ABSTRACT
Pyrethrins are extracted from pyrethrum flowers and are classified into two groups; Pyrethrins I and Pyrethrins II. They are used as a broad spectrum natural insecticide active ingredient in agriculture and public health. Pyrethrins are degradable on exposure to air, moisture and high temperatures. The aim of this research was to investigate the optimum drying temperature, time, light intensity and moisture content of drying pyrethrum flowers for maximum yield of pyrethrins on extraction. Mature pyrethrum flowers from experimental farm, College of Agriculture and Veterinary Sciences, Kabete campus, University of Nairobi were harvested into brown paper bags, divided into sixteen portions and taken to the laboratory. The first six portions of the flowers were dried at varying temperatures of 30, 40, 50, 60, 70 and 80 ºC respectively to total dryness in an oven. The seventh to twelfth portions of flowers were dried in the oven at 30, 40, 50, 60, 70 and 80 ºC for a maximum period of 18 hrs. The thirteenth portion of the flowers were dried in darkness to a constant weight at temperatures below 50oC. The fourteenth portion of the flowers were dried in sunlight to a constant weight at temperatures below 50oC. The fifteenth and sixteenth portions were dried in darkness and in direct sunlight respectively for two weeks. The determination of moisture content of the flowers dried in the oven was carried out at given temperatures at one hour intervals. Hexane was used for solvent extraction after the flowers were ground into fine particles. Analysis of the extracts was carried out using Ultra High Performance Liquid Chromatography and Titrimetric method after refining the extracts. Maximum moisture loss of about 10% was achieved by the flowers at varying times and temperatures. The time recorded for drying at temperatures of 70 and 80 ºC was 18 hours. A percentage yield of 0.84 was obtained when the flowers were dried to constant weight at 30 ºC while drying for 18 hours yielded 0.75. Extractable Pyrethrins II were found to reduce by 8.6% when the drying temperature was raised from 50 to 60 ºC, by 11.3% from 60 to 70 ºC and by 8.5% when temperature was raised from 70 to 80 ºC for flowers dried for 18hrs. Extractable Pyrethrins I were found to reduce by 2.5% when the temperature was raised from 50 to 60 ºC, by 5.2% from 60 to 70 ºC and by 5.5% from 70 to 80 ºC when flowers were dried to constant weight. The percentage reduction of Pyrethrins II when the temperature was raised from 50 to 60 ºC during drying was 8.2%, 12.5% from 60 to 70 ºC and 12.2% from 70 to 80 ºC. Percentage reduction of Pyrethrins I when the drying temperature was raised from 50 to 60 ºC was 6.0%, 5.1% from 60 to 70 ºC and 9.5% from 70 to 80 ºC. Percentage of pyrethrins obtained from flowers dried at 50 ºC for 18 hours was 1.37% and 1.44% when drying was done at the same temperature to constant weight. Total pyrethrins obtained from the flowers dried in darkness was 1.38% and 1.02% when drying was done in direct sunlight. Moisture level for the flowers dried to a constant weight was below 10%. There was a variation in the ratio of Pyrethrins I : II over the temperature range of 30-80oC. The optimum conditions for drying pyrethrum flowers were found to be at a temperature of 50 ºC, a duration of 21 hours in darkness to moisture level of less than 10%.
TABLE OF CONTENTS
DECLARATION i
DEDICATION ii
ACKNOWLEDGEMENTS iii
ABSTRACT iv
LIST OF FIGURES viii
LIST OF TABLES ix
LIST OF ABBREVIATIONS/ACRONYMS x
CHAPTER ONE
INTRODUCTION
1.1 Background of the Study 1
1.2 Statement of the Problem 5
1.3 Objectives 5
1.4 Significance of the Study 6
CHAPTER TWO
LITERATURE REVIEW
2.1 History of Pyrethrum 7
2.2 Production of Pyrethrum 7
2.3 Toxicity of Pyrethrins 8
2.4 Chemistry of Pyrethrins 8
2.4.1 Properties of Natural Pyrethrins 9
2.4.2 Thermochemistry and Photochemistry of Pyrethrins 10
2.5 Environmental Fate of Pyrethrins 11
2.5.1 Solvent Partitioning 11
2.6 Chemical Transformations of Pyrethrins 13
2.6.1 Effects of Pyrethrins on insects 14
2.7 Sampling of Pyrethrum Flowers 14
2.7.1 Methods of Drying Pyrethrum Flowers 15
2.7.2 Extraction of Pyrethrins 17
Instrumentation of Soxhlet extractor 17
2.8 Analysis of Pyrethrins 18
2.8.1 Working Mechanism and Instrumentation of High Performance Liquid Chromatography 19
2.8.2 Working Mechanism and Instrumentation of Gas Chromatography 21
2.8.3 Mercury Reduction Method 25
CHAPTER THREE
MATERIALS AND METHODS
3.1 Flower Sampling 29
3.2 Reagents, Instrumentation and Apparatus 29
3.3 Drying of Flowers 29
3.4 Grinding of Dry Pyrethrum Flowers 30
3.5 Cleaning and Sterilization of Glassware 30
3.6 Preparation of Working Solutions 30
3.7 Extraction of Pyrethrins Using Soxhlet Extraction apparatus 31
3.7.1 Sample Clean-Up for Analysis Using Mercury Reduction Method 31
3.7.2 Separation of Chrysanthemic Acid and Pyrethric Acid 32
3.7.3 Mercury Reduction Method for Analysis of Pyrethrins I 32
3.7.4 Determination of Pyrethrins II 33
3.8 Sample Preparation for Analysis of Pyrethrins Content Using Ultra High Performance Liquid Chromatography 34
3.8.1 Analysis of Pyrethrins Using Ultra High Performance Liquid Chromatography 35
CHAPTER FOUR
RESULTS AND DISCUSSIONS
4.1 Percentage Moisture Loss during Drying of Pyrethrum Flowers 37
4.1.1 Percentage Moisture Lost during Drying in an Oven to Constant Weight 37
4.1.2 Percentage moisture lost during drying in an oven at constant time of 18hours. 37
4.1.3 Percentage Moisture Loss of Flowers after Drying in Direct Sunlight and in the Dark. 39
4.2 Results Obtained from Pyrethrins Analysis Using Mercury Reduction Method 40
4.2.1 Percentage of Pyrethrins on Drying for 18 hours in an Oven 41
4.2.2 Percentage of Pyrethrins on Drying to a Constant Weight in an Oven 42
4.2.3 Percentage of Pyrethrins from Flowers Dried in Direct Sunlight and in Darkness to a Constant Weight. 43
4.2.4 Percentage of Pyrethrins from Flowers Dried for Two Weeks 44
4.3 Comparison of the Percentage of Pyrethrins I and Pyrethrins II Concentrations on Drying for 18 hours in the Oven. 45
4.3.1 Comparison of the Percentage of Pyrethrins I and Pyrethrins II Concentrations. 45
4.4 Results Obtained from Pyrethrins Analysis Using Ultra-High Performance Liquid Chromatographic Method 46
4.4.1 Percentage of Pyrethrins on Drying to a Constant Weight in an Oven Analyzed Using Ultra High Performance Liquid Chromatography 49
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1 CONCLUSION 51
REFERENCES 53
APPENDICES 63
LIST OF FIGURES
Figure 1: Mature flower of Chrysanthemum cinerariifolium 1
Figure 2: Mature flower of Chrysanthemum coccineum 2
Figure 3: A Schematic Diagram of the Chemical Structures of Pyrethrins 9
Figure 4: Photodegradation and Ozonolysis Pathways of Pyrethrins. 14
Figure 5: Drying of Pyrethrum Flowers on the Ground. 15
Figure 6: Drying of Pyrethrum on a Rack. 16
Figure 7: Drying Pyrethrum Flowers on a Rack Covered with a Polythene 16
Figure 8: Soxhlet Extractor. 18
Figure 9: Block Diagram of High Performance Liquid Chromatography 20
Figure 10: Diagram of a Gas Chromatograph 23
Figure 11: Graphical Representation of the Weight Lost During Drying 39
Figure 12: Percentage of Pyrethrins on Drying the Flowers for 18 hours in an Oven 45
Figure 13: Comparison of the Percentage of Pyrethrins I and Pyrethrins II Concentrations Analyzed Using Titrimetric Method. 46
Figure 14: Chromatograms of 50% w/w Pyrethrins Standard 47
Figure 15: Chromatogram of Sample Dried at 50˚C to Constant Weight. 47
Figure 16: Percentage of Pyrethrins on Drying to a Constant Weight in an Oven Analyzed Using Ultra High Performance Liquid Chromatography 49
LIST OF TABLES
Table 1: Aqueous Partition Coefficient of Pyrethrins. 12
Table 2: Drying of Flowers to a Constant Weight. 37
Table 3: Percentage Moisture Lost During Drying in an Oven at Constant Time of 18hours. 38
Table 4: Percentage Moisture Loss of Flowers after Drying in Direct Sunlight and in the Dark 40
Table 5 Titrimetric volumes in determining Pyrethrins I 40
Table 6: Titrimetric Volumes in Determining Pyrethrins II 41
Table 7: Percentage of Pyrethrins on Drying for 18 hours in an Oven 42
Table 8: Percentage of Pyrethrins on Drying to a Constant Weight in an Oven 43
Table 9: Percentage of Pyrethrins on Drying to a Constant Weight 44
Table 10: Percentage of Pyrethrins from Flowers Dried for Two Weeks 44
Table 11: Percentage of Pyrethrins Obtained from Ultra High Performance Liquid Chromatography Analysis 48
Table 12: Percentage of Pyrethrins on Drying to a Constant Weight 49
LIST OF ABBREVIATIONS/ACRONYMS
A.O.A.C - Association of Analytical Chemists BCF- Bioconcentration Factor
ECD – Electron Capture Detector GC – Gas Chromatography
GLPC – Gas Liquid Partition Chromatography HPLC - High Pressure Liquid Chromatography KARI – Kenya Agricultural Research Institute Kp – Partition coefficient
LOD – Limit of detection LOQ- Limit of quantitation
OSHA – Occupational Safety and Health Administration PBK – Pyrethrum Board of Kenya
PI – Pyrethrins I PII – Pyrethrins II
PHP - Potassium Hydrogen Phthalate. RMM – Relative Molecular Mass
SGD – Sustainable Development Goals TF – Titration Factor
TLC – Thin Layer Chromatography
UHPLC – Ultra High Performance Liquid Chromatography WHO – World Health Organization
WSPE – World Standard Pyrethrum extract
CHAPTER ONE
INTRODUCTION
1.1 Background of the Study
Pyrethrum is a plant from Compositae family and has been widely studied for its commercial importance as a source of insecticidal components known as Pyrethrins. There are two main species of pyrethrum; Chrysanthemum cinerariifolium and Chrysanthemum coccineum. C. cinerariifolium is also referred to as the Dalmatian chrysanthemum derived from the region of its origin, Dalmatia. It has white flowers, with yellow center that sprout from the stiff stems as shown in Fig. 1. The stems have blue-green leaves and can grow to about 46-100 cm tall. C. coccineum is known as the Persian chrysanthemum and it is a perennial species native to Caucasus. The species has large white, pink or red flowers as shown in Fig. 2 and grows to a height of about 30-60 cm with leaves resembling ferns. Insecticidal potency substances in its flowers are lower compared to those C. cinerariifolium (Motsoeneng et al., 2015).
Figure 1: Mature flower of Chrysanthemum cinerariifolium
Figure 2: Mature flower of Chrysanthemum coccineum.
C. cinerariifolium is the most planted species in the world for its flower head. Countries producing this species for commercial purposes are: Uganda, Kenya, Ecuador, Japan, Rwanda, Tasmania, Papua New Guinea and Italy (Laugeray et al., 2017).
Pyrethrum grow well in semi-arid conditions and cool winters deliver optimal pyrethrins production (Vadhana et al., 2013). Cool temperatures, high rainfall of above 750mm and high altitude areas with ranges of about 1500-3000m favors the growth of pyrethrum. As the altitude increases, the concentration of pyrethrins increases. In addition, fertile soils that are well drained with favorable organic matter are ideal for pyrethrum flower production. The land should be well tilled to allow easy penetration by roots while all weeds should be removed and ploughing done during dry months to help destroy persistent weeds. Temperature change affects the content of pyrethrin in the flowers of the plant. Pyrethrum plants flowers continuously for about 10 months annually. The first harvesting of mature flowers is done 4 months after planting with follow up harvest every two (Wanjala et al., 2008).
The active substance used as natural insecticide extracted from the flowers of C. coccineum or C. cinerariifolium are referred to as pyrethrins. Pyrethrins is a mixture of six closely related esters namely pyrethrin I and II, Jasmolin I and II and Cinerin I and II. The pyrethrins are used as a broad spectrum natural insecticide in agriculture and public health. They are biodegradable thus making them environmental friendly. The pyrethrin insecticides have been used for several decades with no resistance developed since they get degraded in the environment. Pyrethrins I have got a “knock down” effect while Pyrethrins II have the “kill” effect on insects. The efficacy of pyrethrins is determined by the ratio of Pyrethrins I to Pyrethrins II. The pyrethrins are very efficient when the ratio is 1:1.
Growing of pyrethrum flowers in Kenya brings great economic value since it is an income generating activity for small scale farmers and also earns the country foreign exchange through the export. Pyrethrum growing areas in Kenya are; Kiambu, Nyandarua, Nakuru, Nyamira, Murang’a, Bomet, Kericho, Koibatek, Nandi, Nyeri, Laikipia and Kisii. These areas receive adequate rainfall amounts and the temperatures are cooler.
Processing the flowers to extract the pyrethrin is a process that varies from place to place. Processing starts with picking of the mature flowers that have attained horizontal petals positioning (Hughes et al., 2016). The flowers are then dried to total dryness and then ground into fine particles before commencement of extraction. The dried flowers are then transported to the processing factories and sold for extraction of pyrethrins which are the active components (Ang’endu, 1994). In the pyrethrum industry, drying is necessary to remove the extra moisture. Drying of the flowers is done in open air shortly after picking to avoid fermentation and pyrethrins losses. A number of drying techniques have been employed which are time consuming and labor intensive. For instance, some farmers spread the flowers on polythene sheets in direct sunlight or in a shade for two to three weeks. Farmers growing pyrethrum on large scale use solar driers or roasters to hasten the drying process (Manna et al., 2005). Heat causes rearrangements of the pyrethrins structure to form iso-pyrethrins which are insecticidally inactive. Exposure to air and direct sunlight also leads to degradation of the pyrethrins due to the presence of the UV in the sunlight (El Okda et al., 2017). The described drying methods leads to degradation of pyrethrins in the flowers since drying conditions are not controlled.
Extraction of pyrethrins is done from C. cinerariifolium flowers grown as an agricultural commercial crop in the world. Farmers and suppliers of crude pyrethrum extract use procedures that have not changed since shortly after World War II (Costa, 2015). The flowers are harvested by hand on attaining maturity and full bloom to obtain maximum pyrethrins content then dried. Transportation of the dried flowers is done to the nearest processing facility where grinding is done and solvent extraction commences. Any undissolved substance is filtered from the ground pyrethrum flowers after dissolving using an organic solvent. The solvent used is removed resulting in a crude oleoresin containing about 30% pyrethrins (Wylie et al., 2016). Components of the crude oleoresin (which is black and viscous) produced in a batch are: vegetable matter, waxes and pyrethrins. The produced oleoresin is then ready for refining.
Producers in Tasmania have genetically selected plants to fully bloom within a short span of about two weeks. Harvesting of the flowers is done mechanically rather than using labour intensive, hand-picking method used by farmers in other regions. The flowers are left to dry in the fields, cut from the plant and then separated from the stems. The dry flowers are ground, pelletized and extracted using hexane. Removal of the solvent is then done leaving a crude oleoresin that consistently has had an increased pyrethrins content greater than 35% (Singh, et al., 2012).
Crude oleoresin undergoes further processing to get rid of impurities from the plant matter, which are resins and waxes (Oda et al., 2012). The refining step yields a clear, amber solution of pyrethrins normally diluted with kerosene to a standard concentration for marketing. Refined extract has low staining characteristics and a minimum level of inert insoluble substance (Skolarczyk et al., 2017). Since the objective of refining operations being a light coloured extract, a high recovery of the pyrethrins is important. Major refineries operate batch processes based upon proprietary solvents for extraction to separate the pyrethrins from the unwanted vegetable waxes and resins. Despite the fairly simple solvent extraction operations concept, refiners have realized that the content of the extract vary with the type of solvent used. Research on determining the best solvent mixture should be done to achieve a high quality product with good pyrethrins recovery (Wang et al., 2017). Dilution of the refined pyrethrins to a standard concentration is done based on the analysis method from the Association of Analytical Chemists (AOAC) and Butylated hydroxytoluene (BHT) is added as an antioxidant.
The standard concentration in United States has historically been 20% pyrethrins which has been marketed as a technical grade material. The material was eventually blended with synergists, emulsifiers and solvents to produce insecticide and formulations primarily for consumer product applications (Wylie et al., 2016).
Kenya began to refine oleo resins in the 1970s which produced about 50-60% pyrethrins. Transportation costs for the highly concentrated product was lower compared to that of dilute material. In addition, the concentrated extract appealed pyrethrum users who preferred to formulate technical grade material with a higher purity. The refined pyrethrum concentrate is still being marketed in Kenya today (Kumar et al., 2011). A pyrethrum refining process using carbon (IV) oxide in a super-critical-fluid extraction procedure has been investigated. This process is advantageous since it prevents exposure of the extract to heat when flashing off the solvents used in the conventional procedures operated by major primary pyrethrum refiners (Del Prado-Lu, 2015). Pyrethrum refiners must be able to recognize the differences and respond appropriately to process the material properly to achieve acceptable recoveries and a high quality product (Costa, 2015).
Different analytical methods such as; Gas Chromatography, Ultra High Performance Liquid Chromatography, Titrimetric method and High Performance Liquid Chromatography are used in the analysis of pyrethrins (Holynska et al., 2018).
Pyrethrins content is determined using the listed analytical methods for pyrethrum flowers dried using different methods. Determining the pyrethrins content helps in selecting the optimum drying conditions.
1.2 Statement of the Problem
Drying methods of pyrethrum flowers used by farmers may lead to degradation of pyrethrins since they are biodegradable when exposed to air, direct sunlight, moisture and temperatures above their boiling points. Degradation of pyrethrins leads to losses of income for the pyrethrum farmers due to low quality and quantity of their produce. In addition, sun drying pyrethrum flowers is time consuming, labor intensive and lead to inconsistence of pyrethrins content per flower. Drying the flowers under sunlight is weather dependent and therefore consistent pyrethrin content would not be attained. Moreover, inadequate drying would lead to rotting and fermentation during storage process leading to economic losses. There is a threshold of moisture content beyond which leads to loss of the pyrethrins in the flowers. This research investigated the optimum conditions in relation to light, moisture content and temperature of drying pyrethrum flowers without losing the pyrethrin in them.
1.3 Objectives
General Objective
The main objective is to establish the optimum drying conditions of pyrethrum flowers to retain high pyrethrins content. To realize the main objective, the following specific objectives were undertaken:
I. To establish the threshold moisture content of dried pyrethrum flowers for pyrethrin extraction.
II. To validate the titrimetric method of analysis of pyrethrins from pyrethrum flowers.
III. To determine the effects of temperature on the content of pyrethrins in pyrethrum flowers.
IV. To determine the effects of drying pyrethrum flowers in direct sunlight.
1.4 Significance of the Study
Establishing the optimum drying conditions of pyrethrum flowers ensures minimum loss of pyrethrins. This leads to improved quality of the pyrethrum flowers thus increasing revenue from the produce since farmers are paid depending on its quantity and quality. Improved drying methods would also help save on time and work load required to dry the pyrethrum flowers. This would go a great mile in helping to actualize sustainable development goal (SDG) 1 on fighting poverty and SDG 8 on promoting economic growth. Availability of high pyrethrin content in the manufacture of insecticides will reduce the need to use pyrethroids which are toxic to human and animal life. Usage of pyrethrins in the manufacture of insecticides will help in ensuring a sustainable environment, good health and well-being hence realization of SDG 3. Pyrethrins are biodegradable in the environment and in mammalian tissues hence they do not last long in the environment. Pyrethrins being esters are not toxic to animals. This is because they are swiftly hydrolyzed into harmless products in the gut of animals which are subsequently excreted.
Click “DOWNLOAD NOW” below to get the complete Projects
FOR QUICK HELP CHAT WITH US NOW!
+(234) 0814 780 1594
Login To Comment