Anti-Mutagenic Potential of Gotu Kola (Centella Asiatica) On Onion Root Cells and In Vivo Micronucleus Test of Liver cells

in Rodent

CHAPTER I INTRODUCTION Background of the Study: Cancer is one of the leading causes of death in the Philippines. In fact, according to the journal article, Cancer and the Philippine Cancer Control Program, it ranks third in leading causes of morbidity and mortality after communicable diseases and cardiovascular diseases (Ngelangel & Wang, 2001). Furthermore, cancer rates could further increase by 50% to 15 million new cases in the year 2020, based on the World Cancer Report. This projection may become a reality if the current state of low cancer prevention consciousness among the people persists. Cancer is caused by mutagens, physical or chemical agents that alter the genetic material of cells, usually DNA, of an organism above its natural supposed structure. There is increasing evidence that mutation in somatic cells are not only involved in the carcinogenesis but can also cause genetic disorders like

atherosclerosis, heart diseases and several other degenerative disorders. Since, the mutagens are involved in the initiation and promotion of several human diseases, including cancer, the significance of novel bioactive phytocompounds in

counteracting these pro-mutagenic and carcinogenic effects is now gaining credence. Such chemicals that reduce the mutagenicity of physical and chemical mutagens are referred to as antimutagens. (Bhattacharya, 2010).

The use of antimutagens and anticarcinogens in everyday life is an effective procedure for preventing human cancer and genetic diseases. The action of mutagens can be reduced or prevented in several ways. (Qari) One of the best ways to minimize the detrimental effects of mutagens is by the use of natural antimutagens. These include flavonoids, phenolics, coumarins, carotenoids, anthraquinones, tannins, saponins and many more (Bhattacharya, 2010). Gotu Kola is a perennial plant native to India, Japan, China, Indonesia, South Africa, Sri Lanka, and the South Pacific. It thrives abundantly in most localities in Samar and Leyte especially along the grounds of moist soil. In one of the studies published in the Journal of Medicinal Food (2002), it was concluded that soybeans that contain saponins have anti-mutagenic potential against aflatoxin B1-induced mutagenicity and DNA-adduct formation. The study concluded that soybean saponins possess a significant anti-mutagenic activity. Since Gotu Kola is known to have saponins, as mentioned in one study entitled Studies on phytochemical constituents of six malaysian medicinal plants, the researchers were able to formulate a problem that will focus on the potential of Gotu Kola as anti-mutagenic agent against a proven mutagen, Sodium Azide, using the Allium cepa test method. The potential for Gotu Kola as an antimutagen is not limited to the mutagenicity of Sodium Azide. There is a great possibility of it countering tetracycline hydrochloride, a known mutagen in animals. The researchers therefore included an in vivo rodent erythrocyte micronucleus assay on tetracycline hydrochloride-induced mice to assess its capability as an antimutagen in fields, animal and plant.

Statement of the Problem Main Problems:

1) Does the Gotu Kola extract have an anti-mutagenic effect on onion root
cells that were exposed to Sodium Azide based on the number of mutated cells of the meristematic region of the onion root tips?

2) Does the Gotu Kola extract have anti-mutagenic effect against tetracycline
hydrochloride induced mice based on the number of micronucleated

polychromatic erythrocyte in the Liver?

Sub-Problems: 1. What treatment will be a more effective anti-mutagen on the onion root cells?

a. b. c. d. e. f. g. h.

300 g/mL 25% Gotu Kola Extract 300 g/mL 50% Gotu Kola Extract 300 g/mL 75% Gotu Kola Extract 300 g/mL 100% Gotu Kola Extract 500 g/mL 25% Gotu Kola Extract 500 g/mL 50% Gotu Kola Extract 500 g/mL 75% Gotu Kola Extract 500 g/mL 100% Gotu Kola Extract

2. Is there a significant difference in the number of mutated cells in the meristematic

region of the onion roots between the Gotu Kola extract concentrations in:

a. b.

300 g/mL NaN3 500 g/mL NaN3

3. At what percentage will the Gotu Kola extract, show greater potential as an antimutagen, based on the number of micronucleated polychromatic erythrocyte in the Liver, to tetracycline-induced mice?

a. b. c.
d. Hypothesis

25% Gotu Kola extract 50% Gotu Kola extract 75% Gotu Kola extract 100% Gotu Kola extract

Main Problems: H1: If the Gotu Kola extract has an anti-mutagenic effect on onion root

cells that were exposed to Sodium Azide, then there would be a significant decrease in the number of abnormal cells in the meristematic cells of onion as compared to the control. H2: If the Gotu Kola extract has an anti-mutagenic effect on mice, then there would be lesser micronuclei in its Liver as compared to the control. Sub-Problems: H3: If the concentration of the Gotu Kola extracts increases, then its capability as an anti-mutagen would also increase. H4: If the 100% Gotu Kola extract concentration will be used, then the number of micronucleated PCE in the Liver of the mice would be lesser as compared to the control.

Objectives: General Objective To determine whether the Gotu Kola extract has an anti-mutagenic effect on onion roots cells that were exposed to Sodium Azide and on tetracycline-induced mice.

Specific Objectives

1. To identify which concentration of the Gotu Kola extract will prove to be the more
efficient anti-mutagen in: a. b. 300 g/mL NaN3 500 g/mL NaN3

2.

To verify if there is a significant difference of the number of mutated cells in the

meristematic region of the onion roots between the Gotu Kola extract concentrations in:

a. b.

300 g/mL NaN3 500 g/mL NaN3

percentage

3. To determine what

of the Gotu Kola extract show greater potentials as an

anti-mutagen to tetracycline-induced mice based on the number of micronucleated polychromatic erythrocyte in the liver.

a. b. c.
d.

25% Gotu Kola extract 50% Gotu Kola extract 75% Gotu Kola extract 100% Gotu Kola extract

Significance of the Study Once this study is proven, it will benefit: 1. People who are exposed to high levels of mutagens - This supposed anti-mutagen will serve as a preventive measure to people who are susceptible to cancer and other such diseases. This would help lower, if not eliminate,

the risk for acquiring these mutation related diseases. This would be a better alternative for these people because other ways of prevention such as

chemoprevention is expensive and will require exposure to radiation. The Gotu Kola, on the other hand, is inexpensive and low maintenance. 2. Agricultural Investors and Producers - This discovery will provide them a means in providing them a livelihood. This study will also benefit the producers of Gotu Kola because of their newfound use, which may present to be very profitable. 3. Researchers- This study would provide new grounds for research on the capabilities of the Gotu Kola as an anti-mutagen, widening the knowledge of other researchers on the potential of plants related to the Gotu Kola. This would encourage the researchers to find new ways to incorporate the Gotu Kola in commercial medicines by manufacturing supplements and capsules.

Scope and Limitations This study will focus on the anti-mutagenic potential of Gotu Kola against Sodium Azide on onion root cells and on liver cells in mice. This investigation will also focus on determining what concentration of the Gotu Kola extract is a more effective anti-mutagen. The study will be conducted in PSHS-EVC Laboratory. The Gotu Kola plant and Allium cepa, commonly known as onion, will be bought from a local nursery in Tacloban City and will be identified by an agricultural technician from the Department of Environment and Natural Resources (DENR). The tetracycline hydrochloride will be bought from a local drugstore. The laboratory mice will be acquired from Leyte Marine

Biotoxins Testing Center (LMBTC) in Tacloban City. The mutagen, Sodium Azide (NaN3), will be acquired from Yana Chemodities Incorporated in Quezon City, Manila. The study will use the Allium cepa Test Method to verify the anti-mutagenic properties of Gotu Kola. All equipments that will be used aside from some reagents and mice cages are present in laboratory. Proper protocol in dissection and handling laboratory mice will be followed.

Definition Of Terms Gotu Kola plant to be used in which its extracts would be the antimutagen. The term pertains to the extracts of Centella Asiatica. Azide - this term refers to Sodium Azide (NaN3) that would be used as the mutagen in in vitro experiment of onion root meristematic cells. Allium Cepa Test Method method that uses onion (Allium cepa) for the in vitro experiment of onion root meristematic cells Tetracycline- term refers to the tetracycline hydrochloride that would be used as the mutagen for the in vivo rodent micronucleus assay.

Micronucleus- an acentric chromosome fragment detaching from a chromosome after breakage which does not integrate in the daughter nuclei.

CHAPTER II REVIEW OF RELATED LITERATURE

Gotu Kola (Centella Asiatica) Centella Asiatica, also known as the Gotu Kola or Indian Pennyworth, is found mainly in gardens, thickets, and open, damp grasslands, on rice paddy banks and streams throughout the Philippines. (Philippine Medicinal Plants) Any place that is very damp and very moist could be an appropriate place in which the Centella

Asiatica would thrive. It has seven essential phytochemical constituents namely Tannins, phlobatannins, saponins, flavonoids, terpenoids, cardiac glycosides, and alkaloids (Krishnaiah, Devi, Bono, & Sabatly, 2009). It has also vallarine, asiaticoside, hydrocotylin, pectic acids, steroids, hersaponin, bacogenin, monnierin, and triterpene. (Sathya & Ganga) Because of these chemical components found in the plant, it is used as an herbal medicine for burns, psoriasis, episiotomy, and for external fistulas. It also lowers sugar levels and blood pressure in the body (Hawkins & Ehrlich, 2006)

Sodium Azide Sodium Azide is highly mutagenic to barley, yeast, and other plants, even though it is not known to induce mutagenicity in human lymphocytes because of the enzyme converting azide to non-genotoxic azidoalanine (Ragunathan &

Panneerselvam, 2007). This, as well as it being a point mutagen, is a contributing factor to its efficacy as a factor for inducing chromosomal aberrations (Stanton, Dotson, & Somers). The potential of Sodium Azide as a mutagen to plant tissues increases as its concentration increases. Therefore, the researchers propose that there be three different concentrations of Sodium Azide to be used in this study.

Tetracycline Tetracycline is an antibiotic used for bacterial infections, such as the urinary tract infections, acne, gonorrhea, Chlamydia, and others. (Drugs.com) Tetracycline harmless as it may seem can also cause harm especially to unborn babies. Studies

show that doses of Tetracycline higher than 0.1mL could cause lethal effects on rodents. (Amano) This indicates that tetracycline could cause lethal effects on rodents at very high doses.

Liver Micronucleus Test (Hematoxylin-eosin staining) According to the study conducted by Amano, Golo, and Tan, entitled Antimutagenic effects of Ampalaya plant extracts against tetracycline-induced mice based on PCE production., the micronucleus test is used to determine information about a chemicals ability to disrupt chromosome structure and function. It is an assay used to determine whether the material to be tested underwent more mutation as compared to other compounds. This study will use the in vivo method but instead of using the bone marrow as the source for the micronucleated cells, liver cells of the affected mice would be taken and be stained using hemotoxylin-eosin. Specifically, this study will perform the Micronucleus test in liver cells- assessing the number of micronuclei to normal liver cells. According to the study entitled, Anti-mutagenic effects of Ampalaya plant extracts against tetracycline-induced mice based on PCE production, when the chromatin material is fragmented by the mutagen, some fragments will be left behind after red blood cells eject the nucleus after telophase. During cell division, the DNA replicates and then divides equally between the two cells that are produced. If the process is disrupted, or certain chemicals damage the chromosomes, then the distribution of genetic material between the two cells produced will have a new nucleus that may form its own micronucleus which is clearly visible in the microscope. (Golo, Amano, & Tan)

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The micronucleus is the third nucleus that is formed during meiosis or mitosis. It contains a portion or whole chromosome that was not pulled to the opposite poles during anaphase. This indicates a mutation in the compound. Since the chromosomes shape, size and number are constant in a species, it is easy to see a mutation occurring from looking for differences. (Golo, Amano, & Tan) In addition, micronucleus contains no distinct nucleus so it would be easier to locate and distinguish from the normal cells. If a treated group of mice shows significantly higher frequencies of micro nucleated cells as compared to the untreated control animals, then the chemical is considered a mutagen. This micronucleus test in liver cells will be used in the evaluation of the Gotu Kolas efficacy as an anti-mutagen.

Anti-Mutagens Several authors have suggested that natural antimutagens may belong to any of the following major class of compounds. Major emphasis has been laid on the flavonoids, phenolics, carotenoids, coumarins, anthraquinones, tannins, terpenoids, saponins and several others all of which are secondary plant metabolites. More than 500 compounds belonging to at least 25 chemical classes have been recognized as possessing anti-mutagenic/protective effects (Bhattacharya, 2010). Scientists have found that saponins, among several other class compounds including flavonoids, phenolics, carotenoids, coumarins, anthraquinones, tannins, terpenoids, and several others, can be natural antimutagens.

11

Hematoxylin-Eosin Staining According to the study Hematoxylin and Eosin Staining of Tissue and Cell Sections of Fischer, Jacobson, Rose, and Zeller. Hematoxylin-eosin staining has been used by many since it is still an effective way of recognizing various tissue types and morphological changes that form the basis of contemporary cancer diagnosis. It has also the ability to demonstrate a wide range of normal and abnormal cell and tissue components. Hematoxylin has deep blue-purple stains while Eosin has pink stains. Cells when stained, show blue stained nuclei and pink stained cytoplasm and extra cellular matrix.

http://cshprotocols.cshlp.org/content/2008/5/pdb.prot4986.abstract

RELATED STUDIES: Medicinal foodstuffs. XXVII. Saponin constituents of Gotu Kola (2):

structures of new ursane- and oleanane-type triterpene oligoglycosides, centellasaponins B, C, and D, from Centella asiatica cultivated in Sri Lanka. Matsuda H., Morikawa T., Ueda H., Yoshikawa M. In this study, the researchers isolated ursane and oleananetype triterpene oligoglycosides, saponins B, C, and D, from the aerial parts of urban cultivated Gotu Kola (Centella asiatica (L.)) in Sri Lanka. They also isolated other components of Gotu

12

Kola such as madecassoside, asiaticoside, asiaticoside B, and sceffoleoside A. The plant was tested for the different structures of the component, saponin. Since saponins B, C, and D were isolated from the plant, this conforms the idea that the Gotu Kola does, in fact, contain saponins. Without saponins, the Gotu Kola would not be an applicable variable in our study for there would be little or no antimutagenic properties of the Gotu Kola leaving the study unsuccessful.

Protective effect of soybean saponins and major antioxidants against aflatoxin B1-induced mutagenicity and DNA-adduct formation Jun HS, Kim SE, Sung MK. This study tested the efficacy of saponins from soybeans as an antimutagen against the mutagenic aflatoxin B (1) (AFB (1)) and DNA adduct synthesis through Salmonella typhimurium. The bases for the evaluation of their tested saponin are antioxidants including L-ascorbic acid and alpha-tocopherol, which are reported to be effective as antimutagens. The results of this research concludes that the changes brought about by saponins as an antimutagen were significant compared to those displayed by alpha-tocopherol and L-ascorbic acid countering the mutagenicity by 52%, 64%, and 81% at concentrations of 600, 900, and 1,200. In addition, saponins inhibit the growth af carcinogeninduced mutagenic activity and prevents the initiation of carcinogenesis. This study mentioned, is related in our study in such a way that it gives the researchers information that soybean saponins displayed anti-mutagenic potential against aflatoxin B1-induced mutagenicity and DNA-adduct formation. Furthermore, according to the study, given that these saponins inhibit the growth of carcinogen-induced mutagenic activity and prevent the initiation of carcinogenesis,

13

the Gotu Kola plant, proven to contain saponins, would have a very high chance of success in our study.

Anti-mutagenic potential of Curcumin on chromosomal aberrations in Allium Cepa Irulappan Ragunathan and Natarajan Panneerselvam Research Center and Postgraduate Studies in Botany, the Madura College, Tamil Nadu, India In this study, they used the curcumin from turmeric, a spice and food colouring agent in Asia, as an antimutagen against mutations in the chromosomes in onion roots meristem cells to prevent carcinogens from spreading or beginning. The study used Sodium Azide as the mutagen, causing significant chromosome aberration with increasing concentrations. The study reveals that curcumin has antimutagenic potential against Sodium Azide induced chromosomal aberrations in Allium cepas root meristem cells. In addition, it showed mild cytotoxicity by reducing the percentage of mitotic index in all curcumin treated groups, but the mechanism of action remains unknown. (Ragunathan & Panneerselvam, 2007) With this study, we can assure that our proposed study can use Sodium Azide as the mutagen in the meristem cells of onion root cells. In addition, the researchers would be able to use the allium cepa test method based on this study. Effect of tetracycline on cultured mouse cells T. Tsutsui, M. Umeda, M. Sou, H. Maizumi Department of Pharmacology, Nippon Dental College, Fujimi, Chiyoda-ku, Tokyo 102 Japan

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In this study, it concludes that tetracycline hydrochloride being a mutagen is much more effective to mammals than it is to bacteria. The researchers subjected FM3A cells to the tetracycline hydrochloride and damage to the chromosomes of the cell, aberration, and inhibition of the syntheses of nucleic acids and protein were observed.

Our study proposes to use tetracycline hydrochloride to induce mutation in the cells of mice in order to set the base for our experiment regarding Gotu Kola extract which will then supposedly inhibit the development of this mutation. With the information that tetracycline hydrochloride is a potent mutagen in mice, as mentioned in the mentioned related study, it would be used as the mutagen in our study on the mice to be experimented on.

Hematoxylin and Eosin Staining of Tissue and Cell Sections Andrew H. Fischer, Kenneth A. Jacobson, Jack Rose and Rolf Zeller Hematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. The Golgi zone 15

can be tentatively identified by the absence of staining in a region next to the nucleus. Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed. Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.

CHAPTER III Methodology

I.

Anti-mutagenic Potential of Gotu Kola Extract (Centella

Asiatica) on Onion Root Cells

16

Research Design This research study will have two control set-ups; negative control and positive control, and one experimental set-up. This study will focus on the antimutagenic potential of Gotu Kola extract on onion root cells. For the negative control, three containers containing onion root tips will be exposed to water and will be subjected to microscope examination. For the positive control, a new set of six onion root tips will be exposed to the different concentrations of Sodium Azide; 300g/mL, 500g/mL. After two days, all onion root tips will be subjected for microscope examination. For the experimental set-up, a new set of onion root tips will be exposed to different Gotu Kola concentrations; 25%, 50%, 75%, 100%. After two days, the onion roots will be exposed to different concentrations of Sodium Azide; 300g/mL, 500 g/mL,. After another two days, all onion root tips will be subjected to microscope examinations. Procedure: Extraction of Gotu Kola Leaves of Gotu Kola would be collected from Tacloban City . These leaves will be chosen in such a way that each leaf is free from spots, cuts, tears, and discoloration. The collected leaves would then be washed with running water. In order to remove excess water from leaves, they would be blot dried using tissue paper. After this, all leaves would be placed in a blender with ethanol as the solvent for the extraction with the ratio 1:2 (mass of leaves is to volume of ethanol) and would be blended.

17

The blended leaves would be put in a refrigerator overnight. After 24 hours, the mixture would be filtered with the use of cheesecloth and filter paper, twice. To separate the extract from the solvent- ethanol, a rotary evaporator would be used. Once the extract is free from ethanol, it would be prepared with concentrations 25%, 50%, 75%, 100% and would be stored in three separate vials until further use.

Growing of Onion Roots Onion bulbs which appear to have approximately the same sizes are collected from the Tacloban City Dry Market and from the Marasbaras Public Market. The onion bulbs are to be washed with running water and are to be dried using tissue paper. Using a scalpel, old and dry scales would be removed. Then, the onion bulbs would be placed in small beakers with 50 mL and would be allowed to grow for 2 days in room temperature. Nine onion bulbs will be used for the negative, positive, and experimental set-up all together.

Negative Control Onion roots will be grown in five onion bulbs for two days in distilled water and will be used in this control. All roots of each onion bulb will be put into one vial for storage. Root tips near the meristematic region of the roots are to be cut for microscopic analysis.

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Table for the Negative Control BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells 200/0 BULB 2 (must 200 have cells) 200/0 H2O BULB 3 (must 200 have cells) 200/0 BULB 4 (must 200 have cells) 200/0 BULB 5 (must have

200 200 cells)

200/0

Positive Control Different concentrations of Sodium Azide are to be exposed to two-day grown onion roots which are two cm in length. These concentrations are 300g/mL and 500g/mL. All roots from the five bulbs will be exposed to these concentrations. Roots are to be exposed for 48 hours or 2 days. After that, root tips near the meristematic Region would be cut for microscopic examination.

19

Table for the Positive Control BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells 300 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must 200 have cells) (must 200 have cells) (must 200 have cells) BULB 5 (must have

200 200 cells)

BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells

500 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must (must 200 have cells) (must 200 have cells)

BULB 5 (must have

200 have cells)

200 200 cells)

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Experimental Set-up When the 48-hour onion roots reach two cm in length, they are to be transferred and would be exposed to different concentrations of Gotu Kola extract,100%, 75%, 50%, and 25%, for two days. After the 48-hour extract exposure, the root tips would then be exposed to different concentrations of Sodium Azide, 300 g/mL and 500 g/mL, for another two days or 48 hours.All roots from the five bulbs would then be subjected to microscope analysis. This process is performed in order to confirm whether the Gotu Kola extract would be effective enough to prevent and lessen mutagenic actions of Sodium Azide.

Table for 25% Gotu Kola Extract BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells 300 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must 200 have cells) (must 200 have cells) (must 200 have cells) BULB 5 (must have

200 200 cells)

21

BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells

500 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must (must 200 have cells) (must 200 have cells)

BULB 5 (must have

200 have cells)

200 200 cells)

Table for 50% Gotu Kola Extract BULB 1 (must have cells) 300 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must 200 have cells) (must 200 have cells) (must 200 have cells) BULB 5 (must have

200 200 cells)

22

Number of Healthy cells/Unhealt hy cells

BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells

500 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must (must 200 have cells) (must 200 have cells)

BULB 5 (must have

200 have cells)

200 200 cells)

Table for 75% Gotu Kola Extract BULB 1 (must have cells) 300 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must 200 have cells) (must 200 have cells) (must 200 have cells) BULB 5 (must have

200 200 cells)

23

Number of Healthy cells/Unhealt hy cells

BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells

500 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must (must 200 have cells) (must 200 have cells)

BULB 5 (must have

200 have cells)

200 200 cells)

Table for 100% Gotu Kola Extract 300 g/mL of Sodium Azide

24

BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells

BULB 2 (must

BULB 3 (must

BULB 4 (must

BULB 5 (must have

200 have cells)

200 have cells)

200 have cells)

200 200 cells)

BULB 1 (must have cells) Number of Healthy cells/Unhealt hy cells

500 g/mL of Sodium Azide BULB 2 BULB 3 BULB 4 (must (must 200 have cells) (must 200 have cells)

BULB 5 (must have

200 have cells)

200 200 cells)

Preparation of Slides

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The cut roots would be put into slides. These slides are placed in vials having 37% Formalin Solution for 1 hour. Different vials were used for every concentration in the positive and experimental set-ups. The roots would be then exposed to 2 drops of 1M EDTA Solution for 25 minutes. Excess EDTA solution was removed using tissue paper. Then they were stained with acetoorcein stain for 30 minutes. After that, the roots would now be macerated, using the Squash Technique, and were to be labeled accordingly. Microscopic Examination For the microscopic examination of the onion root cells, the researchers will set the parameters to the meristematic cells of the root tip and will only count the first 1000 mitotic cells that will be identified as normal or abnormal cells. Using the compound microscope at the PSHS-EVC laboratory, the cells will be manually counted.

II.

Anti-mutagenic Potential of Gotu Kola Extract (Centella Asiatica) In

Vivo Rodent Erythrocyte Micronucleus. Research design The methods used by Amano, Golo, and Tan are used also in this study with slight modifications. A dosage of 0.1mL for each Gotu Kola Extract concentration was introduced in mice, which are preliminarily induced with 0.1mL of Tetracycline Hydrochloride, using the Gavage Method. The Anti-mutagenic effect of Different Concentrations of Gotu Kola Extract will be measured using the number of Micronucleated Immature Erythrocytes out of 2000 immature erythrocytes per mouse but instead of using the bone marrow of these mice, liver cells would be taken and be counted for the presence of micronuclei

26

Procedure: Acquisition of experimental animals Mice that will be used in this process would be provided by the Leyte Marine Biotoxins Center from their office in Government Center, Palo, Leyte but only with the factors of age and size taken into consideration. Fifteen mice will be used.

Preparation of crude extracts Leaves of Gotu Kola would be collected from Tacloban City. These leaves to be collected will be chosen in such a way that each leaf is free from spots, cuts, tears, and discoloration. The collected leaves would be then, washed with running water. In order to remove excess water from leaves, and be blot dryed using tissue paper. After this, all leaves would be placed in a blender with ethanol as the solvent for the extraction with the ratio 1:2 (mass of leaves is to volume of ethanol) and would be blended. The blended leaves would be put in a refrigerator overnight. After 24 hours, the mixture would be filtered with the use of cheesecloth and filter paper, twice. To separate the extract from the solvent, ethanol, a rotary evaporator would be used. Once the extract is free from ethanol, it would be prepared with concentrations 25%, 50%, 75%, 100% and would be stored in three separate vials until further use.

Preparation of Test Animals Fifteen mice will be used in this study. These mice are separated into 5 groups. 3 groups will be used for the experimental set-up, one group for the positive control, which would be injected with 0.1mL of tetracycline only, and another group for the negative control, which would not be injected with anything. Each group has 3

27

mice. The mice that would be used in the experimental set-up are made to fast 12 hours prior to the conduct of the Gavage.

Induction of tetracycline Amano, Golo, conducted preliminary inductions of Tetracycline Hydrochloride and Tan in order to find out the tolerable dosage since the weight of each mouse is not specifically determined. They found out that the maximum tolerable amount of tetracycline in mice is 0.1mL. Similarly, the dose of 0.1 mL will be used in this study since factors that were only considered in choosing the mice are the age and the size. 1cc/mL tuberculine syringes would also be used.

Intraperitoneal injection The procedures found in the Laboratory Animal Workshop Manual of the Biological Research and Development Laboratory for Intraperitoneal Injection would be followed. First the mouse will be restrained and the injection site will be wiped with cotton soaked alcohol (70% will be used in this research). Then the needle will be inserted to 1 inch midway of the abdominal cavity. The needle must not hit any visceral organs or blood vessels. Then the plunger of the syringe will be pushed firmLy to inject the solution. Lastly, the needle will be removed. This will be repeated to the experimental and the positive control groups using one tuberculine syringe per mouse.

Induction of Gotu Kola extracts

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Twelve hours after the Tetracycline induction, the Gotu Kola extracts would now be induced using the Gavage Method. The standard procedures that will be followed for the Gavage Method is mentioned in the Laboratory Animal Workshop Manual of the Biological Research and Development Laboratory. The mouse must be restrained and be straightened. The Gavage needle would be inserted in the mouth and throughout its entire length. Then the plunger is pushed to administer the solution in the stomach and is then removed. 0.1 mL would be the only dosage used for each concentration of extract in the experimental set-up.

Hematoxylin and Eosin Staining The laboratory mice after it has been subjected to the different tests, will have their livers removed and washed(following the proper protocol in handling and dissecting). Then samples would then be soaked in Formalin solutions. Samples would then be cut and be placed in slides for staining. The slides would then be placed in manual staining racks and would then be air dried in a 60 degrees C oven for 30 to 40 minutes. Then the following would then be done (in order):

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http://www.bcbiolibrary.icapture.ubc.ca/pathologistsresearchers/docs/BL.LAB.GN.006.01%20Haematoxylin%20and%20Eosin %20Staining.pdf

Data Analysis:

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Micronuclei Scoring The incidence of micronuclei in liver cells indicates that mutation has occurred. Because of this, the number of micronucleated liver cells out of 2000 liver cells is determined. (OECD, 1997).

Analysis: If the average ratio of one experimental set-up is lower compared to that of the Positive control then the action of anti-mutagenesis has occurred. Table for Experimental Set-up 0.1 mL Tetracycline 1 mL of of 1 mL of 25% Concentration Concentration 1st 2nd 3rd 1st 2nd 50% 1 mL of 75% Concentration 1st 2nd 3rd

Mice (number of micronucleus/2000 liver cells)

3rd

1 mL of 100% Concentration 1st 2nd 3rd

Table for Positive Control 0.1 mL of Tetracycline Mice 1st (number of micronucleus/2000 liver cells) Table for Negative Control Mice (number of cells) No induction of tetracycline hydrochloride 1st 2nd micronucleus/2000 liver 3rd 2nd 3rd

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