Philippine Science High SchoolMain Campus

Cytotoxicity of Lagundi Extract on Human Colorectal Carcinoma and Lung Non-Small Cell Carcinoma

Jose Sandino A. BandonilMarc Patrick C. CelonRichard Dean Clod C. dela Cruz

September 2012

Cytotoxicity of Lagundi Extract on Human Colorectal Carcinoma and Lung Non-Small Cell Carcinoma

by

Jose Sandino A. BandonilMarc Patrick C. CelonRichard Dean Clod C. dela Cruz

Submitted to the Faculty of thePhilippine Science High School Main Campusin partial fulfillment of the requirements forScience and Technology Research 2

September 2012ABSTRACT

Cancer is one of the most lethal conditions known to man. According to the latest statistics, it caused 7.6 million deaths, or 13% of worldwide total deaths in 2008; among these, 70% came from low and middle-income countries. To counter this, a cheap and widely accessible drug must be produced. In order to start the research the aforementioned need, this study aims to discover the cytotoxicity of the crude extract of lagundi (Vitex negundo), a common shrub in Africa and South Asia, against human colorectal carcinoma (HCT-116) and lung non-small cell carcinoma (A549) cells. The crude extract will be extracted from 10 mg of powdered lagundi, through the Soxhlet extraction process. This extracts cytotoxicity will then be determined through the MTT assay. The cytotoxicity can be measured through the IC50. The extracts cytotoxicity will be compared with that of Adriamycin PFS a known effective anticancer drug and dimethyl sulfoxide (DMSO). The respective IC50 results taken from the different treatments will be computed through linear regression, given the absorbance of each treated unit. The MTT assay will also be applied, with the same treatments, on Chinese hamster ovarian cells (AA8). This will determine the feasibility of lagundi extract as an anticancer drug, for an extract cytotoxic to cancer cells but ineffective against normal cells will make it possible for such a drug to be produced. Data analysis will be done through one-tailed independent T-test. An value of 0.05 will be used to determine the effectivity of the treatments.

APPROVAL SHEETThis research work entitled, Cytotoxicity of Lagundi Extract on Human Colorectal Carcinoma and Lung Non-Small Cell Carcinoma by Jose Sandino A. Bandonil, Marc Patrick C. Celon, and Richard Dean Clod C. dela Cruz, presented to the Faculty of the Philippine Science High School Main Campus in partial fulfillment of the requirements in Science and Technology 2, is hereby accepted.

Ana Victoria M. LlorenResearch Adviser

ACKNOWLEDGEMENTS

To our parents, who never stopped supporting us physically and mentallyTo our Research adviser, Maam Bick Lloren, for pushing us to our limitsTo Dr. Sonia D. Jacinto, for allowing us to use her facilities for this study and for giving us the scientific know-how we neededTo our friends, for hearing out our concerns when we needed to blow off pressureTo our teachers, who helped us become more efficient workersAnd finally, to Pisay, for arming us with the knowledge and wisdom we needed to accomplish this project

Thank you very much

TABLE OF CONTENTSPage

Approval SheetiAcknowledgementsiiTable of ContentsiiiList of TablesvList of FiguresviIntroduction1Background of the Study1Statement of the Problem1Significance of the Study 2Scope and Limitations 3Review of Related Literature5Cancer5Human Colon Carcinoma6Lung Non-Small Cell Carcinoma7Lagundi (Vitex negundo)8Cytotoxicity Assays9Soxhlet Extraction11Materials and Methods13Process Flowchart13Procurement of Materials and Equipment14Extraction of the Crude Extract14Cytotoxicity Assay for Lagundi Extract15Data Analysis17Results and Discussion18Extraction of the Crude Extract18Cytotoxicity Assay on HCT116 Cells18 Cytotoxicity Assay on A549 Cells19 Cytotoxicity Assay on AA8 Cells19 Data Analysis19Summary and Conclusion21Recommendations22Bibliography23Appendix A: Complete Results with Absorbance Values26

List of TablesTableTitlePage

1HCT116 IC50 Values18

2Complete MTT Results for HCT-116 Cells26

List of FiguresFigureTitlePage

196-well plate after MTT assay on HCT116.19

2HCT116 IC50 Value Comparison20

23

INTRODUCTION

Background of the StudyCancer is one of the deadliest conditions that man has ever encountered. Current statistics compiled by the World Health Organizations International Agency for Research on Cancer show that cancer is currently the world leading cause of mortality, causing 7.6 million deaths or 13% of total 2008 deaths. 70% of these deaths occurred in low and middle-income countries. Current projections predict 13.1 million deaths by 2030. The five main types of cancer worldwide are of the lung, of the stomach, of the liver, of the colorectum, and of the breast, in decreasing order for number of deaths (World Health Organization, 2012). Meanwhile in the Philippines, cancer, or malignant neoplasms, ranks third in the ten leading causes of mortality, and is superseded only by heart diseases and vascular diseases. Cancer has a mortality rate of 49.5% in the Philippines and was the cause of 43,043 deaths in 2006. It can also be noticed that the Philippine cancer incidence and mortality rate has been steadily increasing, except for the years 2001 to 2006, when the mortality rate stabilized at 48.5% 0.3% (Department of Health, 2006). Top cancer sites in the Philippines include the breast, the lungs, the liver, the colorectum, and the cervix uteri, in decreasing number of cases (GLOBOCAN 2008, 2008).

Statement of the ProblemThis project aims to determine the cytotoxicity of lagundi through the testing of its crude extracts. The crude extract will be extracted through Soxhlet extraction, after it will be subjected to rotary evaporation. The crude extract will undergo the MTT assay to determine its cytotoxicity against human colorectal carcinoma (HCT-116) and lung non-small cell carcinoma (A549) cells. While crude extracts from lagundi are already known to be cytotoxic against HCT116 cells, this study will validate such claims. Crude extract treatments will undergo different concentrations: 50 g/mL, 25 g/mL, 12.5 g/mL, and 6.25 g/mL. The cytotoxicity will be measured through the half maximal inhibitory concentration (IC50), which measures the highest concentration of the tested chemical that can kill half the cells in a well. During the assay, temperature and % CO2 level will be kept constant in the incubator. The crude extracts IC50 will be compared to those of Adriamycin PFS a well known anticancer drug and dimethyl sulfoxide (DMSO), the solvent with which the extract was dissolved for assay purposes. However, Adriamycin PFS will be tested with the concentrations 25 g/mL, 12.5 g/mL, 6.25 g/mL, and 3.125 g/mL because of its reputation as an effective drug.The crude extract will also be tested against Chinese hamster ovary (AA8) cells. This is so that the effect of the extract on normal cells can also be discovered. It is ideal that the crude extract is not cytotoxic against AA8 cells. Such a result will mean that lagundi is a possible natural anti-lung cancer drug. It will also be ideal if results have a low variance, so that the effect of the crude extract on humans in vivo will be more predictable.

Significance of the Study Current scientific research on anticancer cures has found several possible anticancer cures. However, no cure has yet been developed that can be mass-produced. This is because available treatments for cancer demand either expertise on cancer treatment or a chemical produced only on small quantities. Such requirements drive up the cost of cancer treatment. A cancer drug that is widely available in the Philippine archipelago and one that can be easily mass produced will lower the price of cancer treatment and make it more accessible to the general populace. An anticancer drug derived from lagundi will do just that. Not only is lagundi a well-known cough medicine, it is also found in almost all parts of the Philippines. Lagundi is promoted by the Department of Health as a cheap cough medicine, and being used in local health centers for the same function. A study that proves the possible anticancer capabilities of the plant will give lagundi medicine a larger market and thus earn Philippine producers millions or even billions of dollars in revenue. This can increase the governments tax revenue, making possible several improvements in infrastructure and in other sectors. In other words, this small project by Philippine Science High School students may serve as a tipping point to usher in several changes in the Philippines.

Scope and LimitationsThis study will be conducted in the Philippine Science High School Main Campus (PSHS-MC) Research Unit and in the Cell Biology Laboratory of the University of the Philippines Institute of Biology (UP-IB). The extraction process for the lagundi crude extract will be done through Soxhlet extraction in the PSHS-MC Research Unit. The crude extract will be taken from 10 mg of powdered lagundi leaves. From the crude extract, 4 mg will be taken and dissolved in 1 mL dimethyl sulfoxide (DMSO). This solution will be used for the MTT assay. 100 L of extract with different concentrations will be transferred to each well during the assays treatment process. The same amount will be transferred to each well for the control treatments Adriamycin PFS and DMSO. The MTT cytotoxicity assay will test the crude extracts effect against HCT116, A549 and AA8 through the measurement of the crude extracts IC50. The lower the IC50, the more cytotoxic the extract is. The ideal IC50 is below 20; however, an IC50 at the range between 20 and 25 is said to still be acceptable for crude extracts. It is because crude extracts might have a compound that retards the effect of the cytotoxic compound. The MTT assay will be done in the UP-IB Cell Biology Laboratory.

REVIEW OF RELATED LITERATURE

CancerCancer is one of the most lethal conditions known to man. To counter this, the World Health Organization (WHO) founded a research agency dedicated to cancer research called the International Agency for Research on Cancer (IARC). Statistics compiled by IARCs GLOBOCAN 2008 show that cancer caused 13%, or 7.6 million deaths, of all total deaths around the world. Of these, 70% occurred in low and middle income countries. Current projections predict 13.1 million deaths in 2030. The five main types of cancer are of the lung, of the stomach, of the liver, of the colorectum and of the breast, in decreasing order in number of deaths (WHO, 2012). In the Philippines, cancer ranks third in the ten leading causes of mortality, counting 43,043 deaths and having a 49.5% mortality rate. The Philippine cancer incidence rate and mortality rate have been increasing, except for the years 2001 to 2006, when the mortality rate stabilized (Department of Health, 2006). Leading cancer sites in the Philippines are in the breast, the lung, the liver, the colorectum and the cervix uteri (GLOBOCAN 2008, 2008).Cancer is merely a disease in which abnormal cells divide uncontrollably instead of undergoing the normal cell cycle. Instead of undergoing apoptosis when they age or are damaged, they continue multiplying in a process called metastasis. The change from a normal cell and tumor cell stems from several events, but the start of these changes occur a cells DNA is either damaged or altered. The damage to the DNA causes mutations that alter the normal cell division and growth process (National Cancer Institute, 2012). These changes usually occur due to four important factors: the organisms genetic material, physical carcinogens such as radiation, chemical carcinogens like asbestos, and biological carcinogens, which include bacterial or viral infections like Hepatitis A. Aging also increases the possibility of cancer development (WHO, 2012). In an effort to remedy cancer, huge efforts are being done to look for possible cures. Several possible natural cures have been found, according to an article by Hafidh, et. al (2009). In the same article are examples of plants that tested positive to cytotoxicity. The list includes garlic (Allium sativum), which has been tested against the colon cancer cell line, and Mangor ginger (Curcuma mangga), assessed to be cytotoxic against the DU-145 (human prostate cancer), NCI-H460 (non-small cell lung cancer), and MCF-7 (breast cancer) cell lines. Hafidh et. al. (2009) also goes on to list possible anticancer chemicals. These chemicals include flavonoids, flavonols, sterols, carotenoids, and phenols. The flavone flavopiridol has in fact tested positive to cytotoxicity against lung non-small cell carcinoma (A549) cells. Inhibition of RNA and protein synthesis in the A549 cells has been pinpointed to contribute to the flavones cytotoxicity (Bible et. al., 1996). Another example would be camptothecin, an extract taken from the tree Camptotheca acuminata. It is known to be cytotoxic against HCT116 and MCF-7 cancer cells (Gupta et al., 1997).

Human Colon CarcinomaColon cancer is a cancer concentrated on the colon and rectal portions of the body. It is one of the most common types of cancer in the world, accounting for 10% of total cancer patients worldwide. Most of these patients are concentrated on developed countries, with Australia and New Zealand having the highest incidence rates in the world, with Western Europe and Southern Europe at second and third highest, respectively. However, while developed countries have higher incidence rates, developing countries have higher mortality rates than the developed countries (GLOBOCAN 2008, 2008). The cancer is characterized by tumor penetration through the bowel wall. Prognosis is done by inspecting for the presence of nodal involvement and distant metastases. Nodal involvement may signal metastases in other regions of the body. Aside from these two, however, cancer stage and race has also been observed to play a role in the survival of the patient (National Cancer Institute, 2012). For in vitro testing of extracts on colon cancer, HCT116 is used. HCT116 is a cancer cell line classified under Biosafety Level 1. The cell line is epithelial, and is adherent to surfaces. Products of cells from this particular cell line include carcinoembryonic antigen and keratin. The cell line has a mutation in codon 13 of the ras protooncogene, which might account for its cancerous behavior (American Type Culture Collection, 2011). This cell line is one of the possible cells that start human colon cancer, which ranks as the fourth most common cause of death from cancer; it is outranked only by cancer of the liver, cancer of the stomach, and cancer of the lung, in increasing order for number of deaths (World Health Organization, 2012).

Lung Non-Small Cell CarcinomaLung non-small cell carcinoma is a type of epithelial lung cancer that arises from lung epithelial cells between the central bronchi to the terminal alveoli. It has three main types: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Squamous cell carcinomas start near a central bronchus, while adenocarcinoma and large cell carcinoma start in peripheral lung tissue. The main risk factor for this type of cancer is smoking; however, exposure to some carcinogenic chemicals, exposure to second-hand smoke, and radiation to the chest or the breast are also important risk factors. Symptoms of the condition may include chest pain, breathing problems, weight and appetite loss, hoarseness, and facial swelling. In the United States, the 5-year survival rate of lung cancer depends on the cancer stage: 49% for local stages, 16% for the regional stage, and 2% for the distant stage. Current treatments cannot cure most patients with non-small cell lung cancer (National Cancer Institute, 2012).For in vitro testing, the A549 cell line is used for the efficacy of certain extracts against non-small cell carcinoma cells. This cell line was started from lung carcinoma cells taken from a 58-year old Caucasian male. The A549 cell line was established by D.J. Giard. Each cell of the cell line divides every 22 hours, and is adherent to the surface where they were established. Studies by M. Lieber, et. al. show that A549 cells could synthesize lecithin from desaturated fatty acids through the cytidine diphosphocholine pathway. The cell line is classified under Biosafety Level 1 (American Type Culture Collection, 2009). According to a recent study by Diaz et. al. (2003), vitexicarpin, a flavone constituent of Vitex negundo leaves, is cytotoxic to some varieties of cell lines.

Lagundi (Vitex negundo)Lagundi is a shrub commonly found in Southeast Asia and Africa. It can be recognized due to its tri- or penta-foliate leaves, which soon gives rise to bluish-purple flowers. It is recognized across Asia as a folk medicine that is able to cure headaches, sinusitis, and venereal diseases. In the Philippines, it is considered as an anticancer in folk medicine (Vishwanathan, et. al., 2010). The shrub has been proven to be an anti-cough medicine, and is endorsed by the Philippine Department of Health. It is currently being considered as a replacement for dextrothemorphan in the public health system. Phytochemical screenings of the plants ethanolic leaf extract have shown that it contains alkaloids, iridoids, phenolic acids, flavonols, and flavonoids (Lagundi/vitex negundo/five-leaved chaste tree: herbal medicinal plants/alternative medicine in the Philippines, 2012). Vitex negundo has been found to have anti-inflammatory and analgesic properties through antihistamine and prostaglandin-inhibiting activities. The plant has also been found to be an antioxidant, an enzyme inhibitor, a hepato-protector, a laxative, and an antivenin. It was also discovered to have a cytotoxic effect on COLO-320 tumor cells. Also, vitexicarpin, one of the flavones found in lagundi leaves, has been found to have cytotoxic potency against a human cancer cell line panel in a study by Diaz et. al. (2003). Lagundi has been tested on Swiss albino mice against Daltons Ascitic Lymphoma (DAL), and on Ehrlich Ascites Carcinoma (EAC) by Dewade et. al. (2010) and Saluja et. al. (2010), respectively.

Cytotoxicity AssaysThe studies on Vitex negundos cytotoxicity done by Dewade et. al and Saluja et. al. was done in vivo. In other words, they were done on live animals. Another type of testing is in vitro, or studies on organisms through the use of its isolated components. Cytotoxicity assays are one of those processes used for in vitro studies. These assays measure how toxic a certain object is to a certain cell. These assays observe cell damage through observable indicators such as color. There are three types of cytotoxicity assays, all of which are classified based on the parameters of the measurements: metabolic function/ATP, membrane integrity, and cell population (NoAb BioDiscoveries, n.d.). There are several cytotoxicity assays that can be used. One is the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, which uses the cells ability to absorb a formazan product through the application of tetrazolium salts. However, the medium used for the assay can distort the results, as well as the presence of albumin (Chen et. al., 2004). The MTS assay is used in some cytotoxicity studies, such as the cytotoxicity measurement of nanoparticles through the use of human skin fibroblasts (Dechsakulthorn et. al, 2007). Another assay is the LDH (lactate dehydrogenase) assay, which tests the viability of a cell through the measure of cell membrane strength. A modification of the LDH assay adds lysis buffer, allowing the measurement of cell viability in percentages (Vanderbilt, 2007). This assay was used on the assessment of the effect of Ritonavir, an HIV protease inhibitor, on human endothelial cells (Zhong et. al, 2002). One more example will be the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This assay is the same as the MTS assay because they both use tetrazolium salts and are both colorimetric in nature. However, the MTT assay is better in terms of reproducible results. Addition of media, cell population of each well, and the amount of MTT dye were discovered to interfere with results (Twentyman et. al., 1987). This assay is widely used, and was used on a study on the effect of a cyclooxygenase-2 inhibitor on non-small cell lung cancer cell lines and on the cytotoxicity of other anticancer agents (Hida et. al, 2000). The half maximal inhibitory concentration (IC50), which is the amount of concentration needed for a solution to have an effect on 50% of the experimental unit, measures the cytotoxicity of the possible anticancer compound. This is done through the interpretation of the graph of the curve of the function of binding as the concentration logarithmically increases (50% of what? How exactly are EC50 and IC50 defined?, n.d.). The above assays can also be used for the testing of the cytotoxicity of extracts.

Soxhlet ExtractionThe extracts that will be used for the above assays can be extracted through Soxhlet extraction. Soxhlet extraction is a method of extraction developed by Franz von Soxhlet on 1879. It involves a still pot, a thimble, a condenser, and a distillation path. The solvent is placed in the still pot, while the extract source wrapped in filter paper will be placed in the thimble. The condenser facilitates the condensation of the solvent-extract mixture, and the distillation path which the mixture passes on its way back to the still pot (Universiti Malaysia Sabah, n.d.). Before the start of extraction, however, there are specific protocols for different types of samples. For dry samples, the sample will be weighed into the thimble. Moist samples must first be dried for 2-3 hours at 10312 C after being weighed before its addition to the thimble. For liquid samples, it should first be mixed with sand and anhydrous sodium sulfate before being added to the thimble (Behr Labor-Technik, n.d.). A common solvent used for Soxhlet extraction is ethyl alcohol. Ethyl alcohol is a colorless liquid with a mild odor. Its boiling point is at 78 C, while the freezing point is -114 C. It is a miscible compound with the chemical formula C2H5OH. Ethyl alcohol is highly flammable and can cause severe eye irritation and moderate skin irritation. When ingested, it may cause gastrointestinal irritation along with nausea, vomiting, and diarrhea. Inhalation may cause nausea, headache, dizziness, and under extreme conditions, unconsciousness or even coma. Mutagenic and reproductive effects can also be detected when in chronic amounts (North American Fire Arts Association, 2001).

MATERIALS AND METHODS

Procurement of equipmentProcurement of powdered lagundiProcurement of AA8 and A549 cellsProcess Flowchart

Extraction of crude extract

Cytotoxicity assay on HCT116 cells

Cytotoxicity assay on A549 cells

Cytotoxicity assay on AA8 cells

Data analysis

Procurement of Materials and EquipmentThe powdered lagundi that will be used for the experiment will be sourced from HerbSeeds, an online seller that was found in Sulit.com. The powdered lagundi will come from Quezon province, and will be shipped to the Metro Manila through LBC. The equipment that will be used for the extraction of the crude extract will be borrowed from the Philippine Science High School Main Campus (PSHS-MC) Research Unit. The A549 and AA8 cells that will be used for testing will be bought from the Institute of Biology of the University of the Philippines. Likewise, the Institute of Biology will provide for the chemicals that will be used as control for the cytotoxicity assay: Adriamycin PFS, also known as Doxorubicin, and dimethyl sulfoxide (DMSO). The cytotoxicity assay will be done in the Cell Biology Laboratory of the Institute of Biology.

Extraction of the Crude ExtractAfter procurement, the powdered lagundi will be extracted through Soxhlet extraction. The Soxhlet extractor is a device developed by Franz von Soxhlet on 1879. It involves a still pot, a thimble, a condenser, and a distillation path. The setup is as follows: the thimble and the distillation path are both in one glass part. The thimble-distillation path part is placed at the top of the still pot, while the condenser is placed atop the thimble-distillation path tube. The still pot contains the solvent, while the thimble contains the extract source wrapped in filter paper. The extraction works by heating the solvent in the still path. As it evaporates and moves up the tube it encounters the thimble. The evaporated solvent brings along with it the chemicals of the crude extract. It soon encounters the condenser, where the solvent mixed with the chemical is condensed in the presence of relatively lower temperatures caused by cold water. The condensed mixture goes down the distillation path and into the still pot, and the process is repeated again (Universiti Malaysia Sabah, n.d.). Powdered lagundi, the extract source, was a dry sample, thus it was weighed before being added to the thimble as said in Behr Labor-Techniks guidelines for Soxhlet extraction. The extraction will use ethyl alcohol as its solvent. The solvent will initially be heated at 78 C; however, the temperature will be lowered later so that the crude extracts chemicals will not be disturbed or destroyed by the boiling action of the solvent. The extraction will go for 18 hours, after which the crude extract will be separated from the mixture by undergoing rotary evaporation until it has been reduced to a paste.

Cytotoxicity Assay for Lagundi Extract After the extraction process for the lagundi extract, it will be subjected to a cytotoxicity assay. The extract will be tested on human colorectal carcinoma (HCT116) and human non-small cell lung carcinoma (A549) cells. The extract will be tested at different concentrations: 25 L/mL, 12.5 L/mL, 6.25 L/mL, and 3.125 L/mL. Adriamycin PFS will be used as positive control due to its proven efficacy against cancer cells, while DMSO will be used as negative control.The start of the assay will be marked by the seeding of the cell line to a 96-well plate. The extract will be mixed with DMSO with a ratio of 4 mg extract per mL of DMSO. The 96-well plate will then be placed in an incubator and left for 24 hours. The incubator will be set at 37 C temperature and at 5% CO2 content for the rest of the assay. While the cells in the well plate are allowed to grow in the incubator, a master dilution plate will be used to set the different concentrations that will be used for treatment. The process is done by adding 150 L of cell culture media to the first well, and 100 L of the culture media to the three remaining wells. 50 L of the extract solution will be mixed with the first wells media. They will then be mixed, and 100 L will be taken and then added to the next well; this will be repeated until the fourth well is done. After the 24-hour period, the well plate is withdrawn from the incubator and treated with the necessary chemicals. There will be three replicates for the extract treatments and DMSO. However, Adriamycin PFS will only have two replicates, because the drug is already known to have a very low IC50. The treatment process is done such that concentrations of the extracts decrease starting from the wells at the edge to the wells at the center. The treatments will be taken from the prepared master dilution plate. After the treatment process, the treated well plate will again be deposited in the incubator for 72 hours. After 72 hours, the well plate will be taken from the incubator and treated with MTT solution. The MTT solution has a concentration of 5 mg MTT for every mL of phosphate buffer saline (PBS) (Minnesota State University, 2007). 20 L of the solution will be added to each well. The 96-well plate will then again be left in the incubator for 3 hours. The absorbance of the each well will then be taken after 3 hours through a plate reader. The basic premise of the assay, however, is that if the yellow MTT turned to purple formazan after the three-hour waiting period, cells in the well are alive. As the color gets closer to purple, the higher the amount of living cells in the well. A program will be used for the computation of IC50 through the input of each wells absorbance. The assay will also be done on Chinese hamster ovarian cells (AA8) to measure the effect of the extract on normal cells.

Data AnalysisAfter obtaining the value of IC50 of each of the samples, we then test their cytotoxicity by comparing it to Doxorubicin, a known anti-cancer chemical. For this, we need to do an independent t-test to compare the extracts effectiveness with that of Doxorubicin.The independent t-test is a statistical test that determines the statistical significance between the difference of results from two groups. The test judges the variability of results relative to the mean difference. The result t is the difference berween means divided by the variability of both groups. To determine the significance of the results, a p-value is derived from the degrees of freedom, and compared to the t-value. If the p-value is less than or equal to the t, then the difference is significant. However, if the results is not large enough, it is not significant enough (The t-test, n.d.).

RESULTS AND DISCUSSION

Extraction of the Crude ExtractTen grams of powdered lagundi was the source of the crude extract. The liquid resulting from the Soxhlet extraction process was dark green and fluid. It also had the distinct smell of ethanol, most likely arising from the ethanol used as a solvent during the extraction process. A vial of this liquid underwent rotary evaporation. After rotary evaporation, the liquid gave rise to a dark, sticky substance: the pure crude extract. The crude extract had no smell.Cytotoxicity Assay on HCT116 CellsFour mg of the crude extract was used for the MTT assay on HCT116. Table 1 shows the IC50 for HCT116 when treated with the lagundi crude extract and the positive control, Doxorubicin, also known as Adriamycin PFS. Table 1: HCT116 IC50 ValuesTreatmentsReplicates (IC50)

123

Doxorubicin1.91511.84502.4492

Lagundi Crude Extract22.569234.358022.6471

A picture of the 96-well plate used for the assays different replicates is shown in Figure 1. The different concentrations are arranged in the following manner: the highest concentration (50 g/mL) is treated to the cells in the edge wells, with the treated concentration going down as the well gets nearer to the middle along the column. The lowest concentration (25 g/mL) is treated in the fourth well from the edge. Each replicate occupies three columns of a well plate, in effect forming a 3x4 rectangle where only one chemical is treated to the cells.

Figure 1: 96-well plate after MTT assay for HCT116. The 3x4 rectangle on the lower right portion of the plate is the part where the test for lagundi cytotoxicity was done.

Cytotoxicity Assay on A549 CellsThe cytotoxicity assay for this cell line is still ongoing.Cytotoxicity Assay on AA8 CellsThe cytotoxicity assay for this cell line is still ongoing.Data AnalysisThe IC50 of lagundi crude extract and Doxorubicin will be compared through the one-tailed T-test. Figure 2 shows a comparison between the IC50 values for HCT116 between the two treatments. Figure 2: HCT116 IC50 Value Comparison

The t-value between the two was 7.74. Thus, it can be said that there is a significant difference between the two values. However, lagundi crude extract can still be said to be cytotoxic on HCT116 cells since the IC50 value is near the ideal value of 20 g/mL.

SUMMARY AND CONCLUSIONS

With respect to the above partial results, it has been decided that lagundi crude extract is a feasible anticancer drug for human colon carcinoma. The average IC50 taken from the MTT assay done in the Institute of Biologys Cell Biology Laboratory is a value enough to give the lagundi crude extract a status as a possible anticancer drug. The study has validated the apparent cytotoxicity of lagundi crude extract against human colon carcinoma cells (HCT116).Cytotoxicity assays for A549 and AA8 cells are still ongoing.

RECOMMENDATIONSIt is recommended that the cytotoxic chemical responsible for the relatively low IC50 in the assay be isolated. The timeframe for the study is too short for the said process, which is said to take months of repeated cytotoxicity testing after every round of separation. The crude extract should also be tested for compounds that may possibly retard the cytotoxicity of the crude extract. Such testing will explain the IC50 results higher than 20 g/mL.Subsequent studies on the same topic may also test compounds from lagundi against other cancer cell lines. The lagundi compounds may also be tested against cancer cells in vivo. This will determine the compounds effect on cancer cells when other body factors are included in the experiment.Crude extracts from other plants may also be tested against the cancer cells used in the study. A combination of plants may help in the creation of an anticancer drug that is a mixture of cytotoxic compounds taken from such plants. Such studies as the ones said above may help in the creation of a cheap and widely-accessible anticancer drug.

BIBLIOGRAPHY

50% of what? How exactly are EC50 and IC50 defined? (n.d.). Retrieved September 15, 2012, from http://www.graphpad.com/support/faqid/1356. American Type Culture Collection. (2009). Product information sheet for ATCC CCL-185. Retrieved July 5, 2012, from http://www.atcc.org/attachments/17395.pdf.Behr Labor-Technik. (n.d.) The extraction procedure. Retrieved July 15, 2012, from http://www.behr-labor.com/pdf/app_extraction_gb.pdf.Bible, K. & Kaufmann, S. (1996). Flavopiridol: A cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Research, 56, 4856-4861.Chen, Y.H., Huang, K.T., & Walker, A.M. (2004). Inaccuracies in MTS assays: Major distorting effects of medium, serum albumin and fatty acids. BioTechniques. Retrieved from http://www.BioTechOnline/MT_assay.pdf.Dechsakulthorn, F., Hayes, A., Bakand, S., Joeng, L., & Winder, C. (2007). In vitro cytotoxic assessment of selected nanoparticles using human skin fibroblasts. Japanese Society for Alternatives to Animal Experiments, 14(Special Issue), 397-400. Department of Health. (2011). Leading causes of mortality. Retrieved June 25, 2012, from http://www.doh.gov.ph/node/198.Diaz, F., Chavez, D., Lee, D., Mi, Q., Chai, H.B., Tan, G., , Kinghorn, A.D. (2003). Cytotoxic flavone analogues of vitexicarpin, a constituent of the leaves of Vitex negundo. Journal of Natural Products, 66(6), 865-867. doi: 10.1021/np0300784.GLOBOCAN 2008. (2008). Colorectal cancer incidence and mortality worldwide in 2008. Retrieved September 15, 2012, from http://www.globocan.iarc.fr/factsheets/cancers/colorectal.asp. GLOBOCAN 2008. (2008). Philippines fast stat. Retrieved June 25, 2012, from http://globocan.iarc.fr/factsheet.asp#KEY.Gupta, M., Fan, S., Zhan, Q., Kohn, K.W., OConnor, P.M., & Pommier, Y. (1997). Inactivation of p53 increases the cytotoxicity of camptothecin in human colon HCT116 and breast MCF-7 cancer cells. Clinical Cancer Research, 1997(3), 1653-1660.Hafidh, R.R., Abas, F., Abdulamir, A.S., Jahanshiri, F., Abu Bakar, F., & Sekawi, Z. (2009). A review: cancer research of natural products in asia. International Journal of Cancer Research, 5(2), 69-82. doi: 10.3923/icjr.2009.69.82.Hida, T., Kozaki, K., Muramatsu, H., Masuda, A., Shimizu, S., Mitsudomi, T., , Takahashi, T. (2000). Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines. Clinical Cancer Research, 2000(6), 2006-2011.Lagundi/vitex negundo/five-leaved chaste tree: herbal medicinal plants/alternative medicine in the philippines. (2012). Stuartxchange.org. Retrieved June 25, 2012, from http://www.stuartxchange.org/Lagundi.html.National Cancer Institute. (2012). General information about colon cancer. Retrieved September 15, 2012, from http://www.cancer.gov/cancertopics/pdq/treatment/colon/HealthProfessional. National Cancer Institute. (2012). General information about non-small cell lung cancer. Retrieved July 5, 2012, from http://www.cancer.gov/cancertopic/non-small-cell-lung/Patient/page1.National Cancer Institute. (2012). Non-small cell lung cancer treatment (PDQ). Retrieved July 5, 2012, from http://www.cancer.gov/cancertopics/pdq/treatment/non-small-cell-lung/healthprofessional.National Cancer Institute. (2012). What is cancer? Retrieved June 25, 2012, from http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer.NoAb BioDiscoveries. (n.d.). Discovery services: Cytotoxicity. Retrieved June 27 2012, from http://www.noabbiodiscoveries.com/assays/invitro/cytotoxicity_studies.pdf.North American Fire Arts Association. (2001). Ethyl alcohol, 70%. Retrieved July 15, 2012, from http://www.nafaa.org/ethanol.pdf Minnesota State University. (2007). MTT proliferation assay protocol. Retrieved August 29, 2012, from http://web.mnstate.edu/provost/MTT%20Proliferation%20Assay%20Protocol%202012.pdf The t-test. (n.d.). Retrieved September 15, 2012, from http://www.socialresearchmethods.net/kb/stat_t.php. Twentyman, P.R. & Luscombe, M. (1987). A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and sensitivity. British Journal of Cancer, 56(3), 279-285. doi: 10.1038/bjc.1987.190.Universiti Sabah Malaysia. (n.d.). Soxhlet extractor. Retrieved June 27, 2012, from http://wwwsst.ums.edu.my/data/file/IW7zkH8WepCp.pdf.Vanderbilt. (2007). Lactate dehydrogenase (LDH) assay. Retrieved July 12, 2012, from http://www.mc.vanderbilt.edu/rout/pdfs/maclaughlin_lab/ldh_assay.doc.Vishwanathan, A.S. & Basavaraju, R. (2010). A review on Vitex negundo L. a medicinally important plant. EJBS, 3(1), 30-42.World Health Organization. (2012). Cancer. Retrieved June 25, 2012, from http://www.who.int/mediacentre/factsheets/fs297/en/.

Zhong, D., Lu, X., Conklin, B.S., Lin, P.H., Lumsden, A.B., Yao, Q., & Chen, C. (2002). HIV protease inhibitor ritonavir induces cytotoxicity of human endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology, 2002(22), 1560-1566. doi: 10.1161/01.ATV.0000034707.40046.02.

APPENDIX AComplete Results with Absorbance Values

Table 2: Complete MTT Assay Results for HCT116cg/mlTrial I (absorbance)Trial II (absorbance)Trial III (absorbance)

DMSO01.7521.8271.9861.6121.8891.9060.8420.5710.352

Doxo250.2070.20.2130.2020.2260.204

12.50.180.3930.1840.320.2060.205

6.250.1760.5270.1750.370.2030.189

3.1250.190.4730.2060.3460.2150.211

IC501.91511.84502.4492

cg/mlTrial I (absorbance)Trial II (absorbance)Trial III (absorbance)

DMSO01.7521.8271.9861.6121.8891.9060.8420.5710.352

Lagundi500.2350.2010.1670.2160.1870.2270.1950.2020.449

251.4690.4420.2051.3531.21.3910.1680.390.153

12.51.8451.8880.1551.7811.4991.2410.4020.5750.354

6.251.0910.3690.1661.0991.2731.0190.4320.3630.65

IC5022.569234.358022.6471