li></ul></ul></ul><ul><ul><ul><li>Derris elliptica and Derris malaccensis contains 4 - 5 percent rotenone in dry roots. </li></ul></ul></ul><ul><ul><ul><li>Lonchocarpus utilis and Lonchocarpus urucu contain 8 - 10 percent rotenone in dry roots. </li></ul></ul></ul>
  11. 11. Phytochemistry <ul><li>Rotenone molecular structure </li></ul><ul><li>Rotenone empirical formula C 23 H 22 O 6 </li></ul><ul><li>Molecular weight of 394.41 g/mol (Schnick, 1974) </li></ul>
  12. 12. Uses <ul><li>Traditionally </li></ul><ul><ul><li>Used for fish poison, remedy for snake bites, skin sores, eczema and forehead relieve headaches. </li></ul></ul><ul><ul><li>Root soaked or boiled with water and applied externally (Tinde Van Andel, 2000). </li></ul></ul><ul><li>Scientifically </li></ul><ul><ul><li>Selective & non-systemic insecticide used on fruit crops and home gardens (WHO, 1992). </li></ul></ul><ul><ul><li>For external treatment to treat tick, lice, scabies and other ectoparasites on pets (EXTOXNET, 1996). </li></ul></ul><ul><ul><li>Fish eradications as part of water body management. </li></ul></ul>
  13. 13. Analysis <ul><li>Small number of compounds (<10 A.i) </li></ul><ul><li>Difficulty in identification and quantification if the photo-degradation occurred consequently decomposes to at least 20 degradation products (Cheng et al.1972). </li></ul><ul><li>Methods used include: </li></ul><ul><ul><li>Thin Layer Chromatography </li></ul></ul><ul><ul><li>High Performance Liquid Chromatography </li></ul></ul>
  14. 14. Compounds <ul><li>Major Isoflavonoids that naturally occurred in derris resin (Rotenoids) </li></ul><ul><ul><li>rotenone (m.p: 163 0 C) </li></ul></ul><ul><ul><li>elliptone (m.p: 159 0 C) </li></ul></ul><ul><ul><li>sumatrol/15-hydroxyrotenone (m.p: 188 0 C) </li></ul></ul><ul><ul><li>malaccol/15-hydroxyelliptone (m.p: 244 0 C) </li></ul></ul><ul><ul><li>toxicarol (m.p: 101 0 C) </li></ul></ul><ul><ul><li>deguelin (m.p: 165 - 171 0 C) </li></ul></ul><ul><ul><li>tephrosin/oxidation product of deguelin (m.p: 198 0 C) </li></ul></ul>
  15. 15. Thin Layer Chromatography <ul><li>The presence of rotenone is identified under the UV light of 254 nm and 365 nm. </li></ul><ul><li>Solvent mixture of Petroleum Eter and Ethyl Acetate (4:2) – MOBILE PHASE. </li></ul><ul><li>CAMAG Linomat 5 (Semi Automatic Sampler Thin Layer Chromatography Device). </li></ul>CAMAG LINOMAT 5 0.63 R f (8) 0.63 Std R f Methanol 95 % ROTENONE Methanol, Under UV light 254 nm ds dc 8 7 6 STD 9 10 ROTENONE Methanol, Under UV light 365 nm ds dc 8 7 6 STD 9 10
  16. 16. High Performance Liquid Chromatography (HPLC) <ul><li>Based on Ronald L. Baron, (1976). </li></ul><ul><li>Using the external standard method for identification of rotenone content. </li></ul><ul><li>Using the Internal standard method for validation - OPTIONAL. </li></ul>Rotenoids resin Calibration solution Rotenone (6) Tephrosin (5) Deguelin (7) 5 µL Injection volume 294 nm UV Wavelength (  ) 0.7 ml/min Flow rate Ambient Column temperature SETTING PARAMETER
  17. 17. Methodology Phases of the experiment Flow diagram and overview of the project There are four major steps involved in all three phases of experiments PRELIMINARY PHASE OPTIMIZATION PHASE VERIFICATION PHASE PRE-PROCESSING PROCESSING/EXTRACTION ANALYSIS OF RESPONSE VARIABLES STATISTICAL ANALYSIS
  18. 18. Preliminary experiments Processing parameters of the extraction of rotenone at the preliminary experiments Fixed processing parameters of the extraction of rotenone at the preliminary experiments Response variables of the extraction of rotenone 0 – 1440 min (30 min interval times) Extraction duration Rough (5 – 2 mm) & Smooth (2 – 0.5 mm) Raw material particles size 10 ml/g & 3.3 ml/g Solvent-to-solid ratio Chloroform, Ethanol & Acetone Types of solvent Factor levels Factor name Ambient temperature (26 0 C – 30 0 C) Extraction temperature 30 g of dried tuba roots Weight of raw material Factor level Factor name mg (A.i) Rotenone content % (w/w) Yield of rotenone Response Values Response Variables
  19. 19. Optimization experiments <ul><li>Based on the preliminary study, an experimental design is developed based on the second-degree polynomial model of the response surface method (RSM). </li></ul><ul><li>3 factors out of 4 factors are chosen with 2 levels range which are the highest level (+α) & the lowest level (-α). </li></ul><ul><li>DESIGN EXPERT software version 6.0 (Stat-Ease. Inc., 2002) is used to design and interpret the significant effect of the experimental results. </li></ul><ul><li>The design of experiments used in the study is a Central Composite Design; CCD with 2 nd Order Model - (2 3 ) with 30 runs including 3 centre point, 2 replicates & 1 alpha point (α). </li></ul><ul><li>TOTAL OF EXPERIMENT = </li></ul><ul><li>[2 n + 3 CP (Centre Point)]  2 Replicates] + [2 n  (α)] </li></ul><ul><li> = (2 3  2 Replicates) + (3 CP  2 Replicates) + [ 2 3  1(α)] </li></ul><ul><li> = 16 + 6 + 8 = 30 experiments. </li></ul>
  20. 20. Optimization experiments Processing parameters of the extraction of rotenoids resin at the optimization phase Factor levels range is denoted as (α): +α = The highest level; -α = The lowest level Fixed processing parameters of the extraction of rotenoids resin at the optimization phase Response variables of the extraction of rotenoids resin at the optimization phase 0.5 mm (- α ) and 5 mm (+ α ) Raw material particles size X 3 10 ml/g (- α ) and 2 ml/g (+ α ) Solvent-to-solid ratio X 2 Ethanol (- α ) and Acetone (+ α ) Types of solvent X 1 Factor levels Factor name Factor Ambient temperature (26 0 C – 30 0 C) Extraction temperature 50 g of dried tuba roots Weight of raw material Exhaustive extraction time (10 – 12 hours) Extraction duration Factor level Factor name Lethal Concentration (LC 50 ) – mg/ml Brine Shrimp Lethality Bioassay mg (A.i) Rotenone content mg (resin) Yield of rotenoids resin Response Values Response Variables
  21. 21. Verification experiments <ul><li>The verification phase is then carried out based on the results obtained from the optimization phase. </li></ul><ul><li>The experiments are carried out in 2 replicates. </li></ul><ul><li>The result obtained will verified the selection of the most appropriate processing parameters. </li></ul>
  22. 22. Tuba Pre-processing Various particles size of tuba roots Rough (5 – 3 mm) Intermediate (2 – 1 mm) Smooth (1 – 0.5 mm) An important aspect of the phytochemical processing is the pre-processing of the herbal material prior to extraction. For the preliminary phase, the procured tuba roots are sifted and separated into 2 main particles size, smooth (2 – 0.5 mm) and rough (5 – 2 mm) in diameter. For the optimization phase, the procured tuba roots are sifted and separated accordingly to the particles size generated from the software.
  23. 23. Dried tuba roots in small pieces (Based on the particles size generated from the software ) 500 ml solvent (Acetone & Ethanol) added to 50 g sample – (10 mL/g) Normal Soaking Extraction for 10 - 12 hours (Ambient temperature) and stored in a dark place Filtered using Altech filter with GAST Laboratory Diaphragm Vacuum Pump & Compressor at 300 mbar. Evaporated in rotary evaporator with vacuum pump at maximum vacuum of 300 mbar (Water bath heater temperature is set to be 40 0 C for 1 hour – Remove  90 % of solvent) Concentrated Liquid Crude Extract - Rotenoids resin Store in a dark place at room temperature and avoid any light and excessive heat. Extraction of rotenoids resin from Tuba Normal Soaking Extraction method Construct the kinetic extraction curves Content determination for 30 min interval
  24. 24. Analysis of Response Variables Product analysis is carried out on the response variables namely the yield of the extraction, rotenone content and the Lethal Concentration (LC 50 ) of the biological activity analysis. PRODUCT ANALYSIS Yield of rotenoids resin (% w/w) Rotenone content (% w/w) Lethal Concentration (LC 50 ) of the biological activity analysis µg/ml (10 mg/ml = 1 %) High Performance Liquid Chromatography (Quantitative) Thin Layer Chromatography (Quantitative)
  25. 25. <ul><li>The eggs of brine shrimp, artemia salinas are readily available in pet shops. </li></ul><ul><li>Upon being placed in seawater, the eggs hatch within 48 hours to provide large numbers of larvae (nauplii) for experimental use </li></ul><ul><li>Allow 2 days for the shrimp to hatch and mature as naulpii. </li></ul><ul><li>Prepare vial for testing; for each fraction, test initially at 1000, 500, 100, 50, 10  g/ml; prepare 2 vials at each concentration for a total 10 vials plus 1 control vial. </li></ul><ul><li>Transfer 500 µL to vials corresponding to 1000, 500, 100, 50, 10  g/ml, respectively. </li></ul><ul><li>Rotenoids resin concentration is the initial concentration and dilution will be prepared accordingly to the amount of test concentration. </li></ul><ul><li>After 2 days (when the shrimp larvae are ready), add about 4 ml of seawater to each vial, count 10 shrimp per vial (50 shrimp/dilution) and adjust the volume with the seawater to 5 ml/vial. </li></ul><ul><li>Place the vials, uncovered and under the lamp. Be sure that the lamp does not overheat vials. 24 hours later count and record the number of survivors. </li></ul>Brine Shrimp Lethality: A rapid General Bioassay for Bio-active Compounds
  26. 26. Lethal Concentration (LC 50 ) of the biological activity analysis Example of the dilution principles for preparing the bioassay concentration 1 ppm = 10 -6 g/ml = 0.001 mg/ml V 0 C 0 = V 1 C 1 (V 0 )(10) = (5)(1) V 0 = 0.5 ml V 1 C 1 = V 2 C 2 (V 1 )(1) = (5)(0.5) V 1 = 2.5 ml V 2 C 2 = V 3 C 3 (V 2 )(0.5) = (5)(0.1) V 2 = 1.0 ml V 3 C 3 = V 4 C 4 (V 3 )(0.1) = (5)(0.05) V 3 = 2.5 ml V 4 C 4 = V 5 C 5 (V 3 )(0.05) = (5)(0.01) V 4 = 1 ml Transfer 500  L of each fraction to the testing vial contains 10 shrimps/vial (5 ml/vial) for a replicate of 2 (2 fractions/vial) 70 ml 5 ml 5 ml 5 ml 5 ml 5 ml Extract sample C 0 = 10.0 mg/ml 1000 ppm (1.0 mg/ml) 500 ppm (0.5 mg/ml) 100 ppm (0.1 mg/ml) 50 ppm (0.05 mg/ml) 10 ppm (0.01 mg/ml) 500  L 500  L 500  L 500  L 500  L 500  L 500  L 500  L 500  L 500  L CONTROL 0.5 ml 2.5 ml 1.0 ml 2.5 ml 1.0 ml
  27. 27. Brine Shrimp Lethality: A rapid General Bioassay for Bio-active Compounds Artemia salina mortality when exposed to extracts of the Derris elliptica Number of artemia salina mortality will be evaluated using the dose-response curves to determine the Lethal Concentration of rotenone that gives 50 % of the mortality. 0 10 50 100 500 1000 Dose ((  g/ml) LC 50 50 % 100 % Number of Mortality
  28. 28. Statistical Analysis <ul><li>The design of experiment (DOE) are used to evaluate the effects of several different factors on a response variable. </li></ul><ul><li>Statistical tool of (ANOVA) is used to analyze the data from the experiments and to make decisions about whether a given factor has a significant impact on the response variable. </li></ul><ul><li>In the optimization phase, the means of statistical analysis involves ANOVA and Response Surface Methodology (RSM). </li></ul><ul><li>The RSM is carried out based on the design of the experiments (DOE) generated by the Design Expert software version 6.0 (Stat-Ease Inc., 2000). </li></ul><ul><li>Contour plot for response surface and the optimum response can be built and the optimum parameters for each variables can be obtained from the response surface. </li></ul>
  29. 29. Preliminary Experiment Results Effects of the plant parts and types of solvent on yield Yield of the Normal Soaking extraction method for different types of solvent
  30. 30. Preliminary Experiment Results Extraction yield model and the effect of extraction duration on yield Kinetic of Normal Soaking Extraction process of Derris elliptica roots: Yield of rotenone % (w/w) Kinetic equilibrium of the rotenone extraction process (logarithmic) Figure A Figure B
  31. 31. Preliminary Experiment Results Effects of the extraction temperature on yield Degradation of rotenone occurred during the concentration process at 50 0 C & 80 mbar of operating temperature and vacuum pressures respectively
  32. 32. Preliminary Experiment Results Result of rotenone yield (mg/g dried Tuba roots) Result of rotenone concentration (mg/ml) Result of rotenone content (mg) Effect of the solvent-to-solid ratio on yield 7.76 Tuba Kapur Kota Johor Lama (3.3 ml/g) 18.57 Tuba Kapur Kota Johor Lama (10.0 ml/g) mg/g dried Tuba roots Name of Samples 3.95 Tuba Kapur Kota Johor Lama (3.3 ml/g) 2.58 Tuba Kapur Kota Johor Lama (10.0 ml/g) Concentration (mg/ml) Name of Samples 233.05 Tuba Kapur Kota Johor Lama (3.3 ml/g) 185.76 Tuba Kapur Kota Johor Lama (10.0 ml/g) Yield (mg) Name of Samples
  33. 33. Preliminary Experiment Results <ul><li>Physical Parameters </li></ul><ul><li>UV Absorbance - HPLC </li></ul><ul><ul><li>Max: approximately 280 - 294 nm </li></ul></ul><ul><li>Fraction of rotenone in dry roots </li></ul><ul><ul><li>1.14 % - 1.65 % of extract w/w: Acetone extract </li></ul></ul><ul><li>Concentration of rotenone </li></ul><ul><ul><li>1.58 mg/mL - 2.87 mg/mL: Acetone extract </li></ul></ul><ul><li>Mass of rotenone in dry roots </li></ul><ul><ul><li>285.07 mg - 870.00 mg: Acetone extract </li></ul></ul><ul><li>Exhaustive extraction time </li></ul><ul><ul><li>10 – 12 hours: Acetone extract </li></ul></ul>
  34. 34. Preliminary Experiments Discussion <ul><li>Longer duration leads to higher yield: </li></ul><ul><ul><ul><li>Max at 10 to 12 hours </li></ul></ul></ul><ul><ul><ul><li>52 % extracted within 30 min & 90% in 8 hrs </li></ul></ul></ul><ul><li>Acetone is the best solvent for obtaining the high rotenone content in fresh raw material as compared to the others two solvents (Chloroform & Ethanol). </li></ul><ul><li>Rotenone content in the root is always higher than in the stem. </li></ul><ul><li>Rotenone is being bio-accumulated at the root. </li></ul><ul><li>The initial high rate of extraction may be due to washing rather than the leaching process, where the bio-active compounds released from within cells by crushing or grinding are extensively quick on dissolving into the bulk solution. </li></ul><ul><li>The fine roots tend to be more superior in rotenone content because of the resin cell tissue that contains the rotenoids (Rotenone and its derivatives) are relatively abundant in roots of small and medium diameters (Francis A., 1943). </li></ul><ul><li>The best solvent-to-solid ratio for the Acetone extracts is 10 mL/g. In the optimization phase, the solvent-to-solid ratio of 10.0 mL/g & 2.0 mL/g will be used to obtain the optimum condition within the parameter values from the preliminary study and literature cited. Increasing the solvent-to-solid ratio increased the rotenone yield in the extraction. The increase of rotenone yield with the increase of the solvent-to-solid ratio is consistent with the mass transfer principles. </li></ul><ul><li>The solvent-to-solid ratio is strongly depends on the extraction conditions, by modifications on the solubility and solute-solvent interactions. </li></ul>
  35. 35. Expected Optimization Results From the ANOVA table simulated by the software, the significant effects of each processing parameters (X,Y) towards the response variables (Z) will be calculated using the F-value and if the effects is significant, the optimum responses can be interpreted, obtained and the conclusion can be made conclusively. The optimum response variables will be summarized including the optimum processing parameters. MAXIMUM YIELD OF ROTENOIDS RESIN = Q % (w/w) MAXIMUM ROTENONE CONTENT = R % (w/w) MAXIMUM LETHAL CONCENTRATION (LC 50 ) = J (µg/ml) = % (10 mg/ml = 1%) The graphs consist of three axes where Z axes represent the response variable while X and Y axes represent three conditions as follows: X: Types of solvent, Y: Solvent-to-solid ratio X: Raw material particles size, Y: Solvent-to-solid ratio X: Raw material particles size, Y: Types of solvent Z: Yield of rotenoids resin, rotenone content & biological activity
  36. 36. Expected Verification Results <ul><li>Confirmation of the optimization. </li></ul><ul><li>Experiments will be carried out based on the most appropriate processing parameters obtained from the optimization experiments. </li></ul><ul><li>The results obtained verified the selection of the most appropriate processing parameters. </li></ul>Expected results of yield, rotenone content and LC 50 of the rotenoids resin based on the most appropriate processing parameters D3 C3 B3 A3 Bioassay LC 50 (%) D2 C2 B2 A2 Rotenone content (% w/w) D1 C1 B1 A1 Yield of rotenoids (% w/w) RESULT (%) Raw material particles size Solvent-to- solid ratio Types of solvent Response Variables
  37. 37. Expected Comparison of the Optimum Response Variables <ul><li>The optimum response variables obtained from the literature, preliminary, optimization and verification experiments will be compared and correlated to each other. </li></ul><ul><li>No literature is found for the Brine Shrimp Lethality (LC 50 ) of rotenoids resin due to different method and systems of testing used by each researcher. </li></ul><ul><li>The analysis of rotenone content for the preliminary, optimization and verification phase will be used a standard method of analysis based on the method from Ronald L. Baron, (1976) and also from the AOAC Official Method, (1983). </li></ul>G3 F3 E3 NA Bioassay LD 50 (%) G2 F2 E2 14 % (SAPHYR, 1986) Rotenone content (% w/w) G1 F1 E1 39 % (SAPHYR, 1986) Yield of rotenoids (% w/w) Verification Optimization Preliminary Literature Response variables
  38. 38. Preliminary Experiments in Progress <ul><li>Other preliminary work in progress </li></ul><ul><ul><li>Fine tuning analysis method and apparatus. </li></ul></ul><ul><ul><li>Calibration of rotenone standard. </li></ul></ul><ul><ul><li>Determination of the effect of raw material particles size on yield. </li></ul></ul><ul><ul><li>Validation on the effect of the temperature on yield </li></ul></ul><ul><li>The preliminary experiment result of raw material particles size will be used for the optimization experiments. </li></ul>
  39. 39. Conclusion <ul><li>Future Work </li></ul><ul><li>Determination of the effect of raw material particles size on yield – Preliminary experiment. </li></ul><ul><li>The optimization phase studies: effect of processing parameters on the response variables. </li></ul><ul><li>The verification phase studies: confirmation of the optimization. </li></ul><ul><li>Correlation between biological activity studies, yield of rotenoids resin and rotenone content. </li></ul>
  40. 40. Conclusion <ul><li>Recommendation </li></ul><ul><li>The condition in which the Derris elliptica is collected play vital part in ensuring high rotenone content. It is believed that if the production of rotenone by Derris elliptica can be understood, the optimum derris plant can be cultivated and therefore increasing the overall yield of the extraction. </li></ul><ul><li>Rotenone dissipation during concentration process should be taken further noticed and yet there is not conclusive evidence to pronounce that by using the high temperature of operating condition can cause major losses of the toxicological constituents. </li></ul><ul><li>To perform an economic optimizations in order to determine an optimal economic process parameters. </li></ul><ul><li>The development of other processing techniques which reduced energy consumption, minimize degradation and maximise the yield of rotenone should be studied further. </li></ul>