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2.0 LITERATURE REVIEW
Toxicity is an expression of being poisonous, indicating the state of adverse effects led by the interaction between toxicants and cells. This interaction may vary depending on the chemical properties of the toxicants and the cell membrane, as it may occur on the cell surface, within the cell body, or in the tissues beneath as well as at the extracellular matrix. The toxic effects may take place prior to the binding of the toxicants to the vital organs such as liver and kidneys. Hence, evaluation of toxic properties of a substance is crucial when considering for public health protection because exposure to chemicals can be hazardous and results to adverse effects on human being. In practice, the evaluation typically includes acute, chronic, carcinogenic and reproductive effects (Asante-Duah, 2002).
As defined by the Organization for Economic Cooperation and Development (OECD), acute toxicity is the occurrence of adverse effects prior to an oral administration of a single dose of a substance in a short period time or of multiple doses given within 24 hours (Hayes, 2001). The adverse effects refer to operational disability or deterioration and biochemical lesions that may disrupt the whole performance of an organism or limiting the function of an organ to response under critical condition. As substance that entering the organism through the oral route under a restricted time and hence resulting in adverse effect is known to be orally and acutely toxic, however the term acute oral toxicity is very often connected to lethality and the determination of LD50 (Walum, 1998).
3.0 MATERIALS AND METHODS
3.1 Methanol extracts preparation
3.1.1 Plant material sample
Fresh sample of Elaeis guineensis leaves was obtained from Kampung Lekir, Sitiawan, Perak, Malaysia in August 2009 and was authenticated by Mr. Shunmugam A/C Vellosamy from Herbarium Unit, School of Biological Science, Universiti Sains Malaysia. The dried parts of the plant including leaves, fruits and flowers were deposited as voucher specimens (with herbarium number 11036) at the Herbarium Unit, School of Biological Science.
3.1.2 Preparation of the crude extracts
The midribs of the Elaeis guineensis leaves were removed before cutting the leaflets into pieces. The sample was then washed thoroughly and rinsed with tap water and dried in oven at 60 °C for two to four days. The leaves sample was sequentially extracted with methanol by approximately adding 100 g of the dried sample (in fine powder form) to 400 mL methanol. The extraction was carried out at room temperature and soaked for four days with intermittent stirring during the first day. The extracts were filtered and the process of extraction was repeated again for a second time by adding another 400 mL to the sample residue. The filtrate from each extraction was combined and concentrated under vacuum by rotary evaporator until dark green methanol extracts produced. The extracts were freeze dried and kept at 4 °C until use.
3.2 Acute oral toxicity study of Elaeis guineensis methanol extracts in mice
The experiment was conducted on 40 healthy Swiss albino mice (males and females) weighing 25 to 35 g and aged 8 to 10 weeks, acquired from the Animal House, Universiti Sains Malaysia. Those mice were distributed into four groups i.e. both two treated groups and two control groups of opposite sex. The experimental procedures relating to the animals were authorized by ----- [Ethical number:-----] before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care (EEC Directive of 1986; 86/609/EEC).
3.2.2 Procedure of acute oral toxicity
The mice used in the experiment were selected at random and marked at the tails for individual identification. Each ten mice of the same sex were kept in a matte plastic cage, with dimension of 17 Ã- 27 Ã- 14 cm. All of the cages were located in a room at temperature approximately 23 °C with constant humidity. The room is regulated with cycles of 12 h of light and 12 h of darkness. The mice were acclimated to the laboratory environment for a week earlier before starting the experiment. Drinking water and food were provided ad libitum through the experiment except for the short fasting period where the drinking water was still in free access but no food supply within 12 h prior to treatment. The acute oral toxicity of Elaeis guineensis methanol crude extracts was evaluated in mice according to the procedure outlined by the Organization for Economic Co-operation and Development (OECD). A single high dose of 5000 mg/kg of crude extracts was administered to both ten male mice and ten female mice through oral route. The extracts were suspended in a vehicle (Tween-20 in distilled water). Following the fasting period, body weight of the mice was determined and the dose was calculated in reference to the body weight as the volume of the extracts solution given to the mice is 10 mL/kg. Another ten male mice and ten female mice were allotted with distilled water and were regarded as the control groups. Food was provided to the mice approximately an hour after treatment. The mice were observed in detail for any indications of toxicity effect within the first six hours after the treatment period, and daily further for a period of 14 days. Surviving animals were weighed and visual observations for mortality, behavioral pattern, changes in physical appearance, injury, pain and signs of illness were conducted daily during the period.
3.3 Clinical analysis
3.3.1 Organs and body weight statistical analysis
Finishing the 14 days period, the whole mice were gently sacrificed. Vital organs such as heart, kidneys, liver, lung and spleen, and also a fragment of the rib cage were isolated and examined for any lesions. All of the individual organs were weighed and their features were compared between both treated and control groups. Statistical analysis to assess the significant difference between both groups was conducted by running a T test using Microsoft Excel spreadsheet application. The level of significance used in this analysis is 5%.
3.3.2 Histopathology of heart, kidneys, liver, lung, spleen, and ribcage
All the vital organs and the rib cages isolated from each individual were fixed in 10% buffered formalin, routinely processed and embedded in paraffin wax. Paraffin sections (5 µm) were cut on glass slides and stained with haematoxylin and eosin. The slides were examined under a light microscope and the magnified images of the tissues structure were captured for further study.
3.4 Brine shrimp lethality test
3.4.1 Hatching shrimp
Brine shrimp eggs, Artemia salina were hatched in a vessel containing sterile artificial seawater prepared by dissolving 38 g table salt in 1 L distilled water. The vessel was kept under an inflorescent bulb and facilitated with good aeration for 48 h at room temperature. After hatching, active larvae (nauplii) released from the egg shells were collected at the bright side of the vessel (near the light source) by using micropipette. The larvae were isolated from the eggs by aliquoting them in small beaker containing the seawater.
3.4.2 Brine shrimp test
The bioactivity of the extracts was monitored by the brine shrimp lethality test (Meyer, et al., 1982) to predict the presence of cytotoxic activity in the compound. The extracts was dissolved in methanol and diluted with artificial seawater. The assay system was conducted by preparing 10 bijoux bottles filled with 2 mL of seawater each and a two-fold dilution was set up to yield a series of concentrations from 100 to 0.195 mg/mL. Potassium dichromate was dissolved in artificial seawater and functioned as a positive control with concentration ranging from 0.1 to 0.9 mg/mL. An aliquot (0.1 mL) containing about 10 to 15 larvae was introduced to each bottle and the setup was allowed to continue for 24 h. the bottles were observed, and the dead larvae from each bottles were counted after 6 and 24 h. Based on the percentage of the mortality, the concentration that led 50% lethality (LC50) to the larvae was determined by using the graph of mean percentage mortality versus the logarithm of concentration (Latha, et al., 1998).
3.4.3 Data analysis
The mean results of mortality percentage of the brine shrimp versus the logarithms of concentrations were plotted using the Microsoft Excel spreadsheet application, which also formulated the regression equations. These equations were later used to calculate LC50 values for the samples tested with consideration of value greater than 1.0 mg/mL suggesting that the compound is nontoxic.
3.5 General aseptic techniques
Aseptic techniques involve practices to minimize the introduction of contamination when conducting a laboratorial work. All of the apparatus and equipments were sterilized before used and surrounding area is made sure to be clean. Sterilization for the equipments and tools was carried out using autoclave at 120 °C for 20 minutes while the workbench was swept with 70% alcohol to prevent contamination on working surface.
3.6 Materials and chemicals
Table 3.1 List of materials and chemicals used during the study
3.7 Apparatus and equipments
Table 3.2 List of apparatus and equipments used during the study
Distilled water machine
4.1 Preparation of methanol extracts of Elaeis guineensis
The Elaeis guineensis leaves were dried and ground before extracted with methanol. Table 4.1 shows the yield of the products in weight and the percentage calculated for the ratio of dried and ground material per fresh samples and methanol extracts per dried and ground material, in respective.
4.2 Lethality and behavioral analysis
The lethality and toxicity effect of the methanol extracts of Elaeis guineensis on the mice appearance and behavioral pattern are respectively shown in Table 4.2 and table 4.3. There was no death among the animal during the observation as also no significant changes in general appearance or behavioral pattern reported. Moreover, all the organs either of the control or the test groups are in good shape and conditions.
4.3 Organs and body weight statistical analysis
The body weight as well as the weights of the vital organs of the animals were calculated and recorded in Table 4.4. There were no significant differences in the changes of each weight.
4.4 Histopathology analysis of heart, kidneys, liver, lung, spleen, and ribcage
The microscopic structures of the organs depicted through Figure 4.1 to Figure 4.11 show unnoticeable differences between the control and test group. There were also no cell degradation or any unfavorable effects observed when viewed under the light microscope using multiple magnification power.
4.5 Brine shrimp lethality test
Brine shrimp lethality of the methanolic crude extracts of Elaeis guineensis are shown in Figure 4.12 and 4.13 and the LC50 values calculated are recorded in Table 4.5. The methanolic crude extracts show positive result, indicating that the samples are biologically active. Crude extracts resulting in LC50 values of less than 1 mg/mL are considered as significantly active while this suggests that the Elaeis guineensis crude extracts have a very low toxicity effect giving the values of LC50 9.00 and 3.87 mg/mL at 6 and 24 hour respectively. Plotting of mortality percentage versus log of concentration for all tests (Figure 4.12 to 4.14) demonstrates an approximate linear correlation. Furthermore, there is a direct proportional relation between the concentration of the extracts and the degree of lethality. This is shown as the maximum mortalities occurred at a concentration of 100 mg/mL whilst concentration of 0.195 mg/mL only caused very minor mortalities. As a positive control, potassium dichromate has proven a significant toxicity effect to the shrimp as its LC50 reached lower than 1.0 mg/mL. Figure 5.15 depicts the morphology of an Artemia salina tested by the crude extracts, showing no physical damage occurred to the shrimp.
Application of Elaeis guineensis leaves as a medicine for alternative therapy brings potential to cure particular illnesses. Therefore, this study is successfully performed to ensure and support that this plant is nontoxic and hence is safe to apply to human. No acute toxicity exhibited when applied orally at concentration of 5000 mg/kg body weight. This has been demonstrated by the normal behavior and histological observations in reaction to the crude extract when applied to the animals. Another test to predict the toxic potential of this plant is the brine shrimp lethality test. The result based on this test also supports that Elaeis guineensis extract is nontoxic and furthermore, it reveals that this plant has responded actively in bioassay activity. These results are important as they could serve as a basis for further studies related to pharmacological research of Elaeis guineensis.
Asante-Duah, K. (2002). Public health risk assessment for human exposure to chemicals (illustrated ed. Vol. Volume 6 of Environmental pollution). Dordrecht: Kluwer Academic Publishers.
Hayes, A. W. (2001). Principles and methods of toxicology (4th ed.). Philadelphia: Taylor & Francis.
Latha, R. M., Geetha, T., & Varalakshmi, P. (1998). Effect of Vernonia cinerea less flower extract in adjuvant-induced arthritis. Gen Pharmacol, 31, 601-606.
Meyer, B. N., Ferrigni, N. R., Putnam, J. E., L.B.Jacobsen, Nichols, D. E., & McLaughlin, J. L. (1982). A convenient general bioassay for active plant constituents. Planta Medica, 45, 31-34.
Walum, E. (1998). Acute oral toxicity. Environmental Health Perspectives, 106.