The experimental determination of the yield of pyrethrins from the Chrysanthemum cinerariaefolium flower is usually carried out with chromatographic techniques; as such lots of methods have been developed over the years [Wang et.al, (1996)]. These include HPLC [Moorman et.al, (1982)], GC [Caniff, (1995)] and SFC [Wenclawiak, (2000)] methods. The objective of this work is to analyze and report on the total pyrethrins (not the six individual constituents), hence GC is selected. Reports on the yield of pyrethrins (of the dry weight using various extraction methods) varies in literature; 0.91 - 1.30% [Kolak et.al, (1999)], 0.60 - 0.79% [Bakaric, (2005)], 0.75 - 1.04% [Bhatt, (1995)], 1.80 - 2.50% [Morris et.al, (2005); Bhatt and Menary, (1984); Fulton, (1999)], 0.50 - 2.0% [Kiriamiti et.al, (2003)], and 0.90 - 1.50% [Pandita and Sharma, (1990)]. However, Casida and Quistad predicted that pyrethrin yield of 3.0% or even more could be obtained [Casida and Quistad, (1995)]. We got, with hexane extraction in a water bath at controlled temperatures and vigorous stirring (with three magnetic stirrers at a speed of 20rpm), pyrethrin yields varying from 0.85 - 3.76% by dry weight of sample. This, to our knowledge, is the first time pyrethrins yield above 3% is reported.
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Key words: Pyrethrins, Solvent extraction, Two-step extraction, Gas Chromatography, Pyrethrin concrete
Pyrethrum flowers are from the Chrysanthemum genus; and due to the size, shape, and colour of its petals and appearance, they are referred to as "painted daisies" or "painted ladies." Its other names are Buhach, Chrysanthemum Cinerariaefolium, Ofirmotox, Insect Powder, Dalmatian Insect Flowers, and Parexan. According to Visiani (1842-1852), it was first recorded in Dalmatia. Other writers believe that the Croatian Pharmacist, Antun Drobac (1810-1882) from was the first to prove its insecticidal activity [Bakaric, (2005)]. Yet there are claims that it was first identified as having insecticidal properties around 1800 in Asia; and that the Crushed and powdered plants were used as insecticides by the Chinese as early as 1000 BC. The flower contains about 1-2% pyrethrins by dry weight, but approximately 94% of the total yield is concentrated in the seeds [Casida and Quistad, (1995)]. The chemical structure of the active ingredients, pyrethrin I and pyrethrin II was identified in 1924 [Coomber, (1948)]. Kenya is the world's main producer today with more than 70% of the global supply [Casida, (1973)]. The natural active 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, (2001)]. Pyrethrins, though insoluble in water, are soluble in many organic solvents [WHO, (1975)]. They are non-volatile at ambient temperatures; non-toxic to mammals and other worm-blooded animals; highly 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 crop protection; domestic insecticides; and the formulations of synthetic pyrethroids. Although pyrethrins are soluble in a number of organic solvents (benzene, hexane, petroleum ether, alcohol, acetone, methanol, chlorinated hydrocarbons, etc) other considerations (practical, economic and environmental concerns) limit the usage. These reduce the options to just a few; one of the qualities of Hexane in the extraction of pyrethrins is its ability to effectively dissolve the active ingredients effectively without dissolving the contaminants present. Removing it from the concrete is also possible at lower temperatures, which limits degradation due to prolonged heating. Its low boiling point is also an added quality. Again, it can be recovered for recycling and reduces the weight of the concrete. Above all, it is inexpensive, accessible and environmentally friendly. It is less toxic, non-corrosive, and non-reactive. Little wonder and rightly so, that in literature, it is the most adopted solvent especially for the processing of plant materials.
We obtained the grounded chrysanthemum (light green with a characteristic smell) sample and two pyrethrum concretes (yellowish in colour) from â€¦ (the company)â€¦ Their pyrethrin content was claimed to be 50.0% (29.50% PYI and 20.50% PYII) and 85.15% (46.33% PYI and 38.82% PYII). We used the concrete with the 85.15% PY content for preparing the six individual standard solutions for the standardization of the analytical method since it has the highest content as such its impurities would be less among the two concretes obtained. We purchased analytical grade hexane (97.0%) and Ethanol (99.7%) from Sinopharm Chemical Reagent Co. Ltd in China, and used directly without any pre-treatment.
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Extraction of the pyrethrins from 100g of the grounded pyrethrum flowers of particle size of about 30mesh, with hexane was conducted in a water bath( YUHUA, DF-101S) in batches at temperatures of 35oC, 40 oC, 45 oC, 50 oC, 60 oC and 70 oC in 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 (We used one stirrer initially but the yield for using three was better hence the choice) at a speed of 20rpm. The hexane was then removed, with a rotary evapourator (YUHUA, RE-2000B) at a temperature of 35 oC and at a speed of 185rpm; from the pyrethrin concrete- also called Crude Hexane Extract (CHE).
The chromatographic analysis was performed in a gas chromatograph with an FID detector, Agilent GC, HP-5 capillary Column, 30mm Ã- 0.25mm id., 0.25um ï¬lm thickness. Each run required about 50mins. The instrument was calibrated with multiple-point standard additions calibration method using the six individual standard samples prepared. The peak area of each component in the sample solutions was within the linear range of the standard. The split/split less injector, in the ratio 20:1, was kept at 250 â-¦C. Nitrogen was used as carrier gas at a ï¬‚ow rate of 1.6ÂµL/min. The injection volume was 0.1ÂµL.
The temperature program started at 180 oC, kept for 11 minutes, heated at 10â-¦C/ min to 200 â-¦C, kept for 8 minutes; heated to 210 â-¦C at 10 â-¦C/min, kept for 18 minutes, then heated to 245Â°C at 30Â°C/min, staying at this temperature for 4 minutes.
2g of the concrete with the 85.15% pyrethrin content was dissolved in 10mL Ethanol, and then made up to a final volume of 100mL with Ethanol. Six standard aliquots; 1mL, 2mL, 4mL, 8mL, 16mL and 32mL of this PY solution was then transferred into a 50mL flask each and diluted with Ethanol again to the mark and mixed. The solutions were filtrated using a 0.45-Î¼m membrane filter and analyzed. Figure 1 shows that all the six pyrethrin components in the pyrethrum samples were well identified and separated with the following elution order: jasmolin-I (~20 min), cinerin-I (~23 min), pyrethrin-I (~24 min), jasmolin-II (~38 min), cinerin-II (~42 min), and pyrethrin-II (~43 min). Based on the results from Fig.1, a standard curve was established (from best fit line) to express the relationship between the areas produced by the GC and the concentration as indicated in Fig. 2 (Table A1 shows corresponding data). The method used to establish the curve was the standard additions.
Figure 1: Chromatographs of the standard samples
Figure 2: Standard curves for PYI and PYII. The Pearson correlation coefficient R2 in each is about 0.99.
Results and Discussion
3.1 Effect of the extraction temperature
Figure 3 shows the GC chromatograms for extracts at different temperatures in a fixed extraction time of 5 hrs. All the chromatograms also clearly indicate that the six pyrethrin components are very similar to those of the standard sample. Figure 4 summarizes the corresponding yields (refers to Table A2 for the data). As the figure and Table A2 show, at 40oC targeted components are extracted more but above this temperature more undesirable components are extracted at the expense of the pyrethrins components which turn to decompose. This is so because pyrethrins are thermo labile and therefore degrade after 40oC [E. Stahl, 1998; C. Gourdon, 2002; W.H.T.Pan, 1994]. It also gave the best PYI: PYII ratio (4.75).
Figure 3: Chromatographs of extracts obtained at different extraction temperatures in 5 hrs
Figure 4: Comparison of the yields of PYI and PYII, and PYI: PYII at various times
3.2 Effect of the stirring
We compared the effect of two stirring methods on the extraction results: the first with one magnetic stirrer and the other with three magnetic stirrers. Results are shown in Figure 5 (or data in Table A3). This figure confirms that stirring improves extraction yield. That in our view facilitated very well the dissolution of the active ingredients and the effective distribution of heat to effect the extraction. The extraction at 40oC in 4hrs was repeated severally and the results were relatively the same.
Figure 5: Comparison of the yields of PYI and PYII (5 hrs, 40oC) under different stirring methods.
3.3 Effect of the extraction time
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A further investigation was done to confirm the optimum extraction time by fixing the extraction temperature of 40oC with three magnetic stirrers. Figure 6 shows the chromatograms for extracts obtained at different extraction times, indicating that all the six pyrethrin components in the pyrethrum samples were well identified and separated.
Figure 6. Chromatographs for extracts from various extraction times at 40oC
As is seen, there is little difference between the 40oC extraction in 5hrs from figure 3 and those in figure 6. This is mainly due to the difference in the sample volume. The sample volume in fig. 3 was concentrated (10ml) prior to the analysis which produced higher peak areas (Table A6) and corresponding concentrations outside the range of the standard (Table A5) while in fig.6, the sample was not concentrated (500ml) prior to the analysis so as to fit the concentrations into the standard (Table A5). This resulted in lower peak areas and accordingly, shifted (reduced) the retention times a bit (Table A6).
Figure 7. Chromatographs for extracts at 40oC in 5hrs from fig3 an d fig 6.
Figure 8 (or data in Table 3) summarizes the results. The figure shows that at 40oC the extraction yield increases steadily from 3hrs to a peak at 4hrs. Within this range, more desired components are extracted but after 4hrs the yield decreases which indicates that either less and less desired components are extracted or decomposed. The drop in yield is consistent from 4hrs to 6hrs the highest yield (3.76) was at 40oC and 4hrs. This means that the optimum time is not 5hrs but rather 4hrs. The ratio of PYI: PYII is best at 6hrs (5.14). From 3hrs to 4hrs, the yield for both PYI and PYII appreciated but the increment in PYI (0.74) is greater than that of PYII (0.38) hence the drop in the ratio. Between 4hrs and 5hrs, there is decrease in both yields of PYI and PYII. Again, the decrease in PYI (0.98) is greater than that of PYII (0.49) and this accounts for the drop in ratio. The same reason accounts for the drop in ratio from 5hrs to 6hrs.
Figure 8 Comparison of the yields of PYI and PYII (40oC) at various times.
Our results confirm that it is actually possible to obtain an extraction yield of pyrethrins above 3% (3.76%) as suggested by Casida and Quistad (1995) in their book. To our knowledge, this is the first time such a high recovery rate is reported. A number of reasons may be attributed to this high recovery rate such as i) extraction procedure, ii) vigorous stirring and above all, vi) the type of sample used.
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