Two Step Extraction Of Pyrethrins From Pyrethrum

Two Step Extraction Of Pyrethrins From genus PyrethrumThe observational mark of the concentration and yield of pyrethrins from chrysanthemum pyrethrum flower is usually carried out with chromatographic techniques and accordingly, a bevy of methods require been developed over the years Wang et.al, (1997). These include laid-back performance liquid chromatography (HPLC) Todd et.al, (2003) Essig and Zhao, (2001b), gas chromatography (GC) Essig and Zhao, (2001a) and super particular fluid chromatography (SFC) Wenclawiak and Otterbach, (1999). GC was chosen for convenience in this study. The first-step involves using n-hexane as termination to distill the pyrethrins from the solid threadb be (grounded and unsieved with particles size of about 30 meshes), and then the south-step, a purification step involves the use of supercritical carbon dioxide as solvent to obtain the pyrethrins from the crude hexane extract (CHE). The hexane p bentages (100g experiment size), in a water ba th at controlled temperatures and vigorous rousing, generated pyrethrins concentrations varying from 69.85 95.50mg/ml and yields of 0.85 3.76% of the dry weight. Extraction efficiencies under several conditions were investigated and the optimum blood line condition was 400C in 4hrs. Compared with the product from the factory, several inapplicable components exist in the CHE. The SFE was carried out with a self built unit ( stemma vessel of 120ml) with a sample size of 40ml of CHE. Concentrations of 57.25 93.79mg/ml and yields (after the second bloodline) of 0.99 2.15% were obtained with the optimum condition being 350C at a pressure of 20MPa in 2hrs. Compared with the product from the factory, this sample contains cardinal extra components (Tricosane and Tetracosane) besides used in insect control.Key words Solvent descent supercritical carbon dioxide pyrethrins two-step beginning crude pyrethrins extractIntroductionPyrethrum flowers are from the Chrysanthemum genus and are known commercially as painted daisies, painted ladies, buhach, chrysanthemum cinerariaefolium, ofirmotox, insect powder, Dalmatian insect flowers, or parexan. It is believed to be recorded first in Dalmatia Visiani, (1842-1852). However, others contend that its insecticidal activity was first proven by Antun Drobac (1810-1882) Bakaric, (2005). Yet there are claims that it was first identified as having insecticidal properties around 1800 in Asia Jeanne, (2009) and that the Crushed and powdery plants were used as insecticides by the Chinese as early as 1000 BC Amrith, (2004). The flower contains about 1-2% pyrethrins by dry weight, but well-nigh 94% of the essence yield is concentrated in the seeds Casida and Quistad, (1995). The chemical structure of the active ingredients, pyrethrins I and pyrethrins II was identified in 1924 Chandler, (1948) Coomber, (1948). Kenya is the worlds main producer today with more than than 70% of the global supply Jones, (1973). The natural activ e ingredients are referred to as Pyrethrins consisting of cinerin I, jasmolin I, pyrethrin I, cinerin II, jasmolin II and pyrethrin II. The first three (chrysanthemic acid esters) are referred to as pyrethrins I (PYI), and the rest (pyrethric acid esters) as pyrethrins II (PYII) Essig and Zhao, (2001a). Pyrethrins, though insoluble in water, are soluble in many radical solvents WHO, (1975). They are non-volatile at ambient temperatures non-toxic to mammals and other worm-blooded animals gamyly unstable in light (photodegradable) biodegradable but toxic to aquatic animals Todd et.al, (2003) Chen and Casida, (1969) WHO, (1975). Their usage is mainly in biological rank protection domestic insecticides Gnadinger, (1936) and the formulations of synthetic pyrethroids Todd et.al, (2003). Although pyrethrins are soluble in a number of organic solvents (benzene, hexane, petroleum ether, alcohol, acetone, methanol, chlorinated hydrocarbons, etc) other considerations (practical, economic an d environmental concerns) limit the usage. These considerations burn the excerpts to just few. One of the qualities of Hexane in extracting pyrethrins is its ability to takeively dissolve the active ingredients minus contaminants. A nonher is that its removal from the concrete is achieved at lower temperatures limiting adulteration due to prolonged heating. Again, its low boiling point is a needed quality and it can be recycled, reduce the weight of the concrete. Above all, it is inexpensive, considered environmentally friendly, little toxic, non-corrosive, and non-reactive traits which make it the dominant solvent adopted, especially for processing plant (biological) materials (products) which are often thermally labile, lipophilic, and non-volatile and are required to be kept and processed at around room temperatures. Carbon dioxide (carbonic acid gas) has a critical temperature of 31oC which makes it particularly an attractive medium for these kinds of tasks. Though other s upercritical fluids (SCFs) show critical temperatures in this critical state and can be adapted as solvents, they are often difficult to handle and obtain in pure state, may be toxic, fickle or ecologically unsafe. Supercritical carbon dioxide (Sc-carbonic acid gas) is by far, the most extensively used due to its non-toxic, inert and non-flammable nature. It is alike natural, inexpensive, plentiful, non-toxic and inflammable and generally environmentally accepted Schneider et.al, (1980). Its most important properties are enhanced density, viscosity, diffusivity, heat capacity and thermal conductivity. soaringer densities contribute to greater dissolution of compounds while low viscosities enable flaccid penetration into samples and facilitation of flow of extracted (targeted) molecules from the source materials with fewer hindrances Dunford et.al, (2003). Diffusivity offers easy and faster transport through samples hence offers better extraction strengths and dissolved ingredie nts are also easily separated from the supercritical solvent by drivel in pressure Fattori et.al, (1988). Sc-CO2, for the above and many reasons used as solvent in extraction saves both era and money while retaining overall extraction precision and accuracy with high purity and healthy products that are of excellent quality Raventos et.al, (2002) Mohamed and Mansoori, (2002). Expectedly, a lot of research is now focused on the extractions of plant materials with supercritical carbon dioxide due primarily to the global growing solvent (organic) regulations and more importantly, the economic benefits (in harm of low operating temperatures faster extractions and easier purifications, and of course better product quality). Stahl and Schutz Stahl and Schutz, (1980) extracted pyrethrins with CO2 and proposed that in the 20C to 40C temperature range decomposition (usually associated with pyrethrins extraction) does non occur. Sims patented in the US, an extraction of pyrethrins using liquid carbon dioxide Sims, (1981) and Wynn and others patented using Sc-CO2 Wynn et al. (1995). Wenclawiak and coworkers compared extracts obtained with ultrasonic (USE) and Soxhlet extractions (SEX) with hexane and Sc-CO2 extractions (SCE) and reported that direct extraction with SCE gave better pyrethrins content Wenclawiak et.al, (1995).2. 0. observational2.1. Materials and ChemicalsGrounded chrysanthemum (light green with a characteristic smell) sample and two pyrethrum concretes (yellow) were obtained from Yunnan Juxiang congenital Plant Products Company in China. The pyrethrins content of the concretes was claimed to be 50.0% (29.50% PYI and 20.50% PYII) and 85.15% (46.33% PYI and 38.82% PYII). Six individual banner solutions (using standard addition method) were prepared (from the 85.15% PY concrete-higher content, less(prenominal) impurities) for standardization of the analytical method. Analytical grade hexane (97.0%) and Ethanol (99.7%) were purchased from Sinopharm Ch emical Reagent Co. Ltd in China, and used directly without any pre-treatment. CO2 (99.0 %) gas was supplied by Xin Hongli Gas Company also in China.2.2. ExperimentsThree various experiments were performedTo establish the standard/calibration curves for determining the components,To implement hexane extraction and steady down the yield of total PY in the grounded sample, andTo implement SFE and determine the yield of total PY in the CHE.2.3. Establishing Standard CurvesThe GC (Agilent) conditions used for establishing the standard curves are as follows tide rip injector with 201 split ratio at 2500C Nitrogen as carrier gas at 1.6mL/min ow rate injection volume of 0.1 L temperature program started at 1800C, kept for 11 minutes, modify at 100C/ min to 2000C, kept for 8 minutes, heated to 210 0C at 100C/min, kept for 18 minutes, then heated to 2450C at 30 0C/min, maintained for 4 minutes FID detector HP-5 Column, 30 mm 0.32 mm id., 0.25 m lm thickness. This column was chosen becaus e it gives the best resolution, identication and quantication for products containing OH and C=O Rosana, (2003). 2g (85.15% concrete obtained from the company) of the extract was transferred into a 100mL flask containing 10mL ethanol, and then made up to the final volume of with ethanol and mixed well. Six aliquots (1mL, 2mL, 4mL, 8mL, 16mL and 32mL) of this solution were transferred into a 50mL flask each and diluted with ethanol again to the mark. We then calculated the concentrations of the PY in each aliquot, considering the percentage of each group (PYI and PYII) in the sample provided (Table A1 in the Appendix), injected (with a micro syringe) 0.1L of each solution into the GC after filtering (0.45-m membrane filter) and recorded the elution quantify and corresponding peak areas (Table A2) subsequently, established the standard curves to express the relationship between the areas produced by the GC and the concentrations (Figure 2).2.4. Hexane ExtractionWe extracted pyrethri ns (from 100g of grounded sample of particle size of about 30mesh) with hexane in a water bath (YUHUA, DF-101S) in batches at different temperatures (35oC, 40 oC, 45 oC, 50 oC, 60 oC and 70 oC) and times (3hrs, 4hrs, 5hrs, 6hrs and 7hrs) in a 1000mL round-bottom flask, installed with a condenser. Agitation was achieved by stirring vigorously with three big size magnetic stirrers at a speed of 20rpm. The hexane was then removed from the pyrethrin concrete with a rotary evapourator (YUHUA, RE-2000B) at a temperature of 35 oC at a speed of 185rpm to obtain concentrated Crude Hexane Extract (CHE). Each concentrated sample was thereafter, filtered (0.45m) and 0.1L analyzed (Tables A3). This method has the good that the solvent is repeatedly recycled and temperature can be controlled. It offers a light coloured product with high recovery rate of pyrethrins heretofore, not only the sought after components are extracted (Figure 3). Other soluble and hydrophobic substances (waxes and pigm ents) are also extracted Kiriamiti et al, (2003). The solvent is removed by vacuum at lower temperature and the waxy thick mass go away is the concrete composed of essential oils and other oil soluble (lipophilic) materials.3.0. Results and handling3.1. ExtractThe extracts (CHE) contain pigments, fixed oils and waxes whose colour is deep yellow with characteristic smell. It also contains several undesired components (Figure 3) compared with the pure sample from the factory (Figure 1).3.2. Effect of Extraction TemperatureTemperature has long been reported to be a crucial factor in the extraction of natural pyrethrins Atkinson et.al, (2004). Pyrethrins are sensitive to temperature (thermo labile) and are therefore, unanimously reported to degrade above 40oC Stahl and Schuzt, (1980) Gourdon and Romdhane, (2002) Wynn et al, (1994). We investigated the effect of different extraction temperatures (40oC, 50oC, 60oC and 70oC) in fixed extraction times (5 hr gave better resolutenesss than 6hr and 7hr). Our results correct to the reports (refer to Figure 4 and Table A3) the best yield (1.42) and PYI PYII ratio (4.75) is at 40oC (but the best PYII yield-0.33 is at 70oC). This suggests that targeted components are extracted effectively at this temperature (40oC), above which two problems occur (separately or simultaneously) one is the extraction of more undesirable components at the expense of pyrethrins and the other is the decomposition of pyrethrins to form iso-pyrethrins Stahl and Schuzt, (1980) Stahl, (1998) Gourdon and Romdhane, (2002) Wynn et.al, (1994) thereby reducing the yield as seen.3.3. Effect of StirringWe compared the effect of two stirring methods on extraction yield the first with one magnetic stirrer and the second with three magnetic stirrers. The results are shown in Table A4, confirming that stirring improves extraction yield by facilitating the dissolution of the active ingredients and the effective distribution of heat. The extractions (at 40oC in 5hr) were repeated severally to ensure reproducibility and accuracy.3.4. Effect of Extraction TimeWe further investigated the effect of extraction time by fixing the extraction temperature at 40oC with three magnetic stirrers to establish the optimum extraction time (our initial time parameters were 5hr, 6hr and 7hr in which 5hr was the best). From Figure 5, the extraction yield increases steadily from 3hr to a peak at 4hr (see data in Table A5). Within this range, more desired components are extracted but after 4hr the yield decreases indicating that with prolonged time, even at the safest extraction temperature (40oC), less and less desired components are extracted and/or they decompose resulting in the decrease in yield. The drop in yield is consistent from 4hr (3.76%) to 6hr (2.15%). This implies that the optimum time (within the times investigated) is not 5hr as initially expected but rather 4hr. However, the ratio of PYI PYII is best in 6hr (5.14). From 3hr to 4hr, the yiel d for both PYI and PYII appreciated but the growing in PYI (0.74) is greater than that of PYII (0.38) hence the drop in the ratio. Between 4hr and 5hr, there is decrease in both PYI and PYII yields. Again, the decrease in PYI (0.98) is greater than that of PYII (0.49) accounting for the drop in ratio. The same reason accounts for the drop in ratio from 5hr to 6hr.3.5. Effect of Concentrating CHEThe effect of concentrating the CHE, on both PYI and PYII yield was analyzed (Table A6). Even though the concentrating temperature (35oC) was downstairs the temperature above which PY degrades (40oC), there was loss in PY yield indicating degradation. This in our view may be due to the exposure of the pyrethrins directly to heat. As more hexane is evapourated, pyrethrins which hitherto, were locked in the solid sample matrix surrounded by hexane and as such shielded from direct heat, is now in direct contact with the heat and since they are sensitive to heat, decomposition is inevitable. Ho wever, the decomposition is small and negligible (about 2.25mg/ml which is about 0.41% of the total yield) due to perhaps the short concentrating time (about 30 min).4.0. Supercritical liquid Extraction (SFE)The CHE is too thick (viscous) to be used directly, coupled with the presents of undesirable components (waxes and pigments). A further treatment, usually with another solvent that only dissolve the desired compounds from the concrete is necessary. Different from other works, this study carried out SFE on the CHE as a purification step. We looked at the effect of time (hr), temperature (0C) and pressure (MPa) on extraction quality and yield. We have not studied the effect of particle size and pre-treatment for information on this area, see the works of Kiriamiti and others Kiriamiti et al, (2002).4.1. SampleWe concentrated the CHE in a rotary evapourator (from 500ml to 40ml at 185rpm in 30 minutes) for the SFE.4.2. Extraction formAt the beginning of the extraction (Figure 6), all the check valves are closed except valve 2. This allows the CO2 gas into the compressor 4 (OLSB by Zheng Zhou Co. LTD, China) to be compressed, and the pressure gauges are allowed to attain equilibrium at a set pressure (10, 15 and 20 MPa). Valve 5 is then heart-to-heart and the compressed fluid (Sc-CO2) is fed into the bottom of the extraction vessel 7 (120ml capacity) for up flow extraction configuration, containing the CHE (40ml) and metal fillings to comfort effective contacting (increase internal mass transfer) which had earlier been heated to a set temperature (350C, 370C and 390C) and allowed to attain constant temperature with the help of the water bath 6. An appreciable time is allowed (5-10mins) for the total and complete dissolution of the crude extract and then valve 8 is opened and maintained until the pressure is in equilibrium again. The pressure reducing valve 9 is opened finally to collect the pyrethrins in the flask 10. A mass flow meter helps to determine the flow rate (1.5L/min). The extraction process is run and stop at set times (1hr, 2hr and 3hr) and the extracts analyzed with the results tabulated (Table A8). The Metal fillings after each run were washed (10ml or 5ml of Hexane) and collected as residues to check for complete extraction.5.0. Results and Discussion5.1. ExtractsThe extracts did not contain visible pigments as was seen in the CHE. The colour was also different light yellow to orange but the smell was similar. It also contained two extra components (Figure 10) which was found (by GC mass spectrometry) to be Tricosane (Peak 6) and Tetracosane (Peak 7). This was as a result of comparison with the pure sample from the factory (Figure 1).We compared the yield of the extracts after solvent extraction, concentrating the CHE and the SFE and noted that there was difference. The yield from the SFE was less due possibly, to the relatively high pressures used. Separation of the Sc-CO2 and the product is achieved by a drop in press ure. These high pressures have the tendency of causing the products to remain in the BPR or along the pipe (between the BPR and the flask in Figure 6) due to clotting as a result of the pressure drop in spite of our use of heating tapes to minimize this effect. This is confirmed by the value of the yield in the residue (0.05%) which is far less compared to the difference between the concentrated sample yield (3.30%) and that of the SFE (2.15%, see Table A10).5.2. Effect of PressureAccording to Kiriamiti and others, the quantity of pyrethrins extracted decreases with decreasing pressure due to (i) the effect of density on the solubility of pyrethrins, (ii) the slightly high density of CO2, (iii) the moderate variation in density with pressure, and (iv) the very low undesirable extracted products Kiriamiti et al, (2002). Our results conclusively conform to this (Table A7). The best extraction pressure was at 20MPa (at 350C and 2hrs). The concentration of PY also increases within this pressure range (from 81.34mg/ml 93.79mg/ml). Similar phenomenon was observed for both 1hr and 3hrs, indicating that more pyrethrins were extracted than the undesirable components within this pressure range (Figure 7).5.3. Effect of Extraction TimeThe quantity of pyrethrins extracted decreases with extraction time at higher temperatures (above 400C), explaining that either pyrethrins decompose at these princely temperatures or more undesirables are extracted instead. From Table A8, the yield and concentration of PY increase from 1hr to a maximum in 2hr (1.35% 2.15% and 90.42mg/ml 93.79mg/ml at 350C and 20MPa). Both however decrease in 3hr (1.24% and 82.30mg/ml, Figure 8). This implies that pyrethrins were extracted faster than the undesirables from 1 to 2hr but as the extraction proceeds, more undesirables were then extracted at the expense of the pyrethrins or which decompose. Therefore, prolonged extraction time rather favours the extraction of undesirables or promotes decompos ition of pyrethrins.5.4. Effect of TemperaturePyrethrins are thermo labile and therefore require being processed at low temperatures. Therefore, high extraction temperature does not only degrade the pyrethrins but also favours the extraction of undesirables (Figure 9). Within the temperature range we investigated, the best yield was at 350C (Table A9).6. 0. ConclusionsPyrethrins are usually purified with organic solvents (ethanol, methanol, acetone, acetonitrile, petroleum ether etc) or their mixtures Kasaj et.al, (1999) Henry et.al, (1999) Duan et.al, (2006) which are generally expensive, flammable and explosive and above all, face strict legislative controls Patrick, (2003). Alternatively, carbon dioxide is used to polish and purification. Sims proposed the use of liquid carbon dioxide Sims, (1981). Similar to our method, Kiriamiti and others reported the extraction of pyrethrins from crude hexane extract (CHE) from batch extraction experiment using carbon dioxide Kiriamiti et.al , (2003) but with different extracting conditions and analysis method (HPLC). It is worth noting that our set up is very simple and less expensive coupled with the fact that our sample, after the SFE, contains two extra components (Tricosane and Tetracosane) not reported so far as part of the purification step. These components are not hazardous Directive 67/548/EEC and have similar characteristics (may cause respiratory and digestive irritations), uses (as insecticides and biopesticides) and effects (they may not be detrimental to the insects but they certainly disrupt their behaviuor patterns and flushes them out for the more deadly pyrethrins I) as pyrethrins II Chemcas.org Chemnet.com PPDB, (2011) Wylie, (1972) Lewis et.al, (1975). We developed a simple but efficient two-step procedure for the extraction of pyrethrins from chrysanthemum (pyrethrum flowers) and investigated the effect of various operating parameters on concentration and extraction yield. Based on the experimental results, we conclude that the two-step extraction of pyrethrins (first with hexane in a water bath and second with SC-CO2 as a purification step) is feasible and effective the optimum extraction condition for high pyrethrins yield (3.76%) for the n-hexane extraction was 400C in 4hr that vigorous stirring facilitated this and that it is possible to achieve extraction yield of 3% or even more envisaged by Casida and Quistad. To our knowledge, this is the first time such a high recovery of pyrethrins is reported. A number of reasons may be attributed to this high recovery i) extraction procedure, ii) choice of solvent, iii) vigorous stirring and above all, vi) the type of sample used. We further conclude that for the SFE (2.15% and 93.79mg/ml) the optimum conditions were 350C, at pressure of 20MPa in 2hr.7.0. ReferencesAmrith S. Gunasekara, (2004) Environmental Fate of Pyrethrins, Environmental monitor Branch, Department of Pesticide Regulation, 1001 I Street, Sacramento, CA 95812Atki nson B. L, Blackman A. J, and Faber H, (2004) The degradation of the natural pyrethrins in crop storage, J. Agric. Food Chem. 52, 280-28Bakaric P, (2005) Buha prirodni insekticid, Gospodarski list 17 41-45Casida J. E and Quistad G. B, (1995) Pyrethrum Flowers Production, Chemistry, Toxicology, and Uses, Oxford University Press, New YorkChandler S. E, (1948) The Origin and archaeozoic History of the Production of Pyrethrum in Kenya, Pyrethrum attitude 1 (1) 10-13Chen Y-L, and Casida J. E, (1969) Photodecomposition of Pyrethrin I, Allethrin, Phthalthrin, and Dimethrin, J. Agr. Food Chem. 17 208-215Coomber H. E, (1948) The Chemical Evaluation of Pyrethrum Flowers, Pyrethrum Post 1 (1) 16-19Directive 67/548/EEC The Dangerous Substances Directive (as amended) is one of the main European Union laws concerning chemical safety.Duan Wei, Zhengguo Li, Guomin Wang, Yingwu Yang, Yingguo Li and Yuxian Xia, (2006) Separation and purification of Natural pyrethrins by reversed phase high perform ance liquid chromatography, Chin. J. of Anal. Chem., vol.34, is.12, pp 1776-1779Dunford N. T, Teel J. A and King J. W, (2003) A Continuous Counter Current Supercritical Fluid Deacidification Process for Phytosterol Ester Fortification in Rice Bran Oil, Food Research planetary 36, 175-181Essig K and Zhao Z, (2001b) rule Development and Validation of a High-Performance transparent Chromatographic Method for Pyrethrum Extract, J Chromatogr Sci 39 (4) 473-480 (8)Essig K and Zhao Z. J, (2001a) Preparation and characterization of a Pyrethrum extract standard. LC/GC 19(7) 722-730Fattori M, Bulley N. R, and Meisen A, (1988) Carbon Dioxide Extraction of Canola See Oil solubility and Effect of Seed Treatment. J. A. O. C. S. 65, 968-974Gnadinger C. B, (1936) Pyrethrum Flowers. 2nd Ed. McLaughlin, Gormley, King, Minneapolis, MinnesotaGourdon C and Romdhane M, (2002) Investigation in Solid-Liquid Extraction Influence of Ultrasound, Chemical Engineering Journal 87, 11-19Jeanne Roberts, (2009) Insecticide treat Mosquito Nets and Malaria Prevention Weighing the Benefits, Naming the Benefactors at www.celsia.comJones G. D. G, (1973) Pyrethrum Production, In Pyrethrum The Natural Insecticide, J. E. Casida (Eds.), Academic Press. New York, NY, 17-21Kasaj D, A. Rieder, L. Krenn and B. Kopp, (1999) Separation and Quantitative Analysis of Natural Pyrethrins by High-Performance Liquid Chromatography, Chromatographia Vol. 50, No. 9/10Kiriamiti H. K, Camy S, Gourdon C and Condoret J.S, (2003) Pyrethrin Extraction from Pyrethrum Flower using Carbon Dioxide, J. Supercrit. Fluids, 26, 193-200Lewis W. J, Richard L. Jones, Donald A. Nordlund and Gross H.R JR, (1975) Kairomones and their use for the management of entomophagous insects II Mechanisms causing increase in rate of parasitisation by Trichogramma spp, J. Chem. Ecol., vol. 1, No. 3, pp 349-360Mohamed R. S and Mansoori G. A, (2002) The Use of Supercritical Fluid Extraction Technology in Food Processing, feature Article, Food T echnology Magazine, June, The World Markets Research Centre, London, UKPatrick Pelerin, (2003) Comparing Extraction by Traditional Solvents with Supercritical Extraction from Economic and Environmental Standpoint, Proceedings of the 6th International Symposium on Supercritical Fluids, TOME 1Raventos M, Duarte S, and Alarcn R, (2002) Application and Possibilities of Supercritical CO2 Extraction in Food Processing Industry An Overview, Food Sci. Technology International, 8 (5), 269-284Rosana V, (2004). Optimization of Gas Chromatographic-mass Spectrometric Analysis for Fatty Acids in Hydrogenated Castor Oil obtained by Catalytic Transfer Hydrogenation. Analytica Chimica Acta 505, 223-226Schneider G. M. V, Stahl E and Wilke G, (1980) Extraction with Supercritical Gases, Verlag Chemie, Deerfield Beach, BaselSims M, (1981) Liquid carbon dioxide extraction of pyrethrins, US Patent no. 4281171Stahl E and Schtz E, (1980) Extraction of Natural Compounds with Supercritical Gases, J. Med. Plan t Res. 40, 12-21Todd G. D, Wohlers D, and Citra M, (2003). Toxicology Profile for Pyrethrins and Pyrethroids, Department of Health and Human Services, fashion for Toxic Substances and Disease Registry, Atlanta, GAVisiani R, (1842-1852) Flora Dalmatica, Lipsiae In Flora Europaea vol 3, Cambridge University pressWang I. H, Subramanian V, Moorman R, Burleson J and Ko J. R, (1997). Direct Determination of Pyrethrins in Pyrethrum Extracts by Reversed-Phase High Performance Liquid Chromatography with Diode-Array Detection. J. of Chrom. 766, 277-281Wenclawiak B. W and Otterbach A, (1999). Supercritical Fluid Extraction Kinetics of Pyrethrins from Flowers and Allethrin from Paper Strips. J. Anal. Chem. 365 8, 472-474Wenclawiak B. W, Krappe M and Otterbach A, (1995) In Situ Transesterification of the Natural Pyrethrins, J. of chrom. A, 785, 263-267World Health Organization, (1975) Data cruise on Pesticides No. 11, Pyrethrins (www.inchem.org/documents/pds/pds/pest11_e.htm)Wynn H. T. P, Chen g-Chin C, Tien-Tsu S, Fong L, and Ming-Ren S. F, (1995) Preparative Supercritical Fluid Extraction of Pyrethrins I and II from Pyrethrum Flower Talanta 42, 1745-1749