The in-Vitro Acetylcholine Antagonism Study of Tomato

Bronchial asthma is a chronic relapsing inflammatory disorder with increased responsiveness of tracheobronchial tree to various stimuli, resulting in paroxysmal contraction of bronchial airways. It refers to a condition of subjects with widespread narrowing of the bronchial airways which changes in severity over short periods of time, either spontaneously or under treatment, and is not due to cardiovascular disease According to the CDC, more than 22 million Americans, including 6.5 million children under 18, suffer with asthma today. Allergy clearly plays an important role in many asthma cases but not in all. Although drug-induced bronchospasm occurs most commonly in patients with known asthma, the agents themselves may produce asthma. The best-known of these is aspirin, but several other anti-inflammatory agents have been implicated.

Tomatoes are commonly used for cooking. They are rich in lycopene and glutamate which is why they are both healthy and delicious. Although tomatoes are perceived as vegetables, fact is, they are fruits. They are made in different forms such as tomato sauce, tomato juice, tomato soup, sun-dried tomatoes, canned tomatoes and many more. They contain such health benefits, one of which is its ability to treat asthma. So many people suffer from this kind of sickness, and sometimes, it causes an individual’s death. The medication for asthma is expensive, thus we, the researchers, decided to investigate whether the tomato juice extract can be used as a substitute anti-asthma medicine.

Statement of the Problem:
Basically, the problem of the researchers in this study is to determine whether there is any scientific basis for the folkloric claim that Solanum lycopersicum (Plum Tomato) possesses anti-asthma (bronchodilating) property comparable to the herbal standard bronchodilator, which is Vitex negundo (Lagundi aqueous) extract.

Objectives:

Specifically, it sought to:

1. Compare the antagonism effect of tomato given in different dose rates with acetylcholine treated duodenum of rats.
2. Determine the linearity effect of increasing dose rates of tomato against supramaximal contracted duodenum.
3. Compare the mean antagonism effect of different dose rates against the supramaximal contracted duodenum.
4. Compute for the ED 50 of the tomato fruit extract.

Significance of the Study:
The study seeks to prove the folkloric claim of tomato fruits or effective bronchodilator. When proven successful, this study will benefit the people who cannot afford to buy expensive anti-asthma medicines since tomatoes are abundant in the locality. Furthermore, there will be now more choices of indigenous plants that can relieve asthma, aside from the Indian Mango and Lagundi leaves.

Scope and Limitation:
This study is limited to the use of the tomato fruit extract of the “Plum” tomato variety. It is also limited to the determination of pharmacologic effect (bioassay) of a drug, an in-vitro bioassay type of study of tomato fruit extract using the duodenum of five (5) ICR Albino rats.

Hypotheses:
Null: There is no significant difference in the bronchodilating of tomato fruit extract and Lagundi.
Alternative: The tomato fruit extract is a better bronchodilator compared to Lagundi.

Chapter III
Methodology
Materials:

1. 2 kilos of tomato fruits
2. Acetylcholine concentration
3. Physiologic saline solution – Tyrode Solution consisting of solutions A and B

A :
Sodium Chloride or NaCl = 80 gms
Calcium Chloride or CaCl = 2 gms
Potassium Chloride or KCl = 2gms
Magnesium Chloride or MgCl² = 0.1gms

B :
Sodium Carbonate or NaHCO³ = 10gms
Sodium Phosphate or NaH2PO4 = 0.5gms

4. 8 liters of distilled water
5. 5 isolated rat duodenum tissue

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Apparatus:
1. Muscle warmer – container of the tissue (broken fluorescent bulb) 2. Oxygenator – oxygen tank, regulator anchor glass tubing 3. Constant temperature bath
4. 2 laboratory thermometers 0-50° C
5. Analytical balance
6. Glassware:
1000ml graduated cylinder
500ml beaker
Tuber culin syringe
Stirring rod
Glass tubing’s Y or T tube
7. Surgical apparatus:
Needle (1round, 1 curve)
Iris scissors (1 curved, 1 straight)
8. Electrical:
2 = 50 W2H bulb
2 = sockets (female)
9. Kymograph – electrical (1)
10. Heart lever
11. Clamps: pinchcock (3), screw clamp (3),
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Hoffman’s screw
12. Iron stand

Procedure:

1. Two kilograms of tomatoes were washed and squeezed off their juice using cloth. The juice extracted was further strained from foreign materials. 2. The Tyrode solutions and acetylcholine were prepared. 3. The Kymograph machine was set up by preheating at 37 degree centigrade and was maintained. 4. The rats were decapitated and excised of mere duodenum at approximate length of 25mm 5. Immerse duodenum specimen in Physiologic Saline Solution and carefully wash off content and cutting fatty tissues attached to the duodenum specimen. 6. Tether by polyester thread the duodenum specimen and anchor it to the lever accordingly.

7. Allow 1 ½ hour stabilization time and wash it off several times until a constant baseline is  obtained. Explore for the supramaximal dose of acetylcholine using dose rates as: 0.2ml, 0.4ml, 0.8ml and 1.6ml only. E.g. 0.2ml from concentration of 1:1,000 acetylcholine solutions. Allow a 1-minute reaction time after washing it to the baseline. 8. Start testing for inhibition and measure in mm amplitude for 3 minutes using dose rates at 0.2ml/50ml tyrode, 0.4ml/50ml tyrode, 0.8ml/50ml tyrode and 1.6ml/50ml tyrode respectively. If from any of those rates cause 50% amplitude of inhibition, end up for another duodenum 9. Compute for the Percent Amplitude Inhibition (Mean Percent Inhibition) rate and plot on a log probit paper and compare it to the standard curve. Test drug passes ED50 standard if it falls within – or + 25% range.

Bibliography:

○http://www.histopathology-india.net/BrAs.htm

○http://www.webmd.com/asthma/guide/bronchial-asthma

○http://respiratory-lung.health-cares.net/bronchial-asthma.php

○Past investigatory projects at the High School Library

○http://www.wikipedia.com/tomatoes.html

Chapter II
Review of Related Literature
Bronchial asthma is a disease caused by increased responsiveness of the tracheobronchial tree to various stimuli. The result is paroxysmal constriction of the bronchial airways. Bronchial asthma is the more correct name for the common form of asthma. The term ‘bronchial’ is used to differentiate it from ‘cardiac’ asthma, which is a separate condition that is caused by heart failure. Although the two types of asthma have similar symptoms, including wheezing (a whistling sound in the chest) and shortness of breath, they have quite different causes.

Bronchial asthma is a disease of the lungs in which an obstructive ventilation disturbance of the respiratory passages evokes a feeling of shortness of breath. The cause is a sharply elevated resistance to airflow in the airways. Despite its most strenuous efforts, the respiratory musculature e is unable to provide sufficient gas exchange. The result is a characteristic asthma attacks,  with spasms of the bronchial musculature, edematous  swelling of the bronchial wall and increased mucus secretion. In the initial stage, the patient can be totally symptom-free for long periods of time in the intervals between the attacks. As the disease progresses, increased mucus is secreted between attacks as well, which in part builds up in the airways and can then lead to secondary bacterial infections.

Bronchial asthma is usually intrinsic (no cause can be demonstrated), but is occasionally caused by a specific allergy (such as allergy to mold, dander, dust). Although most individuals with asthma will have some positive allergy tests, the allergy is not necessarily the cause of the asthma symptoms. Symptoms can occur spontaneously or can be triggered by respiratory infections, exercise, cold air, tobacco smoke or other pollutants, stress or anxiety, or by food allergies or drug allergies. The muscles of the bronchial tree become tight and the lining of the air passages become swollen, reducing airflow and producing the wheezing sound. Mucus production is increased.

Typically, the individual usually breathes relatively normally, and will have periodic attacks of wheezing. Asthma attacks can last minutes to days and can become dangerous is the airflow becomes severely restricted. Treatment is aimed at avoiding known allergens and controlling symptoms through medication. A variety of medications for treatment of asthma are available. People with mild asthma (infrequent attacks) may use inhalers on an as-needed basis. Persons with significant asthma (symptoms occur at least every week) should be treated with anti-inflammatory medications, preferably inhaled corticosteroids, and then with bronchodilators such as inhaled Alupent or Vanceril. Acute severe asthma may require hospitalization, oxygen, and intravenous medications. Tomatoes:

Most people realize that tomatoes are healthy for them that they have a number of important substances that help us stay away from cardiovascular disease, cancers, cataracts and other health concerns. We are finding that tomatoes contain so many vital nutrients and we are coming to  realize how important it is to have tomatoes as part of a health diet. When you eat tomatoes, it is the color in them that makes them so good for you. The color comes from lycopene which is also found to be an incredible antioxidant. Antioxidants block the effect of oxygen free radicals in the cells and therefore, block the damage they can do to cells that cause cancer in the right circumstances.

This is why tomatoes appear to have cancer-fighting properties. The amazing thing about lycopene is that it is about twice a good as many of the other antioxidants in foods. It has been found by research to be effective in preventing breast cancer, lung cancer and prostate cancer. Lycopene is effective against aging as well. A plum tomato (Solanum lycopersicum), also known as a processing tomato or paste tomato, is a type of tomato bred for sauce and packing purposes. They are generally oval or cylindrical in shape, with significantly fewer seed compartments than standard round tomatoes (usually only two) and a generally higher solid content, making them more suitable for processing.

Plum tomatoes are also sometimes favored by cooks for use during the tomato off-season, as they are generally considered more amenable to handling and are therefore available in a state closer to ripe than other supermarket tomatoes. According to researches around the WWW(World Wide Web),tomatoes have the ability to cure asthma. Although, researchers haven’t done any study yet to prove that tomatoes are really capable of curing asthma, and so it remains as a folkloric claim. Lagundi (scientific name: Vitex negundo) is a shrub that grows in the Philippines. It is one of the ten herbal medicines endorsed by the Philippine Department of Health as an effective herbal medicine with proven therapeutic value.

Commonly known in the Ilocos region as dangla, lagundi has been clinically tested to be effective in the treatment of colds, flu, bronchial asthma, chronic bronchitis, and pharyngitis. Studies have shown that Lagundi can prevent the body’s production of leukotrienes, which are released during an asthma attack. Lagundi contains Chrysoplenol D, a substance with anti-histamine and muscle relaxant properties. Even in Japan, lagundi is becoming recognized as an effective herbal medicine, especially since researches have shown that it contains properties that make it an expectorant and it has been reported to function as a tonic as well. More than that, most of the parts of the lagundi plant are associated with medicinal value. The roots of this shrub are also used as treatment for rheumatism, dyspepsia, boils, and leprosy. The leaves, flowers, seeds, and root of Lagundi can all be used as herbal medicine.

Chapter IV
Results and Discussion
The chapter presents the data obtained, and the analysis of this data.
Table 1 shows the mean percent antagonism of the different treatments of tomato against the acetylcholine supramaximal contracted duodenum of rats. Table 1. Mean Percent Antagonism to the Acetylcholine Contracted Duodenum with Different Application of Tomato Treatments.

Treatments Mean Percent Antagonism|
Treatment 133.6
Treatment 248.2
Treatment 367.2
Treatment 490.4
As seen in Table 1, the highest antagonism was obtained with treatment 4 (1.6ml / 50ml PSS) with a mean result of 90.4 and the lowest was obtained with treatment 1(0.2ml/ 50ml PSS) at 33.6.

Source of variance| DF| SS| Ms| F- Ratio| |
Between treatments| 4| 80,701| 20,175.25| 5.75HS| 3,014.77| Between replications| 16| -56,106.36| 3506.64| | | Total| 20| | | | |

Table 3 shows the Analysis of variance. This shows a significant F ratio of 5.75. This means that the different treatments of tomato had different effect on percent antagonism. Figure 1 shows that the increasing dose had a corresponding increase effect on percent antagonism. The ED 50 was found to be 0.8ml / kg.

The effective antagonism effect to the acetylcholine contracted duodenum suggests that the mechanism by which asthmatic attacks caused by acetylcholine contracting the bronchioles is relieved by application of bronchodilating agents. In this study, the antagonism to the acetylcholine medicated duodenum of rats corroborated the phenomenal relief of asthma attacks.

Chapter V
Conclusion:

Based on the result of the study, the following conclusions were drawn:

1. Different dose rates of tomato application had different effect on percent antagonism affect with highest effect of 90.4 (1.6ml / 50ml PSS). 2. the lowest effect was obtained at 0.2ml / 50ml PSS at 33.6 3. Increase of dose rates increases the percent antagonism effect 4. The ED 50 is 0.8 ml / 50ml PSS

Recommendations:

1. It is strongly recommended that replications of this investigatory project should be done to prove consistency of results thus strengthening the conclusion that the extract of the Tomato Fruit are good.

2. It is also recommended that another investigatory project related to this study must be done which aims to determine the Oral LD 50 (Lethal Dose 50) but this time it is a Quantitative Bioassay In-Vivo Type of Experiment. Lethal Dose 50 means the does by which 50% of the experimental animals will die. There is as yet no Animal Toxicity Studies that were done on the tomato fruit.

The In-Vitro Acetylcholine Antagonism Study of Tomato (Solanum lycopersicum) compared to Lagundi (Vitex negundo L) Using Rat Duodenum

An Entry to the Division Science Fair
September 9-10,2010

Cluster I – life Science
Team Category

Fatima Jamsuri
Judiel Bugnay
Josephine Dumlao
(Proponents)

Mrs. Rita Daliwag
(Adviser)
Abstract:

In this study the over-all objective is to determine and compare the bronchodilating effect of Tomato fruit extract with that of Lagundi leaf extract using isolated tissue experinment. This study utilizes an experimental design, which is a quantitative type of bioassay in-Vitro type of experiment. The depressant or relaxing effect(bronchodilator effect)of one hundred percent decoction or aqueous extract of Tomato fruit extract(Solanum Lycopersicum)on Five(5)supramaximally contracted isolated rat duodenums was determined and was compared with that of the depressant or relaxing(bronchodilator effect) of one hundred percent decoction or aqueous extract of Lagundi leaves (Vitex negundo L.) Acetylcholine was used to supramaximally contract the duodenum.

Increasing doses of 100% tomato fruit aqueous extract was applied to the isolated rat duodenum, which was suspended in a physiological salt solution, and the depressant effect was determined which can be measured by the length of relaxation in millimeters starting from the maximum height of supramaximal contraction also in millimeters. The percent inhibition in the entire five(5) rat duodenums was then computed for each of the four(4) dose levels(0.2ml, 0.4ml, 0.8ml, and 1.6ml). Percent inhibition was determined by dividing the length of relaxation by the length of supramaximal for each contraction for each dose level. Finally, the mean percent inhibition for each dose level of five(5) rat duodenums was computed and plotted in a dose response curve. The dose response curve of 100% tomato fruit extract or aqueous extract was compared to that of the standard dose response curve of 100% Lagundi leaves extract or aqueous extract.