Optimization of Neem Seed Oil Extraction Process Using Response Surface Methodology

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1 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Optmzaton of Neem Seed Ol Extracton Process Usng Response Surface Methodology Tunmse Latfat Adewoye 1 and Oladpupo Olaosebkan Ogunleye 1 Department of Chemcal Engneerng, Unversty of Ilorn, P.M.B. 1515, Ilorn -Ngera E-mal: tuntola00@yahoo.com ; adewoye.tl@unlorn.edu.ng Department of Chemcal Engneerng, Ladoke Akntola Unversty of Technology, P.M.B. 4000, Ogbomoso -Ngera Correspondng Author s E-mal: ooogunleye@yahoo.com ; ooogunleye@lautech.edu.ng Abstract A central composte desgn (CCD) consstng three factors (solvent composton, temperature and tme of extracton) at fve levels was used to study the solvent extracton of neem ol from seed usng n- hexane and ethanol. Neem ol yeld, ph, refractve ndex, acd value, odne value and saponfcaton value were evaluated as the responses. Forty-two expermental runs resulted from the CCD wth a mnmum ol yeld of 1% and maxmum of 41%. Response surface methodology was used to analyse the resul of the CCD of the extracton processes. The optmum values for yeld, ph, refractve ndex, acd value, odne value and saponfcaton value from the surface plot was 4.48%, 4.99, 1.56, 1.411g/g, 89.5g/g, and mg/g respectvely. The maxmum predcted percentage yeld was 4.48% at solvent composton of 80.77% n hexane, 4.9 C and 6 hours duraton of extracton. The fve valdaton expermen had optmum ol yeld range between.85% and 7.0% whle ther accompany qualty characterstcs were not sgnfcantly dfferent from the smulated values at p<0.05 Key words: Neem Ol, Response Surface Methodology, Central Composte Desgn 1.0 Introducton Neem (Azadracha ndca) tree s a natve to tropcal South East Asa and t s a member of the Mahogany famly Melaceae (ICIPE, 1995; Okonkwo, 004). Ths tree s popularly known as dongoyaro n Ngera. All par of ths tree are very useful n varety of bologcal actvty. The most famous part of ths tree s the ol obtaned from the kernel of seed. Neem ols have found use wdely n dfferent regon of the globe for medcnal and agrcultural purpose (Ranajt et al., 00; Awad and Shmala,00; Muñoz-Valenzenla et al., 007; Kumar et al., 010). It s used n soap producton, as raw materal for producng commercal pestcdes and cosmetcs, plant protecton, stock and textle protecton, refnng to edble ol, lubrcaton ol for engnes, lamp ol, candle producton (Peter, 000). It s found to be of great health use and t has an effectve ant-germ property whch nclude the use as an nsect repellent and t has shown postve resul as pestcde (Kovo, 006). In Inda, t has been demonstrated that Neem Ol s a potental new contraceptve for women (NRC,199). Among varous methods avalable for obtanng neem ol from seeds are mechancal press, supercrtcal flud extracton and solvent extracton method. Mechancal method of extracton s the most wdely used to extract ol from neem seed. However, the ol produced wth ths method s usually turbd, contans sgnfcant amount of water and metal conten. Extracton usng supercrtcal flud produced very hgh purty ol but the operatng and nvestment cost are very hgh. Extracton usng solvent has several advantages lke hgh ol yeld and less turbd ol than mechancal extracton and relatve low operatng cost compared wth supercrtcal flud extracton (Lauw et al., 008, Soetaredjo et al., 008). It s clear from varous earler researches on solvent extracton (Kovo, 006; Lauw et al., 008; Soetaredjo et al., 008 and Zahed et al., 010) that the fnal neem ol quantty and propertes are all functons of the process varables of the extracton process. There exst a complex relatonshp between the ol yeld and propertes. The purty and fnal propertes of the neem ol extract determne end use. The quest by these researchers to ncrease the ol yeld has resulted on a great compromse on propertes. The crux of the problem has remaned how to combne ol yeld wth desrable qualtes. Hence, the need to apply process optmsaton as an essental tool that has demonstrated hgh effcency n selectng optmal process varables for optmum turnover n many chemcal processes. Among process optmsaton tools that has been effectvely appled to chemcal processes s response surface methodology (RSM) (Sngh et al., 00). RSM s a collecton of statcal and mathematcal technques useful for developng, mprovng and optmzng process and new produc, as well as n the mprovement of exstng product desgns. Ths study was therefore to develop an optmzaton framework for the Neem ol solvent extracton process that addressed the optmal selecton of processes varables that maxmsed the ol yeld subject to the qualty constran requremen. 66

2 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01.0 Methodology.1 Neem seed materal preparaton Neem seeds used n the solvent extracton of neem ol was obtaned from Ilorn, Kwara State, Ngera. The fru were de-pulped to obtan the seeds that were washed thoroughly to remove the drt and mpurty. These seeds were decortcated by wnnowng to remove the hull from the seeds. The weght of the seeds (W 1 ) was measured and then placed n the oven at 50 0 C untl constant weght (W ) was obtaned usng Lauw et al (008) method for dryng. The dred seeds were crushed n a mortar and the sample was expressed on a standard seve screen of the requred mesh sze ( mm ) to obtan the requred partcle szes.. Neem ol extracton and propertes analyss Solvent extracton method usng varous combnatons of n-hexane and ethanol were used to study the effect of extracton parameter on the yeld and propertes of neem ol. Other mportant factors of the extracton process were the partcle sze, partcle to solvent rato, temperature of extracton and tme of extracton (Lauw et al. 008; Kovo 006). For ths study the factors consdered were temperature of extracton, tme of extracton and type of solvent combnatons used for extracton because the two other factor has beng reported by Lauw et al. (008) to have optmum performance at the fne partcle sze ( mm ) and rato 1:5 respectvely, hence were adopted. Each extracton run was set up by measurng 10g of the seed powder nto 50ml of the solvent mxture n a 50ml corked concal flask. Ths mxture was placed n a thermostatc water bath operatng at a preset requred temperature. At the set tme nterval, the samples were taken and centrfuged to separate the sold fracton from the soluton. The extrac were heated and evaporated usng rotary evaporator apparatus to obtan solvent-free ols. The solvent free ol extracted was analysed for the ph, refractve ndex, acd value, saponfcaton value and odne value usng AOAC (000). The percentage of ol extracted was calculated from the equaton: Percentage ol extracted = 100 (1). Expermental Desgn Central Composte Desgn (CCD) of RSM was used for the expermental desgn to optmze the extracton parameters. The CCD conssted of three factors: solvent composton from 0 to100% based on n-hexane, Temperature (0 o C - 50 o C ) and tme of extracton (- 6 hours) and fve level. Varable actual process varable ( ) s related to the coded process varables as shown on Table 1 accordng to equaton 1. ( x x 0 ) X = () x Where, s the dmensonless coded value of the ndependent varables x s the actual value of the ndependent varables at the desgn center pont and x s the varaton ncremen about the center pont. The center pont chosen for the desgn were 50% solvent composton based on n- hexane, 40 o C temperature and 4 hours tme of extracton. The coded, actual values of the varable at varous levels and the responses are gven n the matrx Table. Three replcatons were carred out for all expermental desgn condtons and the average recorded. Forty- two expermental runs were carred out and the order of the experment was fully randomzed to reduce the effect of the unexplanable varablty n the observed responses due to extraneous factor as recommended by Sngh et al (00)..4 Analyss of Data and Response Equatons Regresson models were developed for neem ol yeld and each of the fve propertes of ol as a functon of the three process factors. The Desgn-Expert 6.0 software was used to analyze the extracton data for developng response equatons, for analyss of varance (ANOVA), generate surface plo and determne optmum extracton condtons usng optmzaton toolbox. In multple regresson as n the present case, R, whch s the square of the adjusted coeffcent of determnaton and standard error are the ndces. F statcs shows the sgnfcance of the overall model whle the t statcs tes the sgnfcance of each of the varables of the model. The functon was assumed to be approxmated by a second degree polynomal equaton : () Where s the value of ftted response at the centre pont (0,0,0), and, and are lnear, quadratc and cross product regresson term respectvely. m s the number of factors consdered n the study whch s equal to, Y 1 (Ol yeld,%), Y (ph), Y (refractve ndex), Y 4 (acd value), Y 5 (odne value) and Y 6 (saponfcaton value)..5 Optmsaton and valdaton of the neem ol extracton A nonlnear programmng problem of the form of equatons 4 to 10 was formed from the vector of equaton as follows: 67

3 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Maxmze : Y1 ( X ) (4) Subject to : Y ( X b (5) ) X ) 4 X ) 5 X ) 6 X ) Y ( b (6) Y ( b (7) 4 Y ( b (8) 5 Y ( b (9) 6 X + (10) Where b are the neem ol property requremen. Ths constraned maxmzaton problem was solved usng the Desgn-Expert 6.0 software. Ths problem statement was valdated wth fve (5) randomly generated ol qualty requremen for optmum extracton condtons as shown on Table, the resul obtaned theoretcally were expermented through extracton and subsequent ol analyss. Theses theoretcal and the expermental resul were compared usng t-test at p< Resul and Dscusson..1 Response Equatons for Neem Ol and I Propertes The effect of the CCD on the ol yeld (Y 1 ), the ph(y ), refractve ndex (Y ), acd value (Y 4 ), odne value (Y 5 ) and saponfcaton value (Y 6 ) s as shown on Table that was subsequently used to ft the response equatons for ol yeld and the fve ol propertes. Multple regresson analyss was used as tools of assessment of the effec of two or more ndependent factors on the dependent varables (Boonmee et al, 010). The coeffcen of determnaton R² s a measure of the total varaton of the observed values of the extracted ol about the mean explaned by the ftted model (Shrdhar et al, 010). The factors of the models, ther parameter estmates and the statcs of the estmates for the best functons adopted, takng nto consderaton all man effec, lnear, quadratc, and nteracton for each model are as shown on Table 4. The coeffcen of determnaton (R²) for the responses, yeld, ph, refractve ndex, acd value, odne value and Saponfcaton value were 0.990, 0.890, 0.995, 0.971, 0.99 and respectvely. The coeffcen of determnaton were hgh for response surfaces, and ndcated that the ftted quadratc models accounted for more than 89% of the varance n the expermental data. Base on p values, the regresson coeffcent that were sgnfcant at p < 95% were selected for the models that resulted n Equatons (11) to (16). Analyses of varance (ANOVA) were conducted to evaluate the adequacy and consstency of the models usng F- statc (Shrdhar et al, 010). The analyss of varance of the models s presented n Table 5. As shown on the Table 5, the F- value for the ol yeld (5.1). ph (1.7), refractve ndex (7.8), acd value (74.84), odne value (10.67) and Saponfcaton value (8.7) were sgnfcant at p< 0.05 mplyng good model ft. Solvent rato (x 1 ) had qute hgh lnear postve effec on ol yeld than t had on saponfcaton value, odne value, acd value and refractve ndex but had negatve lnear effect on ph. Solvent rato had negatve quadratc effect on yeld, refractve ndex, and acd value. Temperature (x ) had greater postve lnear effec on ol yeld compared to ph, refractve ndex and acd value. It had negatve lnear effect on odne value. Temperature had negatve quadratc effect on odne value but postve quadratc effect on saponfcaton value. Tme of extracton (x ) had postve lnear effec on yeld, refractve ndex, acd value and saponfcaton value. It also had postve sgnfcant quadratc effect on yeld, refractve ndex, acd and saponfcaton values, whle t had negatve quadratc effect on odne value. The nteracton of the solvent rato and temperature had postve effect on saponfcaton value but negatve effect on odne value and ph but no sgnfcant effec on other responses. The nteracton of solvent rato and tme had negatve effect on the refractve ndex but no sgnfcant effect on all other responses. The nteracton of temperature and tme also had postve effect on ph but no statcal sgnfcant effect on other responses = ) = X1+ 0.5X X. 0.4X1X + 0.5XX R = Yeld ( Y ) =.0+ 7.X + 4X + 1.X 0.8X + 5.7X R (11) ph ( Y (1) Refractve Index( Y ) = X 1.E X X E X + 5E X 0.049X E X R = , = ( Y5 ) = 89.6 X 4.X 4.X 4.5X1X R = Acd ValueY ( ) = X X + 0.0X 61X + 0.X R (14) Iodne Value (15) (1) 68

4 ISSN (Paper) ISSN (Onlne) Vol., No.6, = Saponfca ton Value ( Y) = X X+ 5.4 X X XX R (16). Optmzaton of the Extracton Process The models (,,,,, ) were useful for ndcatng the drecton n whch to change the varables n order to maxmze yeld, ph, refractve ndex, acd value, odne value and Saponfcaton value. The multple regresson equatons were solved usng Desgn Expert 6.0. The maxmum values obtaned were as follows: 4.48, 4.99, 1.56, 1.411, and for yeld, ph, refractve ndex, acd value, odne value and Saponfcaton value respectvely. The optmum ngredent rato (coded) predcted for each response and actual values for optmum response are presented n Table.0. As shown n Table 6, the coded levels are wthn the expermental range and ths ndcated that the selected varables are vald of the selected varables. The regresson equaton was optmzed for maxmum value to obtan the optmum condtons usng Desgn Expert 6.0. The actual value calculated for optmum response as shown n Table 6, were: 80.77% n- hexane solvent, 4.9 C temperature and 6.0 ( hours) tme for yeld, 5.84% n-hexane solvent, C temperature and.5 hours, tme of extracton for the maxmum ph, 8.%, n- hexane C temperature and 5.4 hours, tme of extracton for maxmum refractve ndex, 0.97% n-hexane solvent, 0.86 C temperature and. hours tme of extracton for mnmum acd value, 51.1% n-hexane solvent, 8.4 C temperature and. hours tme of extracton for maxmum odne value and 6.58% solvent rato,.49 C temperature and 4.09 hours tme of extracton for mnmum saponfcaton value. The response surface plo and the accompany contour plo shown n Fgures 1 to 6 for the chosen model equatons shows the relatonshp between the ndependent and the dependent varables. From Fgure1, the response surface ndcates that the percentage yeld of ol ncreases temperature and solvent composton ncrease to optmum condton whle further ncrease led to decrease of percentage yeld of ol. In addton, there was mutual nteracton between the temperature and solvent composton. From Fgure the ph of ol ncreased wth ncrease solvent composton and temperature for a short perod, at a longer perod, ncreasng the temperature of extracton wll have negatve effect on the ph. From Fgures and 4, ncrease n temperature and solvent composton led to ncrease n refractve ndex and acd values respectvely. It can be seen that the solvent composton was more nfluental factor that affect the refractve ndex and the acd value of the extracted ol. From Fgure 5, t can be seen that ncrease n temperature and solvent composton ncreases the odne value. Fgure 6 show that Saponfcaton value decreased wth ncrease n temperature and solvent composton. The hghest Saponfcaton value was obtaned when all the varables were at the mnmum pont wthn the range of study..4 Valdaton of the Optmzaton of the Extracton Process Table 7 shows result of the optmum process varables for each extracton run, correspondng ol yeld and the Neem Ol propertes obtaned from the experment of each valdaton process. The ol yeld range s between.85 % and 7.0%. The correspondng result of the analyss of each extracton were compared wth the smulated propertes usng pared t-test that shows that there are no sgnfcant dfference between the smulated propertes and ther correspondng expermental values at p < 0.05 as presented on Table 8. Ths result attes to the effectveness of ths framework for optmum and effectve ol extracton. 4.0 Conclusons Ths study has clearly demonstrated the applcablty of RSM selectng extracton condtons for neem ol from seed. Ths approach has not only resulted n the maxmum ol yeld through solvent extracton, but has also guaranteed the fulfllment of the propertes requremen of the neem ol The optmum values for yeld, ph, refractve ndex, acd value, odne value and saponfcaton value from the surface plot was 4.48%, 4.99, 1.56, 1.411g/g, 89.5g/g, and mg/g respectvely. The valdaton expermen and ther accompany qualty characterstcs were not sgnfcantly dfferent from the smulated values at p<0.05 References Assocaton of Analytcal Chems (AOAC) (000). Offcal Methods of Analyss of AOAC Internatonal. Washngton Assocaton of Analytcal Chems. 17th Ed, Vol. II Awad O. M. & Shmala A. (00). Operatonal use of neem ol as an alternatve anophelne larvcde. Part A: laboratory and feld effcacy. Eastern Medterranean Health Journal.9(4): Boonmee, K., Chuntranuluck, S., Punsuvon, V. And Slayo P. (010). Optmzaton of bodesel Producton from Jatropha (Jatropha Curcas L.), usng surface response methodology. Kaseart J. Nat. Sc 44: Internatonal Center of Insect Physology and Ecology (ICIPE). (1995) The neem tree: An Affordable, Effcent and Envronmentally-Frendly Source of Pest Control Produc. Internatonal Center of Insect Physology and Ecology. Narob, Kenya. 69

5 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Kovo, A.S (006). Applcaton of full 4 factoral desgn for development andcharacterzaton of nsectcdal soap from neem ol. Leonardo Electronc Journal of Practces and Technologe 8:9-40 Kumar, P. S., Ghosh, D. G. and Pander C. S. (010). Bologcal acton and medcnal propertes of varous consttuent of Azadrachta ndcal (melaceae a overvew). Annals of Bologcal research 1():-4. Lauw, M. Y., Natan F. A., Wdyant P., Ikasr D. Indraswat N. & Soetaredjo F.E.(008). Extracton of neem ol (azadrachta ndca a. juss) usng N-hexane and ethanol: studes of ol qualty, knetc and thermodynamc. ARPN Journal of Engneerng and Appled Scences () :49-54 Muñoz-Valenzuela, S., Ibarra-López A. A., Rubo-Slva L. M., Valdez-Dávla H.& Borboa-Flores J. (007). Neem tree morphology and ol content. Reprnted from Issues n new crops and new uses J. Janck and A. Whpkey (eds.).ashs Press, Alexandra, VA. Natonal Research Councl (NRC)(199). Neem: a tree for solvng global problems. Natonal Academy Press, Washngton, DC Okonkwo, E. M. (004).Employment creaton and opportuntes n manufacturng sub-sectors: Acase for neem tree n Ngera. Bullon, publcaton of Central Bank of Ngera. 8():0-5 Peter, F. (000). Global Neem usage. Deuche Gesellschaft fdür Technsche Zusammenarbet (GTZ) GmbH Eschborn, Germany. Ranajt, K. B., Kausk B. IshtaC. & Uday B.(00) Bologcal actvtes and medcnal propertes of neem (Azadrachta Indcal). Current Scence 8(11): Soetaredjo F.E., Budjanto G. M., Prasetyo R. I., & Indraswat N.(008). Effec of pre-treatment condton on the yeld and qualty of neem ol obtaned by mechancal pressng. ARPN Journal of Engneerng and Appled Scences (5) :45-49 Shrdhar,B.S. Beena, K.V. Anta,M.V. & Paramjeet K.B.(010) Optmzaton and charaterzaton of castor seed ol. Leonardo Journal of Scence 17: Sngh, S., Rana, C. S., Bawa, A. S. & Sexena D. C. (00). Sweet potato-based pasta product: optmzaton of ngredent levels usng response surface methodology. Internatonal Journal of Food Scence and Technology 8:1-10 Zahed, G., Azz S., Hatam, T., & Shekhattar, L. (010). Gray box modelngof supercrtcal Nmbn Extracton from Neem Seeds usng Methanol as Co-Solvent. The Open Chemcal Engneerng Journal 4: 1-0. Table 1: Expermental Incremen, values of coded levels Factors ± Increment X Coded Levels x x 1 (%) ± 5 0(100) 5(75) 50(50) 75(5) 100(0) x ( 0 C) ± x (Hr) ± Table : Neem ol qualty requremen for valdaton Propertes / Runs ph Refractve Index Acd Value Iodne Value Saponfcaton Value

6 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Table : Central composte desgn arrangement and responses Coded Level Actual Values Reponses Expt. No X 1 X X Solvent Temp. Tme (75) (75) (75) (75) (50) (50) (50) (50) (50) (50) (50) (50) (50) (50) (50) (50) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0)

7 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Table 4: Estmated coeffcen of the ftted quadratc equaton for dfferent responses. Estmated Coeffcen Factors Ol Yeld (Y1) ph(y) Refractve ndex (Y ) Acd value (Y4 ) Iodne value (Y5 ) Saponfcaton value Consta nt Coeffcen p-valu e x x x x Coeffcen p-value Coeffcen p-valu e E E E Coeffcen p-value Coeffcen p-value Coeffcen (Y6 ) p-valu e x E x x1x E E x1x E E xx 1.094E E E R Sgnfcant at p < 0.05 level Sgnfcant at p < 0.1 level Table 5: Analyss of varance for the responses Sources of Sum of Mean Adjusted Responses Varaton d.f Squares Square F Regresson Resdual Total Regresson Resdual E-0 Total Regresson 9 7.4E-0 8.5E Resdual.05E E-06 Total E-0 Regresson Resdual 4.91E-0 1.5E-0 Total Regresson Resdual Total Regresson Resdual Total

8 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Table 6: Calculated coded and Actual Value for optmum responses. OPTIMUM RESPONSE LEVEL Coded Values COMPOSITION Actual Values Optmum values Table 7: Optmum Process Varables, Ol Yeld and Physcal Propertes of the Neem Ol for Valdaton Varable / Propertes Optmum Process Varable X 1 (%) X ( 0 C) X (Hours) Smulated Ol Yeld (%) Correspondng Neem Ol Propertes Ol Yeld (%) Ph Refractve Index Acd Value Iodne Value Saponfcaton Value Table 8: Summary of t-test for Propertes of Neem Ol Propertes for Valdaton Source of Varaton t-value p- value ( tal) Remark Ol Yeld ph Refractve Index Acd Value No Sgnfcant Dfference No Sgnfcant Dfference No Sgnfcant Dfference No Sgnfcant Dfference Iodne Value No Sgnfcant Dfference Saponfcaton Value No Sgnfcant Dfference Sgnfcant level at p <

9 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Ol Yeld Ol Yeld Predcto Fgure 1: Response surface plot showng the D effect of temperature and solvent and ther nteracton effect on the yeld of Neem ol. Contour Plot of Fgure 1a Predcto ph ph Fgure : Response surface plot showng the D effect of temperature and solvent composton ther nteractve effect on the ph of Neem ol. Contour Plot of Fgure a Refractve Predcto Index Refractve Index Fgure : Response surface plot showng the D effec of temperature and solvent composton and ther nteractve effect on the refractve ndex of the Neem ol. Contour Plot of Fgure a. 74

10 ISSN (Paper) ISSN (Onlne) Vol., No.6, 01 Acd Value.177 Acd Value Fgure 4: Response surface plot showng the D effec of temperature nteractve effec on the acd value of Neem ol. Contour Plot of Fgure 4a Iodne Value Predcto 89.5 Iodne Value and solvent and ther Fgure 5: Response surface plot showng the effect of temperature and solvent composton and ther nteractve effect on the odne value of the Neem ol. Contour Plot of Fgure 5a Saponfcaton Value Saponfcaton Value Predcto Fgure 6: Response surface plot showng the effec of temperature and solvent composton and ther nteractve effect on the Saponfcaton value of Neem ol. Contour Plot of Fgure 6a 75

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