Stingless Bee Propolis Lab Report

CHAPTER 3
Methods and Procedures This chapter presents the methodology and procedures used to determine the renal protective property of crude extract in stingless bee propolis.
Research Methodology Experimental methods and techniques will be used for the determination of the renal protective property. Systematic procedures will be observed in the determination of the renal protective property of crude extract in stingless bee propolis. Chemical tests will be used to perform to determine present compounds. Biological and histopathological testing will be conducted to determine its renal protective property in high levels of creatinine by Gentamicin induced in rats. The Extraction, physical and chemical tests of crude extract will be done

Extraction of Crude extract Propolis samples were refrigerated about -20c°. after refrigeration the propolis samples were grinded and macerated (30g of propolis, making up the volume to 100 mL with 70% ethanol) and was filtered. The filtrate was evaporated to dryness at 80°C under reduced pressure.
3. Characterization of Crude Extract 3.1 Physical Test 3.1.1 Organoleptic Test
The color, odor and physical state of extract will be determined. 3.1.2 Solubility Test
The solubility test for extract will be performed using distilled water, 80% alcohol, chloroform, hexane and ether.

3.2 Chemical Tests
The crude extract will be dissolved in 20mL of 80% ethyl alcohol. It will be filtered and divided into 5 test tubes and will be labeled as A, B, C, D and E. The test tube A will be kept blank. 3.2.1 Ferric Chloride Test
The sample extraction will be dissolved in 2 ml ethanol. A few drops of 10% ferric chloride solution will be added in test tube F. A green-blue coloration indicates the presence of a phenolic hydroxyl group (Bhatt et. al., 2011) 3.2.2 Sodium Hydroxide Test
Few drops of 10% aqueous sodium hydroxide solution will be added in a test tube E with 2-3 ml of the extract. Formation of intense yellow color that became colorless on the addition of few drops of diluted HCl indicates the presence of flavonoids (Bello et. al.,

Air-conditioned rooms will be used by the animal holding facility and the test animals will be placed under 12 hours dark/12 hours light condition wherein the area will be lighted from 6:00AM to 6:00PM and keeping the lights off for the next 12 hours.
The rats will be acclimatized for one (1) week before the start of the biological tests. Every other day, the cages will be cleaned properly dried and disinfected. The rats will be fed with commercial rat pellets and will be given sufficient water. Crushed rabbit pellets and corn kernel can be an alternative food.
All animal experiments will be performed with prior approval from the Institutional Animal Care and Use Committee (IACUC) of Centro Escolar University. Biological test procedures will be supervised by a professional veterinarian, and a member of the IACUC. 4.2 Renal Toxicity Inducer
On the 5th to 10th day of biological testing, renal toxicity will be induced to fifteen (15) rats, groups 3 to 5 by intraperitoneal (IP) injection of Gentamicin 100mg/kgBW concomitantly with the sample in 5th – 10th