Traditional and alternative medicine in the prevention

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Traditional and alternative medicine is extensively practiced in the prevention, diagnosis, and treatment of various illnesses. It regains public attention from the past 20 years as this type of medicine is easily accessible in some regions. A postulation denoted that by 2010, at least two-thirds of Americans will opt for alternative therapies (Humber, 2002).

In poor countries such as in Asia and Africa, only half of the people are provided with limited essential drugs, hence making the traditional and alternative medicine as the main solution to cure illnesses. In fact, traditional African medicine serves over 80% of the populations in Africa (Elujoba, et al., 2005). Nevertheless, the most common reasons traditional and alternative medicine becomes more favourable are that it is cheaper, ideally acceptable by the patient's belief, and is less paternalistic comparing with the allopathic medicine ("Legal status of traditional medicine, complementary/alternative medicine: a world review," 2001).

Elaeis guineensis or the oil palm is among the plants that widely used by the traditional natives of West Africa. They apply leaf extract and the juice from young petioles to fresh wounds (Iwu, 1993). Whereas, in Liberia, split leaf stem of the oil palm are woven as splints (Bodeker & Burford, 2007). Originated from the West Africa, E. guineensis was first introduced into the then Malaya in 1875 and now being one of the largest commodity exported by Malaysia (Basiron, 2007).

Investigations on functional plants provide evidence on the presence of substances that are potential for human health benefits. However, there should be a vital requirement to determine the toxic effects of some substances contained in the plants (Bellini, et al., 2008).

Therefore, in the present study of the methanol extracts of E. guineensis leaf, it applied both acute oral toxicity test upon animal models (Joshi, et al., 2007) and brine shrimp lethality test (Meyer, et al., 1982) to determine its toxic properties. The acute oral toxicity testing was carried out for both sexes of animals under the Economic Cooperation and Development guidelines (OECD) 423 ("OECD guidelines for acute toxicity of chemicals no. 420," 2001).


2.1 Toxicity concept and history

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, sub-chronic, chronic, carcinogenic and reproductive effects (Asante-Duah, 2002).

Acute toxicity test evaluates the effects within seven days of a single dose while chronic toxicity test requires evaluation for a longer time period, at least six months and possibly up to the lifetime of the animals tested. The effects of both types of exposure can be different and sometimes lead to the opposite toxic effects (Timbrell, 2008). For instance, a single acute exposure can lead to chronic effects when the arising effects delayed. This can be seen when tri-ortho-cresylphosphate responds to cells and eventually causes peripheral neuropathy to the organism (Song, et al., 2009). In fact, the accumulation of chemical such as organophosphorus compound introduced by chronic exposure may results in either both toxic effects (Read, et al., 2007). As concentration is able to remain relatively constant or increase within the organism at a particular target area, this has made the frequency of exposure become another important factor in influencing the toxic effects (Timbrell, 2008).

The mechanism of toxicity begins with the introduction of the toxicant to the organism at the target area. This toxicant then interacts with the target molecules and may alter the biological environment of that particular area and hence will result in cellular dysfunction and damage. Eventually, such condition and dysrepair will develop to toxicity effects (Casarett, et al., 2008).

2.1.1 Acute oral toxicity

Acute toxicity testing intends to gain information on the biological activity of a substance as well as to study the mechanism of its action (Walum, 1998). 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 (Duffus, et al., 2009). 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 determination of a median lethal dose, LD50 (Walum, 1998).

Acute toxicity emphasizes on identification of the lethal dose that kills an organism after a single dose. The test requires a group of organism to be exposed to a series of increasing dose levels until the level reach the limit at which the whole organism die. The value of LD50 of which it is used to classify the toxicity of a substance will be determined based on this test (Duffus, et al., 2009).

The concept of LD50 was first introduced by Trevan in 1927 to aim for the identification of single lethal dose of a substance intended for human use such as digitalis extracts, insulin, and diphtheria toxin that kills half of the animals in a tested group (Botham, 2002; Walum, 1998). The dosed animals are observed within a two weeks period and very commonly that laboratory mice or rats from both sexes are selected to be tested for the classical study of LD50 (Sim, et al., 2010). However, under MEIC program instigated by the Scandinavian Society of Cell Toxicology, a preliminary calculation involving 30 MEIC chemicals suggested that mice give better prediction for human acute lethal dose compared to rats and this was proven by further MEIC evaluation performed on all 50 chemicals (Walum, 1998).

The absolute value of LD50 for a substance varies dependently on different parameters used and these include the methodology details, animal strain, caging, and test-chemical source. Due to the variation, Oliver (1986, cited in Walum, 1998, p. 498) has brought the problem into criticism by summarizing up two major points:

  1. The median lethal dose is a variable of biologic parameter that cannot be compared to constants such as molecular weight or melting point, indicating that an LD50 cannot be described in term of accuracy but precision and this precision can be only relevant for the experiment from which it was derived. Subsequent experiments do not necessarily result in persistent value of LD50;
  2. The value of LD50 refers only to mortality and lack of other clinical expressions of toxicity description.

A more contemporary design for acute toxicity testing aims to gain as much amount of information from a minimum number of animals (Gad, 2007). Hence, OECD provides a guideline ("OECD guideline for testing of chemicals no. 425," 2001) for testing of chemicals by which a limit test can be used efficiently to identify chemicals that are likely to have low toxicity. The limit test uses a dose of 2000 mg/kg and exceptionally 5000 mg/kg is considered when there is a strong probability that results of such test have a direct relevance for protecting human or animal health or the environment. This test is preferably used in situations where the people who will conduct the test have information indicating that the test material is much probably to be nontoxic. This information can be collected from knowledge of similar tested compounds or mixtures or products, with the consideration of the identity and percentage of components that are known to give significant toxic effects.

An instance of acute oral toxicity study was done by Sim et al. in 2010 by which they studied the acute toxicity effects of Pereskia bleo and Pereskia grandifolia in mice. The mice were administered orally with the highest dose of 2500 mg/kg crude extracts from both species and they showed no evidence of adverse effects or mortalities. While in 2007, Ramesh et al. studied the acute oral toxicity of Asiasari radix extract in mice. A predetermined dosages of 1000, 3000, and 5000 mg/kg body weight of A. radix extracts were administered to the mice orally. Also in this study, all the mice were survived and no signs of toxicity were observed.

2.1.2 Oral chronic toxicity

The classical approach to the study of chronic toxicity of compounds has been reported in many documents by the National Academy of Sciences (NAS), Food and Drug Association (FDA), World Health Organization (WHO), and others. This approach requires the study of two or more species of animals exposed to a test compound with appropriate level of doses and route of administration for a time period ranging from several months to years (Committee for the Working Conference on Principles of Protocols for Evaluating Chemicals in the Environment, 1975). The purpose for this study is to refine the description of the toxicity related with long-term administration of high dose of a substance. Usually, the animals used in this study are rats but mice are also applicable (Gad, 2007).

The parameters monitored in a chronic toxicity study include daily observation for stagnancy and mortality, weekly physical examinations, body weight and food consumption, terminal measurement of serum glucose and urea concentrations, and assessment of the activity of serum aspartateaminotransferase, alanine aminotransferase, and alkaline phosphatase. Whereas, hematological parameters such as red blood cells count, white blood cells count, and hemoglobin concentration are often limited to differential smear evaluations. Dead animals from the study are proceed to gross necropsy by which selected tissues from vital organs are collected, weighed, and processed for microscopic examination (Gad, 2007).

Chronic toxicity is measured in an almost similar manner to acute toxicity that is by using TD50 concept. Studying on the effect of chronic toxicity reveals that the compounds may accumulate in vivo and therefore would give a rough estimation of the probable whole-body half-life of the compounds (Timbrell, 2008). In details, the observable toxic responses may be the products of accumulation of the compounds, whose actions of its metabolites in body tissue include subsequent mobilization and redistribution, repeated and additive damaging to the target organs, enzymes, hormones or other biological systems.

One example of chronic oral toxicity study was conducted by Matsuda et al. in 2007 to evaluate the chronic toxicity of Aloe arborescens Miller var. natalensis Berger (ALOE) in the diet at different level of doses to groups of male and female Wistar Hannover rats for a one-year period time. Also in 2007, Mukinda and Syce worked on the study of chronic oral toxicity of the aqueous extract of Artemisia afra in rats. The rats were introduced with 0.1 or 1 g/kg/day of the extract for three months and experienced no significant changes in general behavior, and haematological and biochemical parameters but had a transient decrement in acute systemic toxicity activity.

2.2 Methodology of toxicity bioassay

2.2.1 Animal models

The appropriate toxicity tests using animal models are designed to gain information that is useful for the evaluation of toxic risk extrapolated to humans. Hence, an understanding of the mechanism of the toxic action requires knowledge on basic physiology, cell biology, pharmacology, and biochemistry (Svendsen & Hau, 1994).

Description of animal testing in toxicity research holds on two main principles. First, the effects in the tested animals that caused by the compounds are applicable to humans under proper qualification; and second, the high doses exposure of compounds to the tested animals is necessary and valid for discovering possible hazards in humans. These principles are the outcome of the dose-response concept by which effects will likely to become greater when higher dose is applied (Svendsen & Hau, 1994).

Acute toxicity test on animal models requires the administration of a single dose or more within 24 hour to the tested animals. It is usually employ three to five animals per gender per dose. The doses tested should range from the amount that does not induce adverse reactions to amount that bring major effects or lethality to the animals. Tested animals are observed for 14 days prior to the administration and important descriptions that refer to the toxic effects such as mortalities and clinical signs are recorded. Gross necropsies are performed after the 14 days observation period for the purpose of histopathology analysis (Fox, 2007).

As toxicologists evaluate and analyze the human risk based on the tested animals studied, however, in his work, Garattini (1985, cited in Walum, 1998, p. 499) recognized the problem when extrapolating the animal information to humans and he eventually presented three main points to be addressed:

  1. Different animal species dispose of chemical in various ways; therefore, the concept of doses (e.g. mg/kg body weight) should be replaced by the use of concentrations (mol/blood or tissue volume);
  2. Biotransformation of chemicals may results in metabolites with biologic activity; therefore, it is important to know in precise the chemical species that are responsible for the respective toxicity effects;
  3. Equal concentrations of chemicals do not necessarily satisfy for equal effects among the animal species because the sensitivity of organs, cells, enzyme, or receptors may vary within different species.

Thus, the three aspects should be taken into consideration in reference with other significant factors that also contribute to toxic effect and these include the strain, sex, age, concomitant pathology, hereditary defects, and concomitant exposure to other chemicals.

2.2.2 Brine shrimp lethality test

Brine shrimp lethality test is one of the most common bioassays used to measure toxicity (Rice & Maness, 2004) as well as for other pharmacological activities such as anticancer (Badisa, et al., 2009), antiviral (Bozic, et al., 2010) and pesticidal activities (Rao, et al., 2007).

In this technique, the in vitro lethality of a simple zoological organism such as brine shrimp, Artemia salina, is used as an indication for bioactive reaction (Rahman, et al., 2006). A. salina lives in saline water (Croghan, 1957) and since evolutionary history shows that it is never been exposed to land plants, A. salina shows a great sensitivity to many compounds consisted in the leaf extracts (Rice & Maness, 2004). Very high concentrations of extracts can kill the whole shrimp (100% mortality), whilst if too low concentrations results none of the shrimp dead (0% mortality). Therefore, LC50 is determined to measure the concentration of the extracts that results in 50% mortality of the shrimp (Chowdhury, et al., 2009). The live shrimp are easily distinguished by their swimming, whereas the dead shrimp are motionless and have their appendages extended (Rice & Maness, 2004).

A computer spreadsheet can be used to reckon the proportion of surviving shrimp. In the graph plotted, the percentage of mortality is a function of log of concentration (Chowdhury, et al., 2009). The LC50 value can be determined from the regression line, as the x-value at which y = 50%. High value of LC50 denotes low toxicity (Rice & Maness, 2004).

Data distribution is not expected to be normal because percentage values are restricted in between of 0 to 100. As a result, parametric regression analysis could be invalid (Rice & Maness, 2004) but this can be avoid by applying Probit analysis (Ayo, et al., 2007). Nevertheless, the problem of limited maximum value to 100% usually not significantly abrupt the clear conclusion provided by the graph of the test and hence the results are still acceptable (Rice & Maness, 2004).

2.3 The Oil Palm

Oil palm (Elaeis guineensis Jacq.) is a perennial monocot belonging to the family Palmae and tribe Cocoineae. It gives the highest oil yield per hectare of all the economic oil crops (Corley & Tinker, 2003). It is an important crop for Malaysia and contributes significantly to the national economy (Yusof, 2002). The oil palm tree originated from West Africa where it was growing wild and later developed into an agricultural crop. It was first introduced to Malaya in early 1870's as an ornamental plant. In 1917, the first commercial planting took place in Tennamaran Estate in Selangor, laying the foundations for the vast oil palm plantations and palm oil industry in Malaysia.

Today, 3.88 million hectares of land in Malaysia is under oil palm cultivation producing 14 million tonnes of palm oil in 2004. Malaysia is the largest producer and exporter of palm oil in the world, accounting for 30% of the world's traded edible oils & fats supply (Yusof, 2002). The industryprovides employment to more thanhalf a million people and livelihood to an estimated one million people. Oil palm is a crop that bears both male and female flowers on the same tree, meaning they are monoecious. Each tree produces compact bunches weighing between 10 and 25 kilograms with 1000 to 3000 fruitlets per bunch (Cheah, 1996). Each fruitlet is almost spherical or elongated in shape. Generally the fruitlet is dark purple, almost black and the color turns to orange red when ripe.

Palm trees may grow up to sixty feet and more in height. Thetrunks of young and adult plants are wrapped in frondswhich give them a rather rough appearance. The older trees have smoother trunksapart from the scars left by the fronds which have withere.
A normal oil palm tree will start bearing fruits after 30 months of planting and will continue to be productive for the next 20 to 30 years thus ensuring a consistent supply of oil.Each ripe bunch is commonly known as Fresh Fruit Bunch (FFB). In Malaysia, the trees planted are mainly the tenera variety, a hybrid between the dura and pisifera. The tenera variety yields about 4 to 5 tonnes of crude palm oil (CPO) per hectare per year and about 1 tonne of palm kernels. The oil palm is mostefficient, requiring only 0.25 hectares to produce one tonne of oil while soybean, sunflower and rapeseed need 2.15, 1.50 and 0.75 hectares respectively (Billotte, et al., 2001).

Mature trees of oil palm trees are single-stemmed, and grow to 20 m tall. The leaves are pinnate, and reach between 3-5 m long. A young tree produces about 30 leaves a year. Established trees over 10 years produce about 20 leaves a year.

2.3.1 Scientific classification

Kingdom: Plantae
(unranked): Angiosperms
(unranked): Monocots
(unranked): Commelinids
Order: Arecales
Family: Arecaceae
Subfamily: Arecoideae
Tribe: Cocoeae
Genus: Elaeis

2.3.2 Folk medicine

According to Hartwell (1967-1971), the oil is used as a liniment for indolent tumors. Reported to be anodyne, antidotal, aphrodisiac, diuretic, and vulnerary, oil palm is a folk remedy for cancer, headaches, and rheumatism (Duke & Wain, 1981).

2.4 The importance of medicinal plants

Plants have been a major source for alternative therapy since years ago. The raising acceptance of traditional herbal systems of medicine such as Ayurveda within India and outside regions has revived the ancient tradition of medicine (Kurian & Sankar, 2007). In fact, 65-80% of the population in the developing countries depends solely on the medicinal plants for basic cares of health, more than 80% among the Africans, while 71% in Chile and 40% in Colombia (Agra, et al., 2008).

Medicinal plants and their derivatives are now seek for the opportunity to enter the economic segment as this was proven by World Health Organization (WHO) in 1991 that traditional medicines, mostly plant drugs, supply to the health demands of almost 80% of the world population (Kurian & Sankar, 2007). Moreover, WHO has stated that 74% of 119 plant-derived pharmaceutical medicines applied in modern ways resembles the method used by native cultures (Goldberg, et al., 2002).

Over the years, the plant related trade grows rapidly due to the high recognition of natural products as non-narcotic compound, with relatively fewer side effects and easily available at affordable prices. China is very successful in promoting its own traditional medicine over the countries as this evidence is clear when United States has legitimated more licensed for Chinese medicine providers in the country (Patwardhan, et al., 2005). In India, about 8000 flowering plants, 650 lichens, 650 algae, 200 pteridophytes, and 150 bryophytes contribute in plant medicine sector and thus spot a strong position in the socio-economic, cultural and spiritual aspects (Kurian & Sankar, 2007).

2.4.1 Plants used for hypoglycaemic and diabetes

Several plants have been found out to possess anti-diabetic potential based on the findings from ethno-botanical claims and these include Aegle marmelos (bael fruit), Allium sativum (garlic), Aloe barbadensis (aloe vera), Catharanthus roseus (rosy periwinkle), and Momordica charantia (bitter gourd).

Study conducted on the aqueous extracts of A. marmelos root bark (1 mL/100 g) showed hypoglycemic effect which peaked (44%) at 3 h in normal fasted rats while another test showed that the same extract completely prevented peak rise of blood sugar at 1 h in OGTT. The aqueous extract of the A. marmelos leaves (1 mg/kg for 30 days) is able to control blood glucose, urea level, body weight, liver glycogen and serum cholesterol (Kesari, et al., 2006).

Aqueous homogenates of A. sativum (10 mL/kg/day) administered orally to sucrose fed rabbits (10 g/kg/day) in water for 2 months significantly increased hepatic glycogen and free amino acid contents, decreased fasting blood sugar, triglyceride levels in serum, liver and aorta, and protein levels in serum and liver in comparison to the sucrose control. Oral feeding of A. sativum extracts (100 mg/kg) for 16 weeks showed anti-atherosclerotic effects in STZ diabetic rats. Therefore, A. sativum can prevent diabetic cardiovascular complications (Grover, et al., 2002).

2.4.2 Plants used in cardiovascular ailments

Crataegus monogyna is a thorny shrub found commonly in temperate areas or the northern hemisphere, including North America, East Asia and Europe. The natives use the aerial part, including the flower of C. monogyna to treat asthma, and support cardiac and circulatory functions. The main constituents of the fruit are flavonoids such as vitexin-4-rhamnoside. Clinical and pharmacological analysis suggested that the standardized extracts of C. monogyna (standardized to 4-30 mg) or flavonoids can increase myocardial performance, circulatory perfusion and tolerance under oxygen deficiency condition, producing anti-arrhythmic effects and reduce afterload. Other positive therapeutic effects have also been observed on patients suffering from congestive heart failure, hypertension, tachycardia and arrhythmia.

Venous circulation is one of the problems faced by patients with cardiac-related ailments. Among the plants that have positive significant effects to this illness are Aesculus castanea (horse chestnut) and Gingko biloba (gingko). Conditions such as haemorrhoids, varicose veins and other better flow of blood problems can be relieved by applying these plants which perform anti-inflammatory and antioxidant activity. The presence of saponins in plants induces the anti-inflammatory activity whilst flavonoids and other molecules favour for antioxidant activity.

As A. sativum can cure diabetic problem, it also has been used in traditional medicine to reduce the concentration of blood, treat asthma and bronchitis, and acts as an expectorant, aphrodisiac, anthelmintic, and antifungal. The anthelmintic activity has been attributed to the presence of allicin which is produced after crushing the A. sativum(Ahtar, et al., 2000). Allicin is also an antioxidant which is capable to protect endothelial cells from oxidized LDL damages.

2.4.3 Plants used against the respiratory system problems

Respiratory disorders like colds, asthma and bronchitis can be treated through phytotherapy. As such ailments leading to infections, antibiotic dependent is inevitable. Nevertheless, decongestants (eucalyptus and mint), broncholytics and expectorants (thyme and mint), and demulcents (mallow) can support to relief the colds and flu-bouts.

Asthma can be treated by steroid as well as bronchodilator which can also found as natural origins, Ephedrine and Theophylline (Weinberger, et al., 1975). Ephedrine, the isolated components of Ephedra is contraindicated in the event of asthma as it has no side effects through the long history of its usage.


3.1 Methanol extracts preparation

3.1.1 Plant material sample

Fresh sample of E. 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 E. 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

3.2.1 Animals

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 (in process) 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 × 27 × 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 E. 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 Organsand 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 log of concentration (Islam, et al., 2009).

3.4.3 Data analysis

The mean results of mortality percentage of the brine shrimp versus the log 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




R & M Chemicals


HmbG Chemicals





Potassium dichromate

R & M Chemicals


Labchem, Ajax Chemicals


J. T. Baker

3.7 Apparatus and equipments

Table 3.2 List of apparatus and equipments used during the study






New Deluxe

Distilled water machine


Electronic balance

A & D

Freeze drier













4.1 Preparation of methanol extracts of Elaeis guineensis

The E. 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.

Table 4.1Yield of methanol extract of Elaeis guineensis



Weight (g)

Percentage (%)

Elaeis guineensis

Fresh samples


Dried and ground material



Methanol extracts



aRatio of dried and ground material per fresh samples in percentage; bratio of methanol extracts per dried and ground material in percentage

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.

Table 4.2 Result of the potential toxic effect of the crude extracts of Elaeis guineensis in mice




Crude extractb


Crude extract





aControl group (treatment without crude extract); btest group (treatment with 5000 mg/kg crude extract); cnumber of mice dead/number of mice used.

Table 4.3 General appearance and behavioral observations for control and treated groups


Control group

Test group

6 h

14 day

6 h

14 day

Skin and fur










Mucous membrane





Behavioral pattern



































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.

Table 4.4 Effect of Elaeis guineensis crude extract on organ-to-body weight index (%) in mice





Crude extract


Crude extract


0.54 ± 0.06

0.63 ± 0.07

0.58 ± 0.05

0.61 ± 0.06


1.63 ± 0.04

1.68 ± 0.05

1.61 ± 0.06

1.40 ± 0.08


6.33 ± 0.19

6.43 ± 0.14

6.35 ± 0.17

5.94 ± 0.17


1.21 ± 0.04

1.20 ± 0.05

1.12 ± 0.06

0.90 ± 0.07


0.47 ± 0.06

0.51 ± 0.05

0.48 ± 0.05

0.48 ± 0.08

Body weight (g)

31.22 ± 0.89

31.90 ± 0.70

31.21 ± 0.76

28.08 ± 0.66

Organ body index = (organ weight x 100)/body weight; acrude extract of Elaeis guineensis was administered to mice at a dose of 5000 mg/kg; values are mean ± SD (n = 10) at 5% level of significance.

4.4 Histopathology analysis of heart, kidneys, liver, lung, spleen, and ribcage

The microscopic structures of the organs depicted through 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

Table 4.5 Brine shrimp toxicity expressed as LC50 mg/mL


LC50 mg/mL

Elaeis guineensis (6 h)


Elaeis guineensis (24 h)


Potassium dichromate (24 h)


Brine shrimp lethality of the methanolic crude extracts of E. 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 E. 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 A. salina tested by the crude extracts, showing no physical damage occurred to the shrimp.


The acute oral toxicity of E. guineensis leaf methanol extracts was determined in the present study. The evaluations of the in vivo toxicity were done both qualitatively and quantitatively by performing histopathology study as well as determining the LC50 value using the brine shrimp lethality test. As medicinal plants application increases over the year, experimental screening on the toxicity of the plants is crucial to assure the safety and effectiveness of those natural sources.

In general, in vivo methods are likely to provide an early hint of toxic expression of a compound since applying in vitro cytotoxicity methods could result in limitation of some information. By applying in vivo assays, toxic expressions that may be observed on the tested animals such as pain, distress, allergic reactions, physical changes and behavioural alterations can be detected. However, acute toxicity study is lack of detecting effects on vital functions like cardiovascular, central nervous, and respiratory systems which are not usually assessed during the study.

5.1 Preparation of the crude extracts

The leaves of E. guineensis used during this study were obtained from a mature oil palm tree. The leaves were washed and rinsed thoroughly to avoid contaminants such as insects, microbes, and soils which may affect the results of the study. Since the leaves are relatively harder and thicker compared with most of other plant leaves, the process of drying and maceration took a longer time than that of other leaves, which are 6-8 days and 3-4 days respectively. Dried leaves of E. guineensis were used instead of fresh leaves as this can be beneficial for long term storage and convenient handling (Phrompittayarat, et al., 2007).

Maceration, a traditional method for solvent extraction, was performed in order to get the crude extract although other researches show that other methods are more effective (Ma, et al., 2009; Wang, et al., 2008; Zhang, et al., 2005). Nevertheless, maceration process is more simpler and does not consume much cost compared with other methods (Phrompittayarat, et al., 2007). The solvent used during the maceration was methanol. Also known as wood alcohol, methanol is commonly used as an organic solvent in the laboratory. It is considered as a polar solvent. Methanol bears a hydroxyl group (-OH) that is attached to a methane (-CH3) and it has a greater negative charge than the methane, hence results in an effective polarity of the solvent.

5.2 Acute oral toxicity study on animal models

Investigation of acute toxicity is the first step in toxicological analysis of medicinal plant. Oral acute toxicity testing in mice could be used to evaluate natural remedies for different pharmacological activities, taking into account the basic premise that pharmacology is simply toxicology at a lower dose (Sasidharan, et al., 2008). A toxic substance might elicit interesting pharmacological effects at a lower non-toxic dose. Toxicity results from animals will be crucial in definitively judging the safety of this E. guineensis as and when they are found to have sufficient potential for development into pharmacological products (Moshi, 2007). In addition, experimental screening is important to ascertain the safety and efficacy of natural products and to establish the active component of these natural remedies (Ogbonnia, et al., 2008).

In this study of acute oral toxicity, 40 Swiss albino mice from both sexes were employed to observe the toxicity effects of methanol crude extract of E. guineensis leaf. The route of administration depends on the dosage form in which the compound is available. Based on the historical researches, oral route administration is the most convenient and commonly used when studying about the acute toxicity. The absorption might be slow, but this method consumes lower cost and is painless and harmful to the animals. Since the crude extract is administered orally, the animals should be fasted before taking the dose because food and other chemicals in the digestive tracts may affect the reaction of the compound.

Although there is a problem regarding extrapolating animals data into human, a study has shown that mice give better prediction for human acute lethal dose compared to rats (Walum, 1998). All the procedures was performed based on the OECD guideline ("OECD guidelines for acute toxicity of chemicals no. 420," 2001). Because it is generally known that E. guineensis leaf is edible to animals, hence it is preliminary assumed to be not toxic. Therefore in this limit dose study, a very high doses level of 5000 mg/kg of crude extracts were administered orally to the tested mice ("OECD guideline for testing of chemicals no. 425," 2001).

From the current testing, no mortalities were reported as well as no adverse effects were observed on the tested mice throughout the period of 14 days. All of the mice gained weight and provided no significant changes in behavior. The physical appearances such as skin, fur and eyes were found to be normal and whilst as the body weight of the mice showed increment, this depicts that the administration of the crude extract does not affect the growth of the animals. Thus, this test reckoned that E. guineensis does not deliver acute toxicity effects with LD50 value greater than 5000 mg/kg. In principle, the method of limit test is not intended for determining a precise LD50 value. However, it serves as a suggestion of classifying the crude extract based on the expectation at which dose level the animals are expected to survive (Roopashree, et al., 2009). Therefore, according to the chemical labeling and classification of a cute systemic toxicity recommended by OECD, the crude extracts of E. guineensis were assigned under class 5 (LD50 > 5000 mg/kg) which was the lowest toxicity class.

Prior to the gross necropsy, the whole vital organs removed out from the dead mice were rinsed with saline instead of using water or fixative before fixing with 10% of formalin. This is because the difference in osmolarity between the tissue and the water may result in cellular swelling and rupture. Whereas, rinsing in fixative will directly starts the fixation process and hence causing discolouration and debris coalition to the surface of the tissues. Therefore, using saline is a good solution, by which it is capable of maintaining the structure of the tissues.

Based on the histopathology analysis, all of the tissues of organs seemed to be in good structures with no cellular lesions observed. There were no significant differences when comparing both the slides of the organs from tested animals and the controls, which suggesting that the crude extracts did not interact with the target cells or change the biological systems of the animals. The present results suggest the possibility of this extract as a potential source for the development of pharmacological agent to treat various types of ailment.

5.3 Brine shrimp lethality test

Brine shrimp bioassay is considered as a rapid preliminary screening for the presence of biochemical activity and used to determine the crude extract toxicity. This test is based on the potential of E. guineensis methanol extract to become lethal to A. salina nauplii due to its toxic expression.

According to Meyer et al. (1982), extracts derived from natural products which have LC50 ≤ 1.0 mg/mL are known to possess toxic effects. Whereas in this study, the graphs plotted have shown that the LC50 value of the crude extract is 9.00 and 3.87 mg/mL for 6 and 24 h respectively. Thus, these results remarked an early sign of proving that the methanol extracts of E. guineensis is not toxic. In contrast, the positive control used in this study, potassium dichromate, with LC50 value of 0.30 mg/mL at 24 h, has shown that it exhibits toxic expressions against the brine shrimp. Nevertheless, as toxic compound expressions can be insignificant in vivo, further investigation of this compound by using in vitro method should be pursued.

Based on the collective results, the leaf of E. guineensis exhibits no acute toxic effects against the animals, and hence signifying that this plant is not toxic to human also. This finding may bring hope and opportunity to E. guineensis to become a huge beneficial in both pharmaceutical and economic sectors as further research can be done to explore more values that it may possess.


This suggested that E. guineensis does not cause any apparent in vivo toxicity. The results of the current study concur with the use of this plant by traditional healers especially in Africa. A Word Health Organization survey indicated that about 70-80% of the world's population rely on non-conventional medicine, mainly of herbal source, in their primary healthcare. Our present study showed that E. guineensis does not exhibit any apparent toxicity and may be used as a medicinal agent in known dosages, especially in rural communities where conventional drugs are unaffordable because of the high cost or are unavailable in developing countries. Studies of this type are needed before a phytotherapeutic agent can be generally recommended for use.


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