VIVA M.Eng. 2006 UTM

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VIVA M.Eng. 2006 UTM

  1. 1. “ THE YIELD AND BIOLOGICAL ACTIVITY (LC 50 ) OF ROTENONE EXTRACTED FROM Derris elliptica” SAIFUL IRWAN BIN ZUBAIRI MK033022 Supervisor: Professor Dr. Mohammad Roji Sarmidi Faculty of Chemical and Natural Resources Engineering Universiti Teknologi Malaysia
  2. 2. OUTLINE <ul><li>JUSTIFICATION OF RESEACH </li></ul><ul><li>KEYWORDS </li></ul><ul><li>SIGNIFICANT CONTRIBUTION </li></ul><ul><li>OBJECTIVE & SCOPES </li></ul><ul><li>METHODOLOGY </li></ul><ul><li>RESULTS & DISCUSSIONS </li></ul><ul><li>CONCLUSION </li></ul>
  3. 3. A. JUSTIFICATION OF RESEARCH <ul><li>Crop protections today rely heavily on synthetic pesticides (Coats, 1994) </li></ul><ul><li>Their uninterrupted and massive use has led to several effects: (A) pesticide resistance in pests; (B) eliminations of naturally occurring bio control agents; (C) Insect resurrection and (D) adverse effects on non-target organisms (Copping, 1998; Harris, 1999) </li></ul><ul><li>The growing public alarm about the hazard linked with unwarranted use of synthetic pesticide has revitalized the interest in the use of environmental friendly phytochemical pesticides </li></ul><ul><li>Derris elliptica is one of the plant species to possess an environmental friendly phytochemical known as rotenone </li></ul><ul><li>The problems occurred when there is NO specific research attribute to the best method to extract rotenone for the purpose of producing high quality of phytochemical pesticide </li></ul><ul><li>Thus, most of the roots of Derris elliptica either fresh or dried was extracted at different processing parameter BUT the most appropriate level of processing parameter that contribute to the high yield of rotenoids resin and rotenone content plus toxicity level have not been identified yet </li></ul><ul><li>It is important to identify the most appropriate processing parameter to obtain high yield of rotenoids resin, rotenone content and toxicity level for the production of efficient botanical insecticide </li></ul>
  4. 4. B. KEYWORDS <ul><li>Derris elliptica : ‘Tuba Kapur’-75 % moisture content-25 0 C to 30 0 C surround temperature-low acidity soil-enhance-development-rotenone high content </li></ul><ul><li>Rotenoids resin: The whole extract-5 rotenoids-rotenone+deguelin+tephrosin+toxicarol+ 6  ,12  -rotenolone+ impurities (e.g.: resin, wax etc.) </li></ul><ul><li>Rotenone: Isoflavonoid-major constituent-extremely toxic-cold blooded organism-unstable-light and heat sensitive-degrade-dihydrorotenone+H 2 0 </li></ul><ul><li>Independent variables (IV): Processing parameters/factor [solvent-to-solid ratio (ml/g), raw material particles size (mm ID) & types of solvent] </li></ul><ul><li>Response variables (RV): Dependent variables [yield of rotenoids resin % (w/w), yield of rotenone % (w/w) & biological activity (LC 50 )] </li></ul>
  5. 5. C. CONTRIBUTION <ul><li>Valuable experimental data - promote and enhance for better processing technology in the phytochemical pesticide industry </li></ul><ul><li>Identification of main independent variables/parameters (IV) effect and the significant interaction </li></ul><ul><li>Determination of the correlation to comprehend better processing method to maximum response variables (RV) </li></ul>
  6. 6. D. OBJECTIVE & SCOPES <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 yield of rotenone (rotenone content) </li></ul><ul><li>To study the effect of processing parameters on the biological activity (LC 50 ) </li></ul><ul><li>To study the correlation between the biological activity (LC 50 ) with the yield of rotenoids resin and the rotenone content </li></ul>“ The effect of processing parameters on the yield and its biological activity (LC 50 ) of rotenone extracted from Derris elliptica using a batch solid-liquid extraction process”
  7. 7. E. METHODOLOGY
  8. 8. THE PHASES OF EXPERIMENT <ul><li>PRELIMINARY: Investigate the suitable range of IV </li></ul><ul><li>OPTIMIZATION: </li></ul><ul><ul><li>Using 2 k factorial design with 3 factors at 2 levels including 3 centre points, 2 replicates and 1 alpha point (α) </li></ul></ul><ul><ul><li>RSM of CCD - evaluate the effect between IV & RV (16 sets of exp+6 CP+8  points = 30 experiments) </li></ul></ul><ul><ul><li>Implemented: 1 st order OR 2 nd order polynomial model </li></ul></ul><ul><ul><li>ANOVA - analyze data & whether IV gives significant optimum to RV </li></ul></ul><ul><li>VERIFICATION: Confirmation of optimization by verified the selection of the most suitable range of IV </li></ul><ul><li>COMPARISON: Significant comparison between literature, preliminary, optimization and verification RV </li></ul><ul><li>CORRELATION: Correlation between RV using the Pearson’s correlation coefficient (r) </li></ul><ul><li> </li></ul>
  9. 9. THE PROCESS FLOW DIAGRAM PRE-PROCESSING : Washing, sifting, separating, drying, grinding and storing PROCESSING/EXTRACTION: Dried roots of Derris elliptica were extracted using the Normal Soaking (NSE) Method by setting processing parameter at diff. level generated by Design-Expert  software ANALYSIS OF RESPONSE VARIABLES: Product analysis was carried out on the RV namely: (A) yield of rotenoids resin and rotenone content - HPLC & TLC; (B) Biological activity (LC 50 ) - Brine Shrimp Lethality Study STATISTICAL ANALYSIS: (A) Model adequacy checking of ANOVA (F-distribution table, coefficient of multiple determination; R 2 and lack of fit (B) Pearson’s correlation coefficient; r
  10. 10. F. RESULTS & DISCUSSIONS
  11. 11. PRELIMINARY EXPERIMENT *Selection of parameter – literature review and exploratory experiment a Purity of the solvents were 95.0 % (v/v) b The solvent-to-solid ratio of 3.3 ml/g and 10 ml/g were set up to evaluate the significant effect on the response variables against other ratios from the literature cited (Grinda et al., 1986) and exploratory experiment done by Saiful et al. (2003) respectively c Source: Pagan and Hageman (1949): a) Fine (0.5 mm to 2.0 mm ID); b) Coarse (2.0 mm to 5.0 mm ID) ≤ 1.00 Biological activity, LC 50 (ppm) Brine Shrimp Lethality study (McLaughlin and Rogers, 1998)  1.95 (Saiful et al., 2003) Yield of rotenone dried roots, % (w/w)  9.50 (Saiful et al., 2003) Yield of rotenoids resin in dried roots, % (w/w) Preliminary experiment results Dependent/response variables 0 to 1440 mins (2 hours interval time) Extraction duration Fine particles size & coarse particles size c Raw material particles size 10.0 ml/g & 3.3 ml/g b Solvent-to-solid ratio Chloroform, ethanol & acetone a Types of solvent Factor levels Factor names
  12. 12. OPTIMIZATION EXPERIMENT a Factor levels range is denoted as (α): +α = The highest level; -α = The lowest level. b Ethanol was added with the H 2 O & oxalic acid - A ratio of ethanol (9): H 2 O (1) [Prepare 1.0 mg/ml of oxalic acid solution from the volume (ml) of H 2 O ratio (1)]. c Purity of the solvents are 95.0 % (v/v). d Source: Saiful et al. (2003). e Source: Grinda et al. (1986). f Source: Pagan and Hageman (1949); Maas (1938) and Moore (1940). a Not determined (ND): The model for this response variable (bioassay) generated from the Design-Expert  software version 6.0 (Stat-Ease, 2002) was insignificant in order to obtain the optimum processing parameters. *Selection of the range of optimization parameter – increasing trend for RV in preliminary experiment 0.5 mm ID (-α) and 5.0 mm ID (+α) f Raw material particles size X 3 d 10.0 ml/g (-α) and e 2.0 ml/g (+α) Solvent-to-solid ratio X 2 b Ethanol (-α) and acetone (+α) c Types of solvent X 1 a Factor levels Factor names Factors a ND Biological activity, LC 50 (ppm) Brine Shrimp Lethality study (McLaughlin and Rogers, 1998) 5.99 Yield of rotenone dried roots, % (w/w) 12.26 Yield of rotenoids resin in dried roots, % (w/w) Optimization experiment results Dependent/response variables
  13. 13. EFFECT OF SOLVENT-TO-SOLID RATIO (ml/g), TYPES OF SOLVENT & RAW MATERIAL PARTICLES SIZE (mm ID) ON THE YIELD OF ROTENOIDS RESIN IN DRIED ROOTS, % (w/w) OPTIMIZATION EXPERIMENT
  14. 14. Ethanol extract: A ↓; B ↓ = ↑ Yield of rotenoids resin in dried roots, % (w/w) Acetone extract: A ↓; B ↓ = ↑ Yield of rotenoids resin in dried roots, % (w/w) * A - Solvent-to-solid ratio (ml/g), B - Raw material particles size (mm ID) Types of solvent - Acetone: Yield of rotenoids resin in dried roots, % (w/w) = 12.90-[0.12×solvent-to-solid-ratio] - [0.06 × raw material particles size] Types of solvent - Ethanol + oxalic acid solution: Yield of rotenoids resin in dried roots, % (w/w) = 23.10-[0.12×solvent-to-solid ratio]- [0.06 × raw material particles size]
  15. 15. EFFECT OF SOLVENT-TO-SOLID RATIO (ml/g), TYPES OF SOLVENT & RAW MATERIAL PARTICLES SIZE (mm ID) ON THE YIELD OF ROTENONE IN DRIED ROOTS, % (w/w) OPTIMIZATION EXPERIMENT
  16. 16. Ethanol extract: A ↓; B ↓ = ↑ Yield of rotenone in dried roots, % (w/w) Acetone extract: A ↓; B ↓ = ↑ Yield of rotenone in dried roots, % (w/w) * A - Solvent-to-solid ratio (ml/g), B - Raw material particles size (mm ID) Types of solvent - Acetone: Yield of rotenone in dried roots, % (w/w) = 12.77-[1.15×solvent-to-solid ratio]-[3.32×raw material particles size]+ [0.36×solvent-to-solid ratio×raw material particles size] Types of solvent - Ethanol + oxalic acid solution: Yield of rotenone in dried roots, % (w/w) = 7.56-[1.15×solvent-to-solid ratio] -[2.15×raw material particles size]+ [0.36×solvent-to-solid ratio×raw material particles size]
  17. 17. SUMMARY OF THE OPTIMIZATION EXPERIMENT <ul><li>The selection of the optimization parameter - desirability (0.7) - range of 0 to 1 </li></ul><ul><li>The optimum processing parameters were used as processing parameters in the verification phase </li></ul>Ambient, 26 0 C to 30 0 C Extraction temperature 10 to 12 hours Extraction duration 0.83 mm ID Raw material particles size 4.72 ml/g Solvent-to-solid ratio Acetone, 95.0 % (v/v) Types of solvent Parameter values Processing parameters
  18. 18. BIOLOGICAL ACTIVITY (LC 50 )
  19. 19. VERIFICATION EXPERIMENT 0.83 0.83 Biological activity, LC 50 (ppm) Brine Shrimp Lethality study (McLaughlin and Rogers, 1998)  2.77  2.44 Yield of rotenone in dried roots, % (w/w)  11.33  11.73 Yield of rotenoids resin in dried roots, % (w/w) Replicate 2 Replicate 1 Dependent/response variables Ambient, 26 0 C to 30 0 C Extraction temperature 10 to 12 hours Extraction duration 0.83 mm ID Raw material particles size 4.72 ml/g Solvent-to-solid ratio Acetone, 95.0 % (v/v) Types of solvent Parameter values Processing parameters
  20. 20. COMPARISON 0.83 ND  1.00  1.00 Biological activity, LC 50 (ppm) Brine Shrimp Lethality study: LC 50 of rotenone standard (SIGMA-Aldrich  ), purity of 95 - 98 % (w/w): 17.45 ppm  2.61 5.99  1.95 (Saiful et al., 2003) 14.0 (Grinda et al., 1986) Yield of rotenone in dried roots, % (w/w)  11.53 (pure resin) 12.26 (pure resin)  9.50 (pure resin) (Saiful et al., 2003) 39.0 (non pure resin) (Grinda et al., 1986) Yield of rotenoids resin in dried roots, % (w/w) Verification Optimization Preliminary Literature Dependent/response variables
  21. 21. CORRELATION <ul><li>Pearson’s correlation coefficients (r) were calculated to determine whether any of the response variables were interrelated </li></ul>ND ND Biological activity (LC 50 ) ND -0.46 Rotenone content ND -0.46 Yield of rotenoids resin Biological activity (LC 50 ) Rotenone content Yield of rotenoids resin CORRELATION OF RESPONSES
  22. 22. G. CONCLUSION <ul><li>A batch solid-liquid extraction of Normal Soaking Extraction (NSE) with controlled temperature (major contribution of rotenone dissipation) was proved to increase the rotenoids resin and rotenone content up to 18 % and 25 % respectively (verification versus preliminary) </li></ul><ul><li>Better processing parameters were obtained for the production of high rotenone constituents of phytochemical semi product </li></ul><ul><li>The biological activity (LC 50 ) of rotenoids resin was insignificant in relation to the optimum processing parameters due to inconsistency of rotenone content and as well as the low value of LC 50 which was less than 100 ppm for all treatments </li></ul><ul><li>The presence of other constituents in the rotenoids resin (e.g.: deguelin, tephrosin, toxicarol, 6  ,12  -rotenolone) contributed to the low LC 50 value for all treatments despite of the lower yield of rotenone </li></ul>
  23. 23. THANK YOU FOR YOUR KIND ATTENTION QUESTION AND ANSWER SESSION

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