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CHAPTER IINTRODUCTION1 Problem and Its BackgroundDepression is a chemical imbalance in the brain. It may affect a person’s mood, thoughts, feelings, behaviors, and physical health. Severe depression can result in feeling of hopelessness and helplessness. It is a major mental disease that is ranked a...

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

CHAPTER I INTRODUCTION 1.1 Problem and Its Background Depression is a chemical imbalance in the brain. It may affect a person’s mood, thoughts, feelings, behaviors, and physical health. Severe depression can result in feeling of hopelessness and helplessness. It is a major mental disease that is ranked as the fourth leading cause of disability. Being low in essential nutrients like B vitamins, vitamin D, magnesium, zinc, omega-3 fatty acids and lack of serotonin in the body can cause depression. Deficiency of both vitamins B12 and D are extremely common. Serotonin is a major neurotransmitter that is a major factor in the quality of overall happiness and mood as well as memory, and learning. They are believed to improve mood by blocking brain receptors that reabsorb serotonin. Tryptophan is an amino acid that is the precursor to both serotonin and the sleep hormone melatonin. Tryptophan supplements increase serotonin levels by providing the essential building blocks of serotonin. Tryptophan has been found to be as effective for depression as antidepressant drugs. A high level of tryptophan can be found in Guava, a tropical fruit. Nutrients like Vitamin-A, vitamin-C, vitamin-B6, vitamin-B12, folic acid, carbohydrates, magnesium, iron, placebo, vitamin-D and total omega-3 fatty acids are also present in this fruit. The researchers are seeking for natural antidepressants in 1

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

order to avoid unwanted adverse reactions, as well as improve efficacy that is why the researchers come up with this study. 1.2 Statement of the Problem The study aimed to determine the neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice). Specifically, the study answered the following questions: 1.) Is there a significant difference on the neuro-physiological effect of the Albino mice before and after the application of Psidium guajava (Guava) fruit extract? 2.) Does the Psidium guajava (Guava) fruit extract have a significant effect on the pain perception of the mice? 3.) Can Psidium guajava (Guava) fruit extract be considered as a natural stimulant? 1.3 Hypotheses The following hypotheses were tested to accept or reject the assumption on the neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice). 1.) There is no significant difference on the neuro-physiological effect of the Albino mice before and after the application of Psidium guajava (Guava) fruit extract. 2.) The Psidium guajava (Guava) fruit extract does not have a significant 2

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

effect on the pain perception of the mice. 3.) Psidium guajava (Guava) fruit extract cannot be considered as a natural stimulant. 1.4 Significance of the Study This study aims to determine if the guava can be a natural stimulant. It can help people by giving knowledge about herbal medicine and can benefit the following: Department of Education This study can benefit the department of education by giving knowledge to its people through schools and many other sector of education. Department of Health This department will officially add guava to the list of fruits that can be a natural stimulant. Agriculturists It can help the farmers and agriculturists by giving income to the farmers who plants guava and sell it for their income. Industry To help the experts produce a product with a cheap price but has the same quality in terms of nutrients. 3

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

Medical Practitioner This study can help the department of health by giving information to the doctors and nurses that they can have guava as an alternative to commercialized stimulants in the field of medicine. Food and Nutrition Research Institute The principal research arm of the government in food and nutrition can explore more about this topic and can add this study on their source for the students to easily gain information. Citizens People that cannot afford to buy medicine were benefited by this study. They will also have a knowledge regarding the herbal plants. Future Researchers This can become a reference to researchers who will have a study that is related to this topic 1.5 Scope and Limitation This study was conducted in the laboratory of De La Salle UniversityDasmariñas. Fresh fruits of Guava were bought from a local market in Bacoor and were extracted in the researcher's home. Albino mice were taken from the Food and Drug Administration. These samples were used because it is 4

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

convenient, small, and can adapt well to new surroundings. Also their biological and behavior characteristics closely resemble those of humans, and many symptoms of human conditions can be replicated in mice. The nature of the study used was an open field test and hot plate method to test the neuro-physiological effect of Psidium guajava (Guava) fruit extract to each sample. This study was conducted for about a month or two. Making a product out of this research is not the scope of the study anymore, only the effect of the guava to the mice was being tested. 1.6 Conceptual Framework Guava has been hailed as one of the super fruits due to the numerous health benefits it offers. However, another potential is yet to be described and determined, hence this study was conducted.

Fruit Extract

Neuro-physiological effect to albino mice Tally of locomotion, exploration and anxiety activities of mice

Psidium guajava (Guava)

Figure 1.1 Conceptual Paradigm The figure above shows the conceptual framework wherein the fruit extract of Guava was subjected to albino mice and therefore exposing to open field test and hot plate for the evaluation of behaviors. 5

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

1.7 Definition of Terms The following operational terms were defined within the context of the study:

Albino mice. It is a small mammal of the order Rodentia which is bred and used to determine the neuro-physiological effect of the guava fruit extract as a natural stimulant. Guava. It refers to the fruit that is used by the Researchers to determine its neuro-physiological effect as a natural stimulant to Albino mice. Hot plate method. It was used to determine the reaction time of a certain number of Albino mice. Neurophysiology. It is a branch of physiology and neuroscience that is concerned with the study of the functioning of the nervous system. Open Field Test. It provides simultaneous measures of locomotion, exploration and anxiety. Stimulant. It is a substance that can be raises levels of physiological or nervous activity in the body. Tryptophan. It is an amino acid that is a constituent of most proteins. It is an essential nutrient in Guava.

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

CHAPTER II REVIEW OF RELATED LITERATURE 2.1 Conceptual Literature Open Field Test The Open Field Test (Walsh & Cummins, 1976) provides simultaneous measures of locomotion, exploration and anxiety in mice, where both the quality and quantity of the activity can be measured. The behaviours scored (Brown et al., 1999) includes: Line crossing in which the mice crossed one of the grid lines with all four paws; Center square entries in which the mice crossed one of the red lines with all four paws into the central square; Central square duration or the extent of time the mice spent in the central square; Rearing that can be measured by getting the frequency with which the mice stood on their hind legs in the maze; Stretch attend postures which is measured by getting the frequency of demonstrated forward elongation of the head and shoulders followed by retraction to the original position; Grooming which is the duration of time the mice spent licking or scratching itself while stationary; Freezing which is the duration of a mouse when it is completely stationary; Urination or the number of puddles of urine and defecation or the number of fecal boli produced. Different directions were conducted to test thedifferent parameters in the said test. The exam is a score-based test in each parameter and each score is

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

being tabulated (Bernas, 2005). The number of line crosses and the frequency of rearing are used as measures of locomotors activity and also measures of exploratory behavior and general activity. A high frequency of these behaviors indicates increased locomotion and exploration and/or a lower level of anxiety. The number of central square entries and the duration of time spent in the central square are measures of exploratory behavior and anxiety. A high duration of these behaviors indicates high exploratory behavior and low anxiety levels. Stretch attend postures are "risk-assessment" behaviors which indicate that the animal is hesitant to move from its present location to a new position (Blanchard, 2001) and thus a high frequency of these postures indicates a higher level of anxiety. Espejo (1997) states that grooming behavior is a displacement response and is expected to be displayed in a novel environment. Therefore grooming behavior should decrease with repeated exposure to the testing apparatus. Defecation and urination are often used as measures of anxiety (Lister, 1990). The collected measurements will be used to determine the neurophysiological effect of the extract to the laboratory rats. Gavage feeding Gavage is performed using a ball ended feeding needle. Estimate the distance that the needle needs to be inserted into the rat (usually from the nose to the first rib) and mark it on the needle. Restrain the rat with the rat extended in a straight line to facilitate introduction of the gavage needle. Introduce the needle

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

in the space between the left incisors and molars, and gently direct it caudally toward the right ramus of the mandible. The rat usually swallows as the feeding tube approaches the pharynx, facilitating entry into the esophagus. If the animal struggles or appears to be in respiratory difficulty withdraw the tube and begin all over again. Once the desired position is attained, inject the material and withdraw the syringe. Monitor the animal after the procedure to ensure that there are no adverse effects. Hot plate test The Hot Plate test performs rapid and precise screening of analgesic drug properties on small-laboratory animals. The animal pain sensitivity alterations induced by a specific experimental context change and/or genetic manipulations can also be evaluated through this method. Initially described by Leimback (1953), the hot-plate test evaluates thermal pain reflexes due to footpad contact with a heated surface. During the experiment, the mouse is introduced into an open-ended cylindrical space with a floor consisting of a heated plate. The plate heated to a constant temperature produces two behavioral components that can be measured in terms of their reaction times, namely paw licking and jumping. Both are considered to be supraspinally integrated responses. One particularity of this test is that it can only be performed once in each animal when the jumping response is evaluated. Indeed, when the animal is exposed to the first time to the test, it learns that the experimenter takes it out of the plate as soon as the jumping behavior is done. So, when the animal is place 9

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

again on the plate, it jumps after some few seconds without performing the primary licking responses. Mus musculus Mice (Mus musculus) are an important research tool for modeling human disease progression and development in the lab. Despite differences in their size and appearance, mice share a distinct genetic similarity to humans, and their ability to reproduce and mature quickly make them efficient and economical candidate mammals for scientific study. The laboratory mouse is a small mammal of the order Rodentia which is bred and used for scientific research. Laboratory mice are usually of the species Mus

musculus. Mice

belong to

the Euarchontoglires clade, which

includes humans. Mice are an important research tool for modeling human disease progression and development in the lab. Despite differences in their size and appearance, mice share a distinct genetic similarity to humans, and their ability to reproduce and mature quickly make them efficient and economical candidate mammals for scientific study. By late 18th or early 19th century, albino mice became one of the most commonly used experimental animals in numerous biomedical researches. They are recognized as the preeminent model in numerous fields, including neurobehavioral studies, psychology medicine and other scientific disciplines (Krinke, 2000). During the research experimentation, the behaviors and other activities of the mice were used as a tool to determine the potential of the fruit as a natural stimulant. 10

The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

Guava Psidium guajava (Guava) is a small tropical fruit in the family Myrtaceae. It is native to the Mexico or Central America and was distributed throughout tropical America and Caribbean region. It grows rapidly that the fruit typically measures 4 to 12 centimeters (1.6-4.7 inches) in diameter. Varying between species, the skin can be in any thickness, usually green before maturity, but becomes yellow when ripe. The pulp inside may be sweet or sour and off-white (P. guajava var. pyrifera) to red (P. guajava var. pomifera). The exterior of the fruit is fleshy, and the centre consists of a seedy pulp (Kaneria, et al., 2011). In many countries, guava is eaten raw, whereas in other countries it is eaten with a pinch of salt and pepper. It is known as the winter national fruit of Pakistan. Guava is a popular snack in Taiwan, sold on many street corners and night markets during hot weather. In Mexico, the guava beverage is popular. In the Philippines, ripe guava is used in cooking sinigang. The juice is often used in culinary sauces, as well as artisan candies, dried snacks, fruit bars and desserts. Guava juice is popular in many countries. The fruit is also often prepared in fruit salads. Because of its high level of pectin, guavas are extensively used to make candies, preserves, jellies, jams, and marmalades and also for juices. All parts of this tree, including fruits, leaves, bark, and roots, have been used for treating stomachache and diarrhea in many countries. Leaves, pulp and seeds are used to treat respiratory and gastrointestinal disorders, and as an

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

antispasmodic, anti-inflammatory, as a cough sedative, anti-diarrheic, obesity and in the control of diabetes mellitus. It also possesses anticancer properties (Ryu, 2012). The seeds are used as antimicrobial, gastrointestinal, anti-allergic and anti-carcinogenic activity (Bontempo et al., 2012). The fruit of guava is very rich in Vitamin C, which is substantially higher than what is found in citrus. It is also contains a different vitamins like vitamin b6, vitamin b12, carbohydrates, magnesium, placebo, vitamin C, vitamin D, folate, omega 3 fatty acids, omega 6 fatty acids and tryptophan. The fruit contains a large amount of citric, lactic, malic, oxalic and acetic acids. Nutrients found in the fruit helps in the stimulation of our body. 2.2 Related Studies According to Pareja (2014) in the study of the neuro-physiologicial effect of Stevia rebaudiana to Imprinted Control Mice (ICR) the physiological and behavioral test

for

CNS

shows that

the

three

doses

including

the

frequencies administered to the ICR mice have no significant differences which indicates that it has the same depressant effect. However, in the hot plate method for PNS the ICR mice belonging to T9 have longest time duration of reaction when the mice were subjected to heat and while in the dose, 0.9g of Stevia exhibited the greatest effect, therefore powdered S . proved

to

depress the

CNS

and

to

rebaudiana was

reduce sensitization to the PNS.

Treatment groups used were TO = Water; T1= 0.3 mg/ml of powdered S.

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

rebaudiana T2= 0.6 mg/ml of powdered S. rebaudiana, T3= 0.9 mg/ml of powdered S. rebaudiana, T4= twice a day (T1), T5= twice a day (T2), T6= twice a day (T3), T7= thrice a day (T1), T8= thrice a day (T2) and T9= thrice a day (T3). Results of the Open Field Test at different light intensities in C57 mice seem to favor the use of a light intensity similar to the light the animals are exposed to, for Open Field studies. Nonetheless we cannot rule out the hypothesis that a slightly brighter arena could be better for detecting mutant lines with an expected exploratory divergence. On the other hand there seems to be some divergences in the combined effects of light and treatment between male and female C57 mice, being the results obtained using males generally more consistent and predictable. Finally, our results emphasize the need to perform ethological analysis when studying animals in this maze, as some values obtained in the study could become statistically similar when the animals are in fact experiencing opposite mood states (Arenas and Pintado, 2014). Callaway

and

Geyer

(1992)

describe

that

3,

4

methylenedioxymethamphetamine has a stimulant effects in the nucleus accumbens of rat.

This study examined the behavioral effects in rats of

intracerebral administration of S (+)-3, 4-mcthylenedioxymethamphctamine (SMDMA) using an automated holeboard and open-field apparatus. Administration of S-MDMA into the nucleus accumbens septi produced locomotor hyperactivity. A similar increase in locomotor activity was observed after S-amphetamine

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The neuro-physiological effect of Psidium guajava (Guava) fruit extract as a natural stimulant to Mus musculus (Albino mice)

administration into the nucleus accumbens. These data indicate that S-MDMA actions in the nucleus accumbens are pharmacologically distinct from the primary effects of systemically administered S-MDMA. Behavioral effects of S-MDMA in the nucleus accumbens may result from the catecholamine-releasing properties that S-MDMA shares with S-amphetamine and not via the 5-HT-releasing properties that it shares with MBDB. The hot plate method in the study of Pareja et al., (2014) was done after one week of administration of S.rebaudiana in which the behavior of the mice was observed and examined. In this meth...