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

7. E. METHODOLOGY

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. F. RESULTS & DISCUSSIONS

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. 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. 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. 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. 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. 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]

18. BIOLOGICAL ACTIVITY (LC 50 )

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. 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

23. THANK YOU FOR YOUR KIND ATTENTION QUESTION AND ANSWER SESSION