Chapter III Methodology This chapter discusses the locale of the study, the methods and procedures used

in performing the study, the research design, and research instrument/methods.

Research design The study is basically a qualitative research for the results of the experiment only depict or detect presence or absence of blood in the samples. The study does not cover the relationships of the different amounts of variables and therefore a descriptive design is utilized. The method to be used in the extraction of anthocyanin from red onion is as described by Guevarra in 2005. The skin peelings from red onion is weighed about 0.1 kilograms and treated with 80% ethyl alcohol. Stopper the flask and soak for 48 hours and then the plant extract is aspirated and the plant material is discarded.

Research locale and respondents The process of extraction and the actual experimentation were performed in the Clinical Microscopy Laboratory located at room 302 of Science Building of Far Eastern University (FEU) Manila. FEU is situated at Nicanor Reyes Street, Sampaloc Manila, Philippines.

Sampling design The plant materials are properly identified and authenticated by Ms. Dulce Marie Nisperos, a faculty of Far Eastern University, Manila, Department of Biological Sciences.

The respondents are selected by means of purposive non-probability sampling. The researchers handpicked the subject based on age and health status that will best fit for the study. The respondents are of the age within 18-30 years old and are healthy based on physical appearance. In addition, blood samples are obtained from two healthy individuals through venipuncture with the use of ethylenediaminetraacetic acid (EDTA) tube of 5ml each.

Data gathering procedure I. Collection of red onion bulb peels. Three kilograms of red onion bulbs were obtained from Cloverleaf dry market Balintawak, Caloocan City. The samples are properly identified and authenticated by a member of faculty of Department of Biological Sciences of Far Eastern University. The red onion bulbs were peeled and the peelings were collected. It will yield approximately 0.3 kilogram peels of red onions. II. Preparation and extraction of anthocyanin from red onion peels. Fresh red onions peels were weighed 0.1 kilogram in an Erlenmeyer flask. The samples were soaked in 80% ethyl alcohol and were incubated at room temperature for 48 hours. The red onion extract was gently sucked using aspirator and pipette. To further purify the extract, rotary evaporator was used. Temperature is maintained at 40oC for 1 hour for optimum separation of ethyl alcohol. The anthocyanin extract is mixed with hydrogen peroxide. The ratio for the mixture is 3:1 (anthocyanin extract is 100ml while hydrogen peroxide is 50 ml). III. Collection and preparation of fecal samples and diluted blood One hundred fecal samples are to be obtained from normal healthy respondents given each a specifically modified diet appropriate for the experiment. Among the one hundred, seventy respondents are subjected to diet restriction three days before specimen collection no red meat (e.g., beef, lamb, liver), no more than 250mg vitamin

C a day from supplements, citrus fruits, and juices. The remaining thirty are not diet restricted prior to sample collection. The blood samples obtained from two individuals are subject to dilution with the ratio of 1:20 (blood is 5ml while distilled water is 95ml). Cell lysis is expected for better reaction. Forty fecal samples from the one hundred were added with a small amount of diluted blood at about 20uL. These were thoroughly mixed manually using applicator stick. These samples could resemble fecal occult blood to be used in the study.

Procedure The following procedures were done for the preparation and extraction of anthocyanin in red onion (Allium cepa) bulb peels: 1. The red onion peels were cut into smaller pieces. 2. The peels were soaked in 80% ethyl alcohol and then incubated at room temperature for 48 hours. 3. After incubation, the obtained extract was sucked using an aspirator/pipette. 4. For further purification of the sample, rotary evaporator was used for 1 hour at 40C. By doing so, anthocyanin extract was obtained. 5. 100 ml anthocyanin extract then was mixed with 50 ml Hydrogen peroxide in making the blood detection solution.

In making the RBC solution, blood sample was diluted by: 1. 5ml EDTA anticoagulated blood was collected. 2. The sample was mixed with 95 ml of distilled water. A hypotonic solution such as distilled water caused the lysis of red blood cells.

For the preparation of fecal occult blood samples and detection testing procedures, the following steps were done: 1. 100 normal fecal samples were obtained. 2. These samples were divided into 3 groups: 30 false positive samples, 30 negative samples (control) and 40 samples for testing. 3. For the false positive samples: 30 samples were obtained from people with unrestricted diet. 100% anthocyanin extract + hydrogen peroxide were added to each sample. 4. For the negative sample or control: 30 samples were obtained from people subjected to diet restriction. 100% anthocyanin extract + hydrogen peroxide were added to each sample. 5. For the samples employed for testing: 40 samples were obtained from people subjected to diet restriction. A minute amount of diluted blood was added to each sample using an applicator stick. 100% anthocyanin extract + hydrogen peroxide were added to each sample.

IV. Test for detection of blood The 100ml pure extract of anthocyanin from red onion is mixed with 50ml hydrogen peroxide. One ml of the solution is added to a test tube each for every sample. A pinch of fecal sample is added to the test tubes and the reaction is observed and recorded after 5 minutes.