1. SAIFUL IRWAN ZUBAIRI Supervisor: Prof. Dr. Mohammad Roji Sarmidi The Yield and Biological Activity of Rotenone Extracted From Derris elliptica

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. 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

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

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. 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. 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)

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. 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

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. 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. 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. 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

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

41. THANK YOU FOR YOUR KIND ATTENTION QUESTION AND ANSWER SESSION NATURAL BIO-PESTICIDE LABORATORY , CEPP, UTM SKUDAI, JOHOR - 2004.