“THE YIELD AND BIOLOGICAL ACTIVITY (LC50) OF ROTENONE

EXTRACTED FROM Derris elliptica”

SAIFUL IRWAN BIN ZUBAIRI SAIFUL IRWAN BIN ZUBAIRI

MK033022MK033022

Supervisor: Professor Dr. Mohammad Roji SarmidiSupervisor: Professor Dr. Mohammad Roji Sarmidi

Faculty of Chemical and Natural Resources EngineeringFaculty of Chemical and Natural Resources Engineering

Universiti Teknologi MalaysiaUniversiti Teknologi Malaysia

OUTLINE

A.A. JUSTIFICATION OF RESEACHJUSTIFICATION OF RESEACHB.B. KEYWORDSKEYWORDSC.C. SIGNIFICANT CONTRIBUTIONSIGNIFICANT CONTRIBUTIOND.D. OBJECTIVE & SCOPESOBJECTIVE & SCOPESE.E. METHODOLOGYMETHODOLOGYF.F. RESULTS & DISCUSSIONSRESULTS & DISCUSSIONSG.G. CONCLUSIONCONCLUSION

A. JUSTIFICATION OF RESEARCHCrop protections today rely heavily on synthetic pesticides (Coats, 1994)Crop protections today rely heavily on synthetic pesticides (Coats, 1994)

• Their uninterrupted and massive use has led to several effects: (A) pesticide Their uninterrupted and massive use has led to several effects: (A) pesticide resistance in pests; (B) eliminations of naturally occurring bio control agents; (C) resistance in pests; (B) eliminations of naturally occurring bio control agents; (C) Insect resurrection and (D) adverse effects on non-target organisms (Copping, Insect resurrection and (D) adverse effects on non-target organisms (Copping, 1998; Harris, 1999)1998; Harris, 1999)

• The growing public alarm about the hazard linked with unwarranted use of The growing public alarm about the hazard linked with unwarranted use of synthetic pesticide has revitalized the interest in the use of environmental friendly synthetic pesticide has revitalized the interest in the use of environmental friendly phytochemical pesticidesphytochemical pesticides

• Derris ellipticaDerris elliptica is one of the plant species to possess an environmental friendly is one of the plant species to possess an environmental friendly phytochemical known as rotenonephytochemical known as rotenone

• The problems occurred when there is The problems occurred when there is NONO specific research attribute to the best specific research attribute to the best method to extract rotenone for the purpose of producing high quality of method to extract rotenone for the purpose of producing high quality of phytochemical pesticidephytochemical pesticide

• Thus, most of the roots of Thus, most of the roots of Derris ellipticaDerris elliptica either fresh or dried was extracted at either fresh or dried was extracted at

different processing parameter different processing parameter BUTBUT the most appropriate level of processing the most appropriate level of processing parameter that contribute to the high yield of rotenoids resin and rotenone content parameter that contribute to the high yield of rotenoids resin and rotenone content plus toxicity level have not been identified yetplus toxicity level have not been identified yet

• It is important to identify the most appropriate processing parameter to obtain high 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 yield of rotenoids resin, rotenone content and toxicity level for the production of efficient botanical insecticide efficient botanical insecticide

B. KEYWORDS• Derris ellipticaDerris elliptica:: ‘Tuba Kapur’-75 % moisture content-25 ‘Tuba Kapur’-75 % moisture content-25 00C C

to 30 to 30 00C surround temperature-low acidity soil-enhance-C surround temperature-low acidity soil-enhance-development-rotenone high contentdevelopment-rotenone high content

• Rotenoids resin:Rotenoids resin: The whole extract-5 rotenoids- The whole extract-5 rotenoids-rotenone+deguelin+tephrosin+toxicarol+rotenone+deguelin+tephrosin+toxicarol+66αβαβ,12,12αβαβ--rotenolone+rotenolone+impurities (e.g.: resin, wax etc.) impurities (e.g.: resin, wax etc.)

• Rotenone:Rotenone: Isoflavonoid-major constituent-extremely toxic- Isoflavonoid-major constituent-extremely toxic-cold blooded organism-unstable-light and heat sensitive-cold blooded organism-unstable-light and heat sensitive-degrade-dihydrorotenone+Hdegrade-dihydrorotenone+H2200

• Independent variables (IV): Independent variables (IV): Processing parameters/factor Processing parameters/factor [solvent-to-solid ratio (ml/g), raw material particles size [solvent-to-solid ratio (ml/g), raw material particles size (mm ID) & types of solvent](mm ID) & types of solvent]

• Response variables (RV): Response variables (RV): Dependent variables [yield of Dependent variables [yield of rotenoids resin % (w/w), yield of rotenone % (w/w) & rotenoids resin % (w/w), yield of rotenone % (w/w) & biological activity (LCbiological activity (LC5050)])]

C. CONTRIBUTION

Valuable experimental data - promote and Valuable experimental data - promote and enhance for better processing technology in the enhance for better processing technology in the phytochemical pesticide industryphytochemical pesticide industry

Identification of main independent Identification of main independent variables/parameters (IV) effect and the variables/parameters (IV) effect and the significant interaction significant interaction

• Determination of the correlation to comprehend Determination of the correlation to comprehend better processing method to maximum response better processing method to maximum response variables (RV)variables (RV)

D. OBJECTIVE & SCOPES

• To study the effect of processing parameters on the yield of rotenoids resin

• To study the effect of processing parameters on the yield of rotenone (rotenone content)

• To study the effect of processing parameters on the biological activity (LC50)

• To study the correlation between the biological activity (LC50) with the yield of rotenoids resin and the rotenone content

““The effect of processing parameters on the yield and its The effect of processing parameters on the yield and its biological activity (LCbiological activity (LC5050) of rotenone extracted from ) of rotenone extracted from

Derris Derris elliptica using a batch solid-liquid extraction process” using a batch solid-liquid extraction process”

E. METHODOLOGY

THE PHASES OF EXPERIMENT• PRELIMINARY:PRELIMINARY: Investigate the suitable range of IV Investigate the suitable range of IV

• OPTIMIZATION:OPTIMIZATION: • Using 2Using 2kk factorial design with 3 factors at 2 levels factorial design with 3 factors at 2 levels including including

3 centre points, 2 replicates and 1 alpha point (α)3 centre points, 2 replicates and 1 alpha point (α) • RSM of CCD - evaluate the effect between IV & RV RSM of CCD - evaluate the effect between IV & RV

(16 sets of exp+6 CP+8 (16 sets of exp+6 CP+8 αα points = 30 experiments) points = 30 experiments)• Implemented: 1Implemented: 1stst order order OROR 2 2nd nd order polynomial modelorder polynomial model• ANOVA - analyze data & whether IV gives significant ANOVA - analyze data & whether IV gives significant

optimum to RVoptimum to RV

• VERIFICATION:VERIFICATION: Confirmation of optimization by verified the Confirmation of optimization by verified the selection of the most suitable range of IVselection of the most suitable range of IV

• COMPARISON:COMPARISON: Significant comparison between literature, Significant comparison between literature, preliminary, optimization and verification RVpreliminary, optimization and verification RV

• CORRELATION:CORRELATION: Correlation between RV using the Pearson’s Correlation between RV using the Pearson’s correlation coefficient (r) correlation coefficient (r)

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 (LC50) - Brine Shrimp Lethality Study

STATISTICAL ANALYSIS: (A) Model adequacy checking of ANOVA (F-distribution table, coefficient

of multiple determination; R2 and lack of fit(B) Pearson’s correlation coefficient; r

F. RESULTS & DISCUSSIONS

PRELIMINARY EXPERIMENT

≤ 1.00Biological activity, LC50 (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

*Selection of parameter – literature review and exploratory experiment

0 to 1440 mins (2 hours interval time)Extraction duration

Fine particles size & coarse particles sizecRaw material particles size

10.0 ml/g & 3.3 ml/gbSolvent-to-solid ratio

Chloroform, ethanol & acetoneaTypes of solvent

Factor levelsFactor names

aPurity of the solvents were 95.0 % (v/v)bThe 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) respectivelycSource: Pagan and Hageman (1949): a) Fine (0.5 mm to 2.0 mm ID); b) Coarse (2.0 mm to 5.0 mm ID)

OPTIMIZATION EXPERIMENT

0.5 mm ID (-α) and 5.0 mm ID (+α)fRaw material particles sizeX3

d10.0 ml/g (-α) and e2.0 ml/g (+α)Solvent-to-solid ratioX2

bEthanol (-α) and acetone (+α)cTypes of solventX1

aFactor levelsFactor namesFactors

aFactor levels range is denoted as (α): +α = The highest level; -α = The lowest level.bEthanol was added with the H2O & oxalic acid - A ratio of ethanol (9): H2O (1) [Prepare 1.0 mg/ml of oxalic acid solution from the volume (ml) of H2O ratio (1)]. cPurity of the solvents are 95.0 % (v/v).dSource: Saiful et al. (2003).eSource: Grinda et al. (1986).fSource: Pagan and Hageman (1949); Maas (1938) and Moore (1940).

aNDBiological activity, LC50 (ppm)

Brine Shrimp Lethality study (McLaughlin and Rogers, 1998)

5.99Yield of rotenone dried roots, % (w/w)

12.26Yield of rotenoids resin in dried roots, % (w/w)

Optimization experiment results Dependent/response variables

aNot 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

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

DESIGN-EXPERT Plot

Yield of rotenoids resin in dried rootsX = A: Solvent-to-Solid RatioY = B: Raw material particles size

Actual FactorC: Types of solvent = Ethanol

21. 8233

22.009

22. 1947

22. 3803

22. 566

Yie

ld o

f rot

enoi

ds re

sin

in d

ried

root

s

3.62

4. 81

6. 00

7.19

8.38 1. 41

2. 08

2. 75

3. 42

4. 09

A : S o l ve n t-to -S o l i d R a t i o

B : R a w m a te ri a l p a rti c l e s s i ze

DESIGN-EXPERT Plot

Yield of rotenoids resin in dried rootsX = A: Solvent-to-Solid RatioY = B: Raw material particles size

Actual FactorC: Types of solvent = Acetone

11.624

11.8097

11. 9953

12.181

12.3667

Yie

ld o

f rot

enoi

ds re

sin

in d

ried

root

s

3.62

4. 81

6.00

7.19

8. 38 1. 41

2. 08

2.75

3. 42

4. 09

A : S o l ve n t -to -S o l i d R a ti o

B : R a w m a te ri a l p a rt i c l e s s i ze

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]

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

DESIGN-EXPERT Plot

Yield of rotenone content in dried rootsX = A: Solvent-to-Solid RatioY = B: Raw material particles size

Actual FactorC: Types of solvent = Ethanol

-0.87441

-0.109015

0.656381

1.42178

2.18717

Yie

ld o

f rot

enon

e co

nten

t in

drie

d ro

ots

3.62

4.81

6.00

7.19

8.38

1. 41

2. 08

2.75

3.42

4. 09

A : S o l ve n t-to -S o l i d R a ti o

B : R a w m a te ri a l p a rt i c l e s s i ze

DESIGN-EXPERT Plot

Yield of rotenone content in dried rootsX = A: Solvent-to-Solid RatioY = B: Raw material particles size

Actual FactorC: Types of solvent = Acetone

0.346433

1.69678

3. 04712

4.39747

5.74782

Yie

ld o

f rot

enon

e co

nten

t in

drie

d ro

ots

3.62

4. 81

6.00

7.19

8. 38

1.41

2. 08

2.75

3. 42

4.09

A : S o l ve n t-to -S o l i d R a t i o

B : R a w m a te ri a l p a rt i c l e s s i ze

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]

SUMMARY OF THE OPTIMIZATION EXPERIMENT

2.48 32.69

Yield of rotenoids resin in dried roots = 12.26 % (w/w)

1 2

Types of solvent = Acetone (2)

2.00 10.00

Solvent-to-solid ratio = 4.72 ml/g

0.50 5.00

Raw material particles size = 0.83 mm ID

0.17 9.82

Yield of rotenone in dried roots = 5.99 % (w/w)

Ambient, 26 0C to 30 0C Extraction temperature

10 to 12 hoursExtraction duration

0.83 mm IDRaw material particles size

4.72 ml/gSolvent-to-solid ratio

Acetone, 95.0 % (v/v)Types of solvent

Parameter valuesProcessing parameters

• The selection of the optimization parameter - desirability (0.7) - range of 0 to 1 • The optimum processing parameters were used as processing parameters in the

verification phase

BIOLOGICAL ACTIVITY (LC50)

VERIFICATION EXPERIMENT

0.830.83Biological activity, LC50 (ppm)

Brine Shrimp Lethality study (McLaughlin and Rogers, 1998)

≅ 2.77 ≅ 2.44Yield of rotenone in dried roots, % (w/w)

≅ 11.33≅ 11.73Yield of rotenoids resin in dried roots, % (w/w)

Replicate 2Replicate 1Dependent/response variables

Ambient, 26 0C to 30 0C Extraction temperature

10 to 12 hoursExtraction duration

0.83 mm IDRaw material particles size

4.72 ml/gSolvent-to-solid ratio

Acetone, 95.0 % (v/v)Types of solvent

Parameter valuesProcessing parameters

COMPARISON

0.83ND≤ 1.00≤ 1.00Biological activity, LC50 (ppm)

Brine Shrimp Lethality study:LC50 of rotenone standard (SIGMA-Aldrich), purity of 95 - 98 % (w/w): 17.45 ppm

≅ 2.615.99≅ 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)

VerificationOptimizationPreliminaryLiteratureDependent/response variables

CORRELATION

NDNDBiological activity (LC50)

ND-0.46Rotenone content

ND-0.46Yield of rotenoids resin

Biological activity (LC50)Rotenone content

Yield of rotenoids resinCORRELATION OF RESPONSES

Pearson’s correlation coefficients (r) were calculated to determine whether any of the response variables were interrelated

G. CONCLUSION• A batch solid-liquid extraction of Normal Soaking Extraction (NSE) A batch solid-liquid extraction of Normal Soaking Extraction (NSE)

with controlled temperature (major contribution of rotenone with controlled temperature (major contribution of rotenone dissipation) was proved to increase the rotenoids resin and dissipation) was proved to increase the rotenoids resin and rotenone content up to 18 % and 25 % respectively (verification rotenone content up to 18 % and 25 % respectively (verification versus preliminary)versus preliminary)

• Better processing parameters were obtained for the production Better processing parameters were obtained for the production of high rotenone constituents of phytochemical semi productof high rotenone constituents of phytochemical semi product

• The biological activity (LCThe biological activity (LC5050) of rotenoids resin was insignificant in ) of rotenoids resin was insignificant in relation to the optimum processing parameters due to relation to the optimum processing parameters due to inconsistency of rotenone content and as well as the low value inconsistency of rotenone content and as well as the low value of LCof LC5050 which was less than 100 ppm for all treatments which was less than 100 ppm for all treatments

• The presence of other constituents in the rotenoids resin (e.g.: The presence of other constituents in the rotenoids resin (e.g.: deguelin, tephrosin, toxicarol, deguelin, tephrosin, toxicarol, 66αβαβ,12,12αβαβ-rotenolone) contributed -rotenolone) contributed to the low to the low LCLC50 50 value for all treatments despite of the lower yield value for all treatments despite of the lower yield of rotenone of rotenone

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QUESTION AND ANSWER SESSION