Abstract: Background and Objective: A major concern in the development of insecticides is their safety to non-target species. This study aimed to analyze the safety of botanical oils with insecticidal properties on albino rats. Materials and Methods: Twenty-eight rats were divided into 7 groups of 4 rats each. The control group (A) received feed and water only, Groups: B1, B2 and B3 received 1500, 3000 and 5000 mg kg–1 b.wt., of C. occidentalis oil extract, while groups C1, C2 and C3 received 1500, 3000 and 5000 mg kg–1 b.wt., of E. milii oil extracts. Oils were extracted from both plants via soxhlet extraction; extracted oils were administered to albino rats orally once a week for 2 weeks. Thereafter, animals were sacrificed and serum analyzed for kidney and liver function markers. Results: Both oils caused a significant (p<0.05) increase in AST and ALP activities and decrease (p<0.05) in ALT activity, creatinine and urea concentrations at 3000 mg and 5000 mg kg–1 b.wt., relative to the control. Cassia occidentalis oil also caused significant (p<0.05) decrease in albumin concentration at 5000 mg kg–1 b.wt. Conclusion: The oils may be considered safe for insecticidal applications, since lower concentrations are required for insecticidal action.
INTRODUCTION
Insects are major enemies of human, livestock and agricultural crops all over the world. They transmit pathogens that cause malaria, dengue fever, yellow fever and leishmaniasis resulting in low life expectancy throughout the world. Nearly half of the world’s population is infected with at least one type of insect-borne pathogen or the other. Malaria alone affects up to 300 million persons yearly and thrives more in areas of poverty and low economic growth1. Insecticides are important tools used for the control of insect pests and reduction in the damages they cause, in order to increase agricultural crop yield and improve the quality of life for humans, domestic animals and livestock. The sustainability of insect pest control is a very interesting topic currently receiving attention in the agric and agric-related literature2. A study in 2001 has favoured an organic management approach3. Several governments have taken initiatives to develop environmentally friendly agricultural practices4. These sustainability programs continuously emphasize the importance of developing organic insecticides for pest control as they assumed that natural insecticides present less risk to the environment than synthetic insecticides, which agrees with the general opinion of the public5.
The synthetic pesticides currently in use such as the organophosphate and organochlorine insecticides have been associated with various forms of cancer, neurological disorders and lung irritations in humans6. Agriculturists who apply these insecticides in farms come in contact with these dangerous pesticides and may be prone to nervous system damages. “Pesticide drift" may also occur as pesticides are sometimes carried by wind and water to non-target areas. They penetrate groundwater, pollute streams and harm wildlife7. Cypermethrin; one of the active ingredients in most synthetic insecticides has been reported to cause toxicity to the reproductive system in male rats. After 15 days of continual dosing, both androgen receptor levels and serum testosterone levels reduced significantly8. Long-term exposure in adulthood is found to induce dopaminergic neuro-degeneration in rats and postnatal exposure enhances the susceptibility of animals to dopaminergic neuro-degeneration if rechallenged during adulthood9. Genetic damage, chromosomal abnormalities, increases in bone marrow and spleen cells are some of the diseases associated with exposure to cypermethrin10. Cypermethrin has also been implicated as a possible human carcinogen and is linked to an increase in bone marrow micronuclei in both mice and humans11. Studies have also shown that residue from cypermethrin can last for 84 days in the air, on walls, the floor and on furniture12 relative to natural pesticides which are degraded shortly after application. Again, being a broad-spectrum insecticide, it kills both beneficial insects and animals together with the target pests13. It is believed to affect the insect’s nervous system and is found in many household insecticide products. Contact with the skin may cause irritation, while inhalation may cause shortness of breath. It has a low solubility in water, making it unlikely to contaminate water sources in run off14.
There is thus need for a credible alternative to the available synthetic pesticides particularly in the developing countries where there are inadequate occupational safety standards, protective clothing and washing facilities, insufficient enforcement, poor labelling, illiteracy and insufficient knowledge of pesticide hazards15. Higher plants are rich sources of novel natural substances that can be used to develop environmentally safe products for insect control16. Many secondary metabolites from various plants are popular for their insecticidal efficacies and are sometimes used domestically to kill or minimize the impact of insect pests17. The discovery of bioactive secondary metabolites from plants which are toxins to herbivores that attack them opened the vista for their assessment as insecticides. These secondary compounds represent a large reservoir of chemical structures with biological activity18. Essential oils from the leaves of E. milii and C. occidentalis have been reported to possess reasonable levels of insecticidal efficacy against insect pests19. Venkatesan et al.20 also reported that crude extract from Cassia occidentalis showed potential growth regulation and a moderate level of adulticidal activity against the urban malaria vector; Anopheles stephensis. The safety of these oils towards non-target species have however not been reported.
Euphorbia milii also known as crown of thorns, Christ plant or Christ thorn is a low-growing evergreen shrub with very thorny grooved stems and branches. It belongs to the family: Euphorbiaceae, phylum: tracheophyta, class: magnoliopsida, order: euphorbiales, genus: Euphorbia specie: milii 21. Legends associate it with the crown of thorns worn by Christ. It is not indigenous to Nigeria, but is believed to have been imported to Nigeria from India22. It is currently found all over the world as widely grown ornamental specie23. A characteristic feature of all Euphorbia species, including the crown of thorns, is the presence of milky latex which is secreted by the plant through broken stems, roots and leaves22. Found in all parts of the plant, the latex is usually poisonous and probably developed in order to protect the plant from herbivores.
Cassia occidentalis also called coffee weed is a small tree that belongs to the phylum: tracheobionta, class: magnoliopsida, order: fabales, family: Fabaceae, genus: Cassia
and Specie: Occidentalis. The specie gives off a foul odour when damaged. It is indigenous to Brazil, it is also found in warmer climates and tropical areas of south Central and north American. In East Africa, it is commonly known as ant bush, arsenic bush or Negro coffee. In Nigeria, it is known as Nigerian senna or stinking weed24, Akidi ogbara by the Igbos, Dora rai by the Hausas and Aboo rere by the Yorubas25.
The motivation behind this study was to explore the possibility of formulating a natural insecticide of botanical origin that can effectively replace the more toxic synthetic insecticides currently in use. The objective was to expose albino rats to different concentrations of essential oils extracted from the leaves of E. milii and C. occidentalis with the view to ascertain the effect of these oils on biochemical markers of liver and kidney toxicity.
MATERIALS AND METHODS
This study was carried out at the Chemical Pathology Unit of the University of Calabar Teaching Hospital, Ezeagbor, Cross Rivers state, Nigeria. It lasted for six months from October, 2017 to April, 2018.
Equipments: Soxhlet extractor Manufactured by B.BRAN Scientific and Instrument Company England, Thermo Scientific Rotary evaporator Model R-300 USA, Electric blender AKAI TOKYO JAPAN Model No: BDOO11DA-1033M made in PRC, weighing balance Symmetry Cole-Parmer Instrument Co., USA. Auto Chemical analyzer, Selectra Pro 5 Elitech Clinical Systems, France. Echotherm Heating dry bath (Toney Pines Scientific 2713 Loker Avenue West Carlsbard. CA29010. Model IC20XR. SERIAL NO: 08021302 USA.
Reagents and chemicals: All reagents and chemicals used were of analytical grade, they include; n-hexane, AST, ALP, ALT, albumin, urea and creatinine assay kits (ELI Tech Clinical Systems SAS-Zone Industrielle-61500 SEES FRANCE.
Collection and identification of plant samples: The E. milii and C. occidentalis plants were earlier identified by a botanist in the Department of Biological Sciences (Botany), College of Natural Sciences, Michael Okpara University of Agriculture, Umudike Abia state. Leaves were thereafter harvested in the desired quantity from No 4 Edim Otop close, off victory way, Satellite town Calabar, Cross Rivers state. The plants appeared healthy, leaves bright green in colour and flowers intact at the time of the harvest.
Experimental animals: A total of 28 rats were used. Rats were monitored from birth and separated from male rats at 4 weeks before attaining sexual maturity. They were adult female nulliparous and non-pregnant weighing between 130 and 150 g. The rats were 8 weeks old as at the time of this research and weighed between 150 and 175 g. The animal ethics procedures were complied with during the whole experimental process as the study procedures were approved by the Faculty of Basic Medical Sciences (FBMS), University of Calabar, Animal Ethics Committee Ref no: FAREC/GP/005/16. Rats were fed standard rat chow and tap water ad libitum with regulated temperature and humidity and a 12/12 h light-dark cycle.
Soxhlet extraction of oils from plants: The dried leaves (dried for a period of 2 weeks under shade) of E. milii and C. occidentalis were pulverized using an electric blender, put in an air-tight container and used subsequently for soxhlet oil extraction. Oils were obtained by continuous extraction in Soxhlet apparatus for 16 h using n-hexane as solvent according to the method of Association of Official Analytical Chemists26. For each 50 g of ground leaves processed, 4 g equivalent of E. milii oil and 5 g of C. occidentalis oil was obtained, this amounts to 8 and 10% oil yield, respectively. The procedure was run four times to obtain 16 g of E. milii and 20 g of C. occidentalis used for the study.
Effect of plant oils on biochemical indices of rats: Exactly 5000 mg kg–1 b.wt., of each extract was used as the limit dose; this dose was selected based on previous studies conducted by Silva et al.27. Cassia occidentalis showed LD50 higher than 5000 mg kg–1. While Euphorbia schimperiana; a plant in the Euphorbia family, at a limit dose of 5000 mg kg–1 b.wt., caused no mortality or clinical signs of toxicity during fourteen days post dose28. Rats were sacrificed under light ether anaesthesia. Twenty eight rats were divided randomly into 7 groups containing four rats each. The control group (A) received rat feed and water only, Groups; B1, B2 and B3 received 1500, 3000 and 5000 mg kg–1 b.wt., of C. occidentalis oil extract, while groups C1, C2 and C3 received 1500, 3000 and 5000 mg kg–1 b.wt., of E. milii oil extracts. The administrations were done orally once a week for 2 weeks.
Blood sample collection for serum biochemical analysis: At the end of 2 weeks, rats were fasted overnight and sacrificed under light ether anaesthesia. Blood samples were drawn via cardiac puncture using a 2 mL sterile syringe. About 1 mL of blood was collected and dispensed into plain test tubes which were allowed to stand for complete clotting. The clotted blood was centrifuged and the serum aspirated. Determination of urea, creatinine, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were done using biochemical assay kits manufactured by ELI Tech Clinical Systems SAS-Zone Industrielle-61500 SEES France.
Determination of serum biochemical indices: The colorimetric determination of albumin was done by the method of Doumas et al.29. The ALT activity was determined by the method already reported by Henley30. The AST activity according to the method of Bergmeyer et al.31. Serum creatinine concentration was determined according to the method previously published by Toro and Ackermann32. Serum ALP activity was determined according to the method of Haussament33. While urea was determined according to the Berthelot method as described earlier by Fawcett and Scott34 and modified by Patton and Crouch35.
Statistical analysis: Data was presented as mean±standard error of mean (SEM). Statistical analysis was performed using one way analysis of variance (ANOVA) in the statistical package for social sciences (SPSS) for windows, version 20.0 (SPSS Inc., Chicago IL, USA).
RESULTS
Biochemical indices of rats administered E. milii Oil: In Table 1, a significant (p<0.05) decrease in ALT activity, creatinine and urea concentrations and increase (p<0.05) in ALP and AST activities relative to the control at 3000 and 5000 mg kg–1 b.wt., was observed.
Biochemical indices of rats administered C. occidentalis oil: In Table 2, significant (p<0.05) decrease in albumin, creatinine, urea concentrations and ALT activity and increase (p<0.05) in AST and ALP activities relative to the control was observed at 5000 mg kg–1 b.wt.
DISCUSSION
Results from this study showed that oil extracts from the leaves of E. milii and C. occidentalis may be safe for use by humans as insecticidal agents, with its toxicity manifesting only at very high concentrations (3000 and 5000 mg kg–1 b.wt). No significant (p>0.05) change in most biochemical parameters was observed at 1500 mg kg–1 b.wt., indicating that these extracts may be safe for insecticidal application by man. This agrees with the report of Nuhu and Aliyu36, who reported slight toxicity in the use of crude extracts from C. occidentalis as concotion for liver ailments. Another researcher also reported that methanolic extracts of Euphorbia schimperiana, a plant in the same genus, exhibited slightly toxic effects on mice28.
Elevated serum levels of alkaline phosphatase (ALP) activity as observed in this study have been associated with chronic kidney disease (CKD)37. While elevated aspartate transaminase (AST) activity is linked to; liver disease or injury, ischemic/toxic damage to the liver, acute viral hepatitis, medications, acute biliary obstruction and alcohol abuse38,39.
SWAN (a synthetic insecticide) caused significantly lower body weight gain and produced; diarrhoea, loss of appetite, eye discharge and mortality even at a low dose40 of 5 mg kg–1 day–1 E. milii and C. occidentalis did not cause any mortality even at 5000 mg kg–1 b.wt., per week. This agreed with the report of a researcher that acute and sub-acute administration of hydroalcoholic extract of C. occidentalis is not toxic in male and female Wistar rats suggesting safety for use by humans27.
| Table 1: | Biochemical indices of rats administered E. milii oil |
Values are expressed as the Mean±SEM (n = 4), *Significantly different from control at p = 0.05, aSignificantly different from 1500 mg kg–1 at p = 0.05, bSignificantly different from 3000 mg kg–1 at p = 0.05 | |
| Table 2: | Biochemical indices of rats administered C. occidentalis oil |
Values are expressed as the Mean±SEM (n = 4), *Significantly different from control at p = 0.05, aSignificantly different from 1500 mg kg–1 at p = 0.05, bSignificantly different from 3000 mg kg–1 at p = 0.05 | |
According to their report, the extract did not produce any hazardous symptoms or death in the acute toxicity test showing LD50 higher than 5 g kg–1. In yet another study by Isah et al.41, sub-acute administration of up tp 3000 mg kg–1 b.wt., of aqueous leaf extract of Cassia coccidentalis did not produce significant effects on kidney of albino rats thereby suggesting a non-lethal effect on kidneys. In the current study however, even though the oils did not cause any significant (p<0.05) change in kidney function markers at 1500 mg kg–1, it caused a significant (p<0.05) decrease in urea and creatinine concentrations at 3000 mg kg–1 b.wt. Low levels of creatinine in the body could be a sign that the liver or muscle is not working properly42. This could be caused by muscle diseases such as muscular dystrophy; characterized by muscle weakness, muscle stiffness, pain and decreased mobility. Since it is produced in the liver, an unhealthy liver will be unable to make the normal amount of ceatinine42. Liver diseases arising from; poor liver function, interference with creatine production can also cause low creatinine concentration43. Reduced plasma/serum urea can be caused by decreased urea production, urinary urea excretion or a combination of the two44. Pathological cause of reduced urea concentration is largely confined to advanced liver disease45. This simply meant these oils may cause liver and kidney damage at very high concentrations. This agreed with the report that crude latex of E. milii caused a higher frequency of minor malformations only at very high doses which are embryolethal and maternally toxic. They concluded that this plant possess no teratogenic hazard or at least the possibility is of a considerably low order of magnitude46.
The significant (p<0.05) decrease in ALT activity corroborated with a previous study which revealed that C. occidentalis extract significantly reduced serum levels of ALT suggesting hepatoprotection of extracts47. While the significant (p<0.05) decrease in albumin concentration at 5000 mg kg–1 of C. occidentalis oil may indicate; liver failure, heart failure, kidney damage, protein losing enteropathy, malnutrition or chemotherapy48. However given the low concentrations of active compounds usually required for insecticidal action on pests, (for instance SWAN; a synthetic insecticide, contains 0.45% tetra-methrin and 0.15% beta-cypermethrin) these oils may be considered safe alternatives to the harmful insecticidal chemicals currently in use.
CONCLUSION
The oils may be considered safe for use as insecticidal agents since toxicity indices are only manifested at very high concentrations. They could gradually replace the more toxic synthetic chemicals in the management of pests and disease vectors. Farmer in the rural and urban settings should be encouraged to embrace the use of natural insecticides to replace the more toxic synthetic ones in order to improve life and increase longevity.
SIGNIFICANCE STATEMENT
This study discovered the safety of plant oils that could be beneficial for agricultural pest control and increased productivity and economic growth. This study will help the researcher to uncover the critical areas of biopesticide toxicity that many researchers were not able to explore. Thus a new theory on essential oils as biopesticides may be arrived at.
ACKNOWLEDGMENT
The authors are grateful to the management of the University of Calabar, Cross Rivers state, Nigeria, for providing the funds used for this research. Grant number UC/DD.27/vol.67.