Biochem Research

8/3/2019 Biochem Research

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Effects of

Agarichus-Blazae Mushroom on

Cell Viability on

Prostate Cancer Cell Line

Our Lady of Fatima University

Biochemistry DepartmentSection E

October 7, 2011



OBJECTIVES General Objective:

- To explore the the biological significance of agarichus-blasae to humanity, its benefits for patients suffering from cancer of the prostate.

Specific Objectives: - To evaluate agarichus-blasae’s possible apoptoticproperties against cancer cells.- To observe agarichus-blasae’s biological relation to liver cancer cell prevention and destruction throughthorough laboratory sampling and experimentation.

- To conceptualize, if not identify credible agarichus- blasae’s preparation that will facilitate prostate cancer cellinhibition.

- To provide recommendations for the improvement anddevelopment of cancer research.



SIGNIFICANCE OF THE STUDY This research does not only contribute in knowledge productionin general, but in making breakthroughs as well. The purpose ofthis research is to gain a better understanding of and/or perspective on Agaricus-Blazae. From its definition, this researchwill also discuss the sources of Agaricus-Blazae and how does it

able to produce apoptotic effects on cancer cells at the sametime evaluate the level of its effectiveness being the main focusof the study. This research is especially relevant in the field ofScience or specifically in Medicine by contributing andpresenting a potential cure for cancer, one of the most fataldiseases in the world. Experts in this field can eventually conductfurther studies based on this research on other perspectives as

well. Not only exclusive to the field of Medical Sciences,Environmentalists can also benefit from this research byacquiring relevant knowledge, become aware that many of our natural resources are of great value, and be encouraged oftheir preservation. As for the masses, this research will be verybeneficial by providing useful reference for different types ofdocuments.



SIGNIFICANCE OF THE STUDY The study will be most significant for the people who are in the lower

class especially those who are in the undeveloped regions of thecountry where medical facilities are least available. It is a fact thatPhilippines, being a third world country, have very limited resourcesand goods are overpriced including medical expenses. As a result,many people resort in untested, ineffective, alternative medicine inwhich side-effects could be fatal. By understanding and evaluatingcarefully, this research will provide and name a potential effectivecure for cancer at a low cost because our country has abundantsources of ABM. Aside from being an addition to other potential

cures for cancer, this study will also be significant in discouragingpeople from using untested substances.



WHY AGARICUS?

It is best known mushroom in the westernworld

Originating in Brazil

Used for CANCER, type 2 diabetes, highcholesterol, liver disease, blood streamdisorders, digestive problems

Also used to boost the immune system for physical and emotional stress



WHY AGARICUS?

Insufficient evidence for cancer treatmentside effects developing research suggests

that taking agaricus mushroom mightreduce some effects.

Efforts to study the health properties of thistasty mushroom is found to be effective

breast cancer inhibitor through animalexperiments, but also it is best cancer fighter



WHY AGARICUS?

Even more astounding for those mice fedwith mushroom as a preventative agent,

injected with powerful cancer-causingSarcoma 180, 99.4 % of them showed notumor.



WHY AGARICUS?

Most experiments done are in animals,rarely have cell lines

At the Medical department of TokyoUniversity and Tokyo College ofPharmacy ; mice with cancerous tumorswere fed with Agaricus mushroom. The

cancerous tumors were eliminated in 90%of the mice.



METHODOLOGY



PLANT EXTRACTION1. The Agaricus blazae were to be grounded

into powder and be dried.

2. Dried powder (100 g) of Agarichus-Blazaewas extracted in soxhlet apparatus with 1000 mlof 95% ethanol. The soxhelation process wascarried out until the solvent was found to becolorless.

3. The dark brown ethanolic extract was thenfiltered, concentrated using a rotaryevaporator.



METHODOLOGY: EXTRACTION

SAMPLEGROUNDED and

PULVORIZED

EXTRACTION USINGSOXHLET

APPARATUS

FILTRATION ANDCONCENTRATIONCRUDE EXTRACT



CELL LINEThe cell line will be obtained from

Saint Luke’s Medical Hospital. The cell will

be cultured in a growth medium (pH 7.4)supplemented with 10% Fetal bovine serum(FBS) and antibiotics, penicillin (100 units/ml)and streptomycin sulfate (100 μg/ml).



ANTICANCER ASSAY

(MTT Assay)1. Cells will be seeded into 4 wells of a 96-well micro

titer plate at 2 x 104 cells per well with 100 μl growth mediumand then incubated for 24 h at 37 °C under 5% CO2.

2. Then the medium will be removed while freshgrowth medium containing the agarichus-blasae extract isadded at 100, 50 and 25 μg/ml (Dependent variable). Water and (anticancer medicine) for your negative and positivecontrol respectively.

3. After 3 days of incubation at 37 °C under 5% CO2,the medium will be removed while 0.1 mg/ml MTT [3-(4, 5-dimethyl thiazole-2yl) - 2, 5-diphenyl tetrazolium bromide]reagent will be added.

4. After 5 h incubation, the MTT will be removedbefore adding 100 μl DMSO to each well and gently shaken.

5. The absorbance was then determined by ELISAreader at 516 nm.

6. Control wells will receive only the media withoutthe extract.



TEST

SAMPLE

POSITIVE

CONTROL

NEGATIVE

CONTROL

INCUBATIONWITH

AGARICUS(100/50/25ug/ml) FOR 3

DAYS

INCUBATION

WITHCISPLATINFOR 3 DAYS

INCUBATION

WITH WATERFOR 3 DAYS

MTT ASSAY MTT ASSAY MTT ASSAY

ELISA READER 516 NM



APOPTOTIC ASSAY

TRYPAN BLUE ASSAY

1. Centrifuge an aliquot of cell suspension .

2. Resuspend the cell pellet in 1 ml PBS or serum-free complete medium

3. Mix 1 part of 0.4% trypan blue and 1 partcell suspension, allow mixture toincubate ~3 min at room temp



TRYPAN BLUE ASSAY

4. Apply a drop of the trypan blue/cellmixture to a hemacytomer

5. Count the unstained (viable) and stained(nonviable) cells separately.



TIME SCHEDULE J ul y

A u g u s t

S e pt em b er

O ct o b er

N ov em b er

D e c em b er

J an u ar y

F e b r u ar y

M ar ch

Review of

literature /

planning Submission

and

consultation

of research

proposal

Finalization

of the

research

proposal



TIME SCHEDULE J ul y

A u g u s t

S e pt em b er

O ct o b er

N ov em b er

D e c em b er

J an u ar y

F e b r u ar y

M ar ch

Experimenta

tion time Collection of

data Data

analysis Finalization

of writtenreport

Final Oral

Defense

Documents

Biochem Research