RESEARCH PLAN

Bioactivity profiling of naturally occurring Philippine strain of

Xylaria hongkongensis mycelia

________________________________

John Andrei A. Delos Santos

Researcher

Bernadette Hipolito

Research Adviser
 Bioactivity profiling of naturally occurring Philippine strain

of Xylaria hongkongensis mycelia

In the development of drugs, scientist eyed fungi as notable source because of their

abundance and low cost-production. Among the many, Penicillium species like Penicillium

chrysogenum also known as Penicillium notatum is the source of an antibiotic called penicillin.

Lovastatin isolated from an oyster mushroom, Pleurotus ostreatus and red yeast rice Aspergillus

terreus is use for lowering cholesterol and prevention of cardiovascular disease. Cyclosporine,

produced by an aerobic fungi Tolypocladium inflatum is for rheumatoid arthritis, psoriasis,

nephrotic syndrome and as immunosuppressant in organ transplant to prevent rejection.

Significant mushrooms like reishi, shiitake, lingzhi, and chaga are included in Chinses cuisine

and are considered medicinal. Search and discovery for new species of fungi and its family

member is apical in the field of medicine as they can be source effective substances for the

treatments of different diseases.1

Just recently, in Hong Kong, China, a new species of Xylaria will be discovered. It is

distinguished by its conical stromata, small spores and having teleomorphic characters. It will be

named Xylaria hongkongnesis.2 However, in the Philippines, similar species will be discovered

and isolated in the laboratory at Central Luzon State University. The Philippine strain of Xylaria

hongkongnesis is not yet explored compared to other species. Hence, this study aims to find out

1
Linda Crampton “Penicillin, Lovastatin, and Cyclosporine: Medicines From Fungi” Owlcation August 30,
2019
2
Alvin Ming Chak Tang, Regent Yau Ching Lam & Mike Wing Keung Leung “Xylaria hongkongensis sp.
nov. from an urban tree” Mycotaxon Volume 128, pp. 37–40 April–June 2014
the bioactivities of this fungus’ mycochemicals usind different standard and scientifically

established preliminary procedures

Statement of the Problem

This study entitled “Bioactivities of Philippine occurring Xylaria hongkongensis” aims to

answer the following:

1. What are the mycochemicals present in Xylaria hongkongensis crude extract using thin

layer chromatography?

2. Does the Philippine occurring X. hongkongensis possess antioxidant property using DPPH

radical scavenging assay?

3. Does the Philippine occurring X. hongkongensis can inhibit the growth of E.coli and

S. aureus using disc diffusion assay?

4. What is the effect of the varying concentration of the Philippine occurring

X. hongkongensis in brine shrimps?

5. What is the effect of varying the concetrations of X. hongkongensis on the embryonic

development of zebra fish (Danio rerio)?

6. What is the effect of the varying concentration of the Philippine occurring X.

hongkongensis in Allium cepa root’s meristematic cells?

This study will be guided by the following hypotheses:

1. There is no significant difference on the mortality of brine shrimps when exposed in

various concentrations of Philippine occurring Xylaria hongkongensis.

 2. There is no significant difference on the development of Zebra fish (D. rerio) exposed in

various concentration of Philippine occurring Xylaria hongkongensis

3. There is no significant difference on the mitotic and mitotic phase indices in Allum cepa

root’s meristematic cells exposed in various concentration of the Philippine occurring

Xylaria hongkongensis.

Significance of the study

By conducting this study, the researcher discovered the potential medical and therapeutic

uses of Xylaria hongkongensis. This study finds the potential antioxidant, antimicrobial,

embryotoxic, teratogenic, cytotoxic and genotoxic properties of Xylaria hongkongensis. By

knowing these potential uses of Xylaria hongkongensis, new drugs might be develop in the near

future using this kind of mushroom.

Scopes and limitation

This project is limited to the use of Xylaria hongkongensis Philippine strain mycelia

isolated and characterized from the Center for Tropical Mushroom and Development (CTMRD)

Central Luzon State University (CLSU), Science City of Muñoz, Nueva Ecija. The researcher

focused on evaluating the potential of Xylaria hongkongensis ethanolic extract by identifying the

present bioactive compounds; quantifying the anti-oxidant and phenolic content; and evaluating

the antibacterial property against E. coli and S. aureus, cytotoxic (Brine Shrimp Lethality

Assay), teratogenic and embryotoxic (Zebra fish (D. rerio) Assay) and genotoxic (Onion Root

Chromosomal Aberration Assay) properties.

Mass production of fungus will be done at Bioassay Center, CTMRD, CLSU, Nueva

Ecija. Dried and milled sample will be sent to the Chemistry Laboratory Center for Natural
Sciences at St. Mary’s University in Bayombong, Nueva Vizcaya for ethanol extraction,

mycochemical screening, antioxidant and phenolic content analysis. Antibacterial, brine shrimp

lethality, teratogenic and embryotoxic property screenings will be done in CLSU while the

evaluation of genotoxic and chromosomal and aberrations assay will be conducted at

Biolaboratory, San Miguel National High School, San Miguel, Bulacan. Research-established

models will be used and considered because of their affordability and reliability in terms of

preliminary medicinal evaluation.

 Methodology

Mass Production of X. hongkongensis

Ethanolic Extraction

Mycochemical Screening

Quantification of Anti-oxidant and Total Phenolic Content

Anti-bacterial Assay Teratogenicity and Brine Shrimp Onion Root Tip

Embryotoxicity Lethality Assay Chromosomal
Assay Aberration Assay
1. Sterilization and
1. Spawning of D. 1. Spawning of 1. Exposure of A. cepa
media
rerio A.salina cysts to treatments
preparation
2. Collection and 2. Preparation of 2. Macro- Root analysis
2. Preparation of
screening of treatments 3. EC50 determination
treatments
viable eggs 3. Exposure to 5. Microscopy
3. Paper Disc
3. Preparation of treatments 6. Chromosomal
Diffusion Assay
treatments 4. Mortality Scoring
4. Exposure to determination
treatments on A. salina
5. Evalutaion of nauplii
teratogenic and
embryotoxic
property of
Xylaria
hongkongensis

Will bete Disposal

Data Analysis

Figure 1. Flow Chart of Activities

Mass Production of Xylaria hongkongensis

Forty (40) sterile bottles will be filled with 40 ml mature and aseptically filtered coconut

water. The bottles will be covered with cotton plugs, autoclaved at 15psi, 121°C for 20 minutes

and allowed to cool down. One square centimetre block of pure culture of Xylaria hongkongensis

will be cut and carefully impregnated to the liquid coconut media. The fungi will be incubated at

37 ° C for 15 days.

After 15 days of culturing, the fungi will be harvested, dried for five days and

powdered using a domestic blender. Milled Xylaria hongkongensis will be sent to the Chemistry

Laboratory Center for Natural Sciences at St. Mary’s University in Bayombong, Nueva Vizcaya

for ethanol extraction, mycochemical screening, antioxidant and phenolic content analysis

Mycochemical Screening of Xylaria hongkongensis

Mycochemical screening will be carried out to detect the secondary metabolites present.

The plant extract will be spotted on marked and labelled TLC (thin layer chromatography) 7 x 4

cm, and will be developed in the acetate-methanol (7.3) mixture in the developing chamber. The

spots for a certain metabolite will be visualised on the TLC plates and will be exposed under UV

light and hot plate to check the separation of different compounds.

For typical visualisation of secondary metabolites, vanillin-sulfuric acid reagents will be

utilized. This solution can determine the presence of Phenols, Sterols, triterpenes and essential

oils. Methanolic potassium hydroxide will be used to test anthraquinones, coumarians, and

anthrones while phenolic compounds and tannins will be detected through the use of potassium

ferricyanide-ferric chloride reagent. Dragendorff’s reagent will be used to spot alkaloids and

Antimony (III) chloride will be used to detect the presence of flavonoids.

Quantification of Anti-oxidant (DPPH Radical Scavenging Capacity) and Total Phenolic
Content

Antioxidants are defined as man-made or natural substances that may inhibit or delay

some types of cellular damage from potentially harmful molecules called free radicals. 3 Our

body use some free radicals to fight bacteria and viruses. In the other hand, free radicals can

damage the cells and might contribute to health problems like heart disease, some types of cancer

and cataract.4

DPPH Radical Scavenging Capacity. Xylaria hongkongensis concentrated extract will

be used to make a stock solution and aliquot will be taken to make 1000ppm dilution and

1000ppm of Catechin as control (1mg/ml). 1ml of prepared stock solution will be mixed with

4ml of 0.1mM DPPH solution in separate plastic cuvette. Reactions will be done in triplicate.

The prepared mixtures will be incubated in the dark at 37°C for 30 minutes. The absorbance

reading will be monitored at 517 nm using a UV VIS spectrophotometer. A lower absorbance of

the reaction mixture indicated higher free radical scavenging activity. The radical scavenging

activities will be compared to the activity of the control Catechin. The ability to scavenge the

DPPH radical will be calculated using the formula:

% of Radical Scavenging Effect = [(Acontrol – Asample) / Acontrol] x 100

Where Acontrol is the absorbance of the control which is the DPPH without the test sample.

Asample is the absorbance of the test sample containing the mixture of DPPH and the sample.

Catechin will be used as the positive control.

3
NCCIH, “Antioxidants: In Depth. National Center for Complimentary and Integrative Health”, (2013),
Accessed from https://nccih.nih.gov/health/antioxidants/introduction.htm
4
Healthdirect. “Antioxidants. Healthdirect”. (2018), Accessed from
https://www.healthdirect.gov.au/antioxidants
 Total Phenolic Content. The total phenolic content in Xylaria hongkongensis extract

will be detected with the Folin-Ciocalteu reagent. Gallic acid will be used as a standard and the

total phenolic content will be expressed as mg/g Gallic Acid Equivalents (GAE). Concentrations

of 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 and 1mg/ml of Gallic acid will be prepared in

methanol. Concentration of 1mg/ml of plant extract will be also prepared in methanol and 0.5 ml

of each sample will be introduced into test tubes and mixed with 2.5 ml of a 10 fold diluted

Folin-Ciocalteu reagent and 2ml of 7.5% sodium carbonate. The tubes will be covered with

parafilm and allowed to stand for 30 minutes at room temperature before the absorbance will be

at read at 760nm spectrophotometrically. All determination will be performed in triplicates. The

Folin-Ciocalteu is sensitive to reducing compounds including polyphenols, thereby producing a

blue color upon reaction.

Anti- bacterial Activity of Xylaria hongkongensis

Escherichia coli are bacterium that is normally found in humans and animal’s intestines.

However, some types of E.coli can cause intestinal infections like diarrhoea, abdominal pain, and

fever which can lead to other complications if not treated.5

Staphylococcus aureus is a non-moving small round shape type of bacteria. It is one of

the five most common causes of infections after injury or surgery. This bacterium can cause

many types of health problems like minor skin infections, boils, cellulitis folliculitis, carbuncles,

skin syndrome and abscesses, lung infections or pneumonia, brain infections or meningitis, bone

5
Jill Seladi-Schulman. “E. coli infection”, (2017), Healthline, Accessed from
https://www.healthline.com/health/e-coli-infection
infections or osteomyelitis, heart infections or endocarditis and generalized life threatening blood

infections or Toxic shock syndrome (TSS).6

Preparation of Test Organisms. Pure E. coli and S. aureus culture will be obtained

from Department of Biological Sciences, Central Luzon State University. The bacteria will be

grown in Nutrient Agar (NA) for 48 hours and transferred in nutrient broth and incubated for 24

hours. It will be sterilized to 1.5 x 108 cells/ml using 0.5 McFarland standards respectively. This

served as the bacterial inoculants.

Media Preparation and Sterilization. Thirteen (13) grams of Mueller-Hilton agar will

be suspended in 500 ml distilled water in an Erlenmeyer flask. The suspension will be boiled

until homogenized. The homogenized media will be sterilized using an autoclave at 121°C, 15

psi for 20 minutes. After sterilization, approximately 20ml of the media will be aseptically pour

in sterile petri dish and allowed to solidify. The plate stand in an inverted position for 24 hours

prior to use.

Preparation of Paper disc. Whattman filter paper no. 1 will be punched to produce discs

measuring 6mm in diameter. The discs will be placed in a petri plates and sterile in autoclave at

15 psi for 30 minutes.

Preparation of treatments. Three sterilized paper discs will be placed in three petri

dishes, impregnated and labelled with Treatment 1 (Xylaria hongkongensis), Treatment 2

(Streptomycin) and Treatment 3 (95% Ethanol).

6
Ananya Mandal, “What is staphylococcus aureus?”, (2018) News Medical Life Sciences, Accessed from:
https://www.news-medical.net/health/What-is-Staphylococcus-Aureus.aspx
 Disc Diffusion Assay. The disc diffusion assay is a method of testing the antibacterial

properties of substances using a channel paper disc. The paper disc is saturated with substance to

be tested and aseptically place in bacteriological media containing the test organism to which

zone of inhibition will be measured.7

An amount of 0.2 ml from the bacterium inoculum will be spread evenly in the assay

plates using sterile cotton swab. The filter paper discs treated with test substances are placed

equidistantly using sterile needles. The plates are labelled properly and incubated at room

temperature for 24 hours in an inverted position. The zone of inhibitions will be measured using

a ruler in millimetre.

Teratogenicity and Embryotoxicity Assay

The transparency of the zebra fish embryos makes it easy for identification of cardiac and

skeletal deformities. It exhibits similarity to higher form of vertebrates which makes them a

standard model for toxicity testing8 According to studies, 84 percent of the genes known to be

associated with human disease have a counterpart in the zebra fish genome and 70 percent of

protein-coding human genes are related to zebra fish genes. These findings highlight the

importance of the zebra fish model in human disease research.9

Spawning of Danio rerio. An oxygen-saturated glass aquarium will be used for

spawning matured female and male zebra fish with a ratio of 1:2. Adult zebra fish will be

7
Jan Hudzicki, "Kirby-Bauer disk diffusion susceptibility test protocol." (2009).
8
Alizza Yvette B. Memita, Sheena D. Macalinao, Lea Esperanza G. Reyes, Ernesto E. Damian Jr. And
Rich Milton R. Dulay “Toxicity And Teratogenicity OF Dieffenbachia amoena Leaf Extract” IJBPAS, August 2018,
7(8): 1591-1600
9
Jayanth A, Muralidhar S.Talkad, “Neuroprotective Activity Of Stachytrapheta Indica On Rotenone
Induced Parkinson’s Disease In Zebra Fish” World Journal Of Pharmacy And Pharmaceutical Sciences Volume 3,
Issue 7, 940-955, 2014
isolated using a plastic mesh and covered with black plastic for 12 hours to induce spawning.

After incubation in the dark, the fishes will be exposed to light to start fertilization. After 30

minutes, the fertilized eggs will be siphoned out of the aquarium. Embryos will be rinse thrice

using embryo water and observed under a stereomicroscope to examine uniformity and normal

condition.10

Preparation of Treatments. An amount of 0.1 ml of X. hongkongensis will be measured

and dissolved in 9.9 ml of embryo water to produce a stock solution of 10, 000 ppm and serially

diluted to produce 10,000, 1000, 100, 10 and 1 ppm, respectively. Table 1 shows the treatments

prepared including the positive control (1.5% ethanol) and negative control (embryo water).

Table 1

Concentrations used for Teratogenicity and Embryo toxicity Assay

Treatments Concentrations (ppm)

Treatment 1 0
Treatment 2 1
Treatment 3 10
Treatment 4 100
Treatment 5 1,000
Treatment 6 10,000
Positive control 1.5% Ethanol
Negative Control Embryo water

10
Rich Milton R Dulay,., Sofronio P. Kalaw, Renato G. Reyes, and Esperanza C. Cabrera. "Embryo-toxic
and teratogenic effects of Philippine strain of Lentinus tigrinus (tiger sawgill basidiomycetes) extract on zebrafish
(Danio rerio) embryos." Annals of Biological Research 5, no. 6 (2014): 9-14.
 Exposure to treatments. Three ml each of the prepared treatments of Xylaria

hongkongensis and controls will be placed into a 24-well ELISA plate. Into each well, four

embryos at segmentation phase (12 hpf) will be transferred. The plates will be incubated at 26°C

± 1°C. At 12, 24, 36 and 42 hpt the mortality and hatchability of the embryos will be recorded.

At 36 hpt, embryos’ heart beat will be recorded. Teratogenic effect of Xylaria hongkongensis

will be examined using a stereo microscope under 40X magnification at 60 hpt. Teratogenic

(malformation of head, tail and heart, scoliosis, deformity of yolk, and growth retardation), lethal

(coagulation, tail not detached, no somites, and no heart-beat), and normal are the parameters for

the morphological endpoint evaluation of zebra fish. Coagulated embryos and no visual heartbeat

embryos are considered dead. 11

Brine Shrimp Lethality Assay (BSLA)

Brine Shrimp Lethality Assay (BLSA) is used for preliminary cytotoxic assay of a

specific substance and is dependent on its capacity to kill nauplii.12 It is used as guide for the

detection of antitumor and pesticidal compounds and furthermore an indicator for general

toxicity.13 The availability of cheap brine shrimp eggs and the ease and cheapness of performing

the assay makes it a top method.14

11
Rich Milton R Dulay,., Sofronio P. Kalaw, Renato G. Reyes, and Esperanza C. Cabrera. "Embryo-toxic
and teratogenic effects of Philippine strain of Lentinus tigrinus (tiger sawgill basidiomycetes) extract on zebrafish
(Danio rerio) embryos." Annals of Biological Research 5, no. 6 (2014): 9-14.
12
Sahely Sarah Quazi, , Fatema Chowdhury Anny, and Mir Misbahuddin. "Brine shrimp lethality
assay." Bangladesh Journal of Pharmacology 12, no. 2 (2017): Online-Jun.
13
Lilybeth F. Olowa and Olga M. Nuñeza “Brine Shrimp Lethality Assay of the Ethanolic Extracts of
Three Selected Species of Medicinal Plants from Iligan City, Philippines” International Research Journal of
Biological Sciences, November 2013 Vol. 2(11), 74-77
14
H. A. El-Fadal., Sherif Mohamed El-Kadi, Mostafa Maher El-Moghazy, Ahmed Ali Soliman, and
Mahmoud Salama Mahmoud El-Haysha. "Correlation Between Active Components of Rocket (Eruca sativa) as
Cytotoxicity (Brine Shrimp Lethality Assay)." American Journal of Biomedical Science and Engineering 3, no. 2
(2017): 20-24
 Spawning of Brine shrimp cysts. Nineteen grams of Sodium chrloride (NaCl) will be

dissolved in 500 mL of distilled water to produce 38% artificial sea water (ASW) suitable for the

spawning brine shrimps (Artemia salina ). Two milligram of brine shrimp cysts will be allowed

to hatch in the ASW for 48 hours in constant aeration and illumination. After 48 hours, nauplii

will be separated from the shells using a micropipette.

Preparation of Treatments. Various concentrations of X. hongkongensis (10,000, 1,000,

100, 10, and 1 ppm) will be prepared. Table 2 shows the treatments including the positive

(Potassium dichromate) and negative (ASW) controls.

Table 2

Treatments used for Brine Shrimp Lethality Assay

Treatments Concentrations (ppm)

Treatment 1 10000
Treatment 2 1000
Treatment 3 100
Treatment 4 10
Treatment 4 1
Positive Control (Potassium dichromate)
Negative control ( Artificial sea water)

Exposure to Treatments. Ten brine shrimps will be introduced in each test tube

containing four mL each of test solutions including the controls. The mortality of the nauplii will

be observed and recorded after 24 hours of exposure. The procedure will be done in triplicates

and in three trials.

 Mortality determination on A. Salina. After 24 hours of exposure to the different

treatments, the mortality of the brine shrimp will be counted and recorded. By dividing the

number of dead nauplii by the total number of brine shrimp and multiplying it to 100 will give

the percent mortality. To ensure that the mortality of nauplii will be due to the bioactive

compounds and not by starvation, Abbot`s formula will be used.

Abbot`s Mortality Formula

% Mortality Due to Treatment = y-x x 100

100-x

Where: x = numbers of dead brine shrimps in the treatment

y = number of dead brine shrimps in the control

Onion Root Tip Chromosomal Aberration Assay

Allium cepa assay is a practical and economical anticancer pre-screening system15. It is

used to determine the genotoxic effect of bio-active compound on onion cells.16

Preparation of treatments. Serial dilution will be used in making 1000, 500, 250 and 125

μL/ml of Xylaria hongkongensis. Table 3 shows the treatments prepared including the positive (5

mg/L Maleic hydrazide) and negative controls (Distilled water with 1% DMSO).

15
Geirid Fiskesjo, “The allium test-as a standard in environmental monitoring. Hereditas” 102:99-112.
(1985)
16
Cresencio C. Cabuga Jr., Julene Joy Z. Abelada , Rene Rose Q. Apostado , Brent Joy H. Hernando, John
Erick C. Lador, Owen Lloyd P. Obenza, Christian James R. Presilda, Honelyn C. Havana “Allium cepa test: An
evaluation of genotoxicity” Proceedings of the International Academy of Ecology and Environmental Sciences,
2017, 7(1): 12-19
Table 3.

Preparation of treatments for ORTCAA

Treatments Concentrations ( μL/mL)

Treatment 1 1000
Treatment 2 500
Treatment 3 250
Treatment 4 125
Treatment 5 Positive control (5 mg/L Maleic hydrazide)
Treatment 6 Negative control (Distilled water with 1% DMSO)

Macroscopic Analysis. Macroscopic analysis will be carried out to find out the effect of

Xylaria hongkongensis on A. cepa’s root length and to compute the EC50 half maximal lethal

dose, the effective concentration to inhibit the root lengths exposed in the negative control.17

Identical 18 onion bulbs will be purchased from San Miguel Public Market. Onions’ dry

scales will be peeled off. Old roots will be removed using a scalpel to expose the root primordia.

The bulbs will be exposed to X. hongkongensis different concentrations, positive control and

negative control in 72 hours. After three days, the root lengths will be measured using a ruler and

submerged to Farmer’s fluid (three parts Ethyl alcohol and one part Glacial acetic acid) in 24

hours for microscopic analysis.

Microscopic analysis. Fixed onion root tips will be carefully placed in a slide and cut by

2mm. A drop of 1N HCl (Hydrochloric acid) will be used to hydrolyse the roots for 4-5 minutes.

17
Peter Firbas & Tomaz Amon, “Chromosome damage studies in the onion plant Allium cepa L..
Caryologia. International Journal of Cytology, Cystosystematics and Cytogenetics.”2014 67:1, 25-35
Aceto-carmine will be added to the treated slide to stain the chromosomes. The slide will be set

over the flame of an alcohol lamp and covered using a cover slip. The cells will be pressed and

excess stain will be expelled. A clear nail polish will be used to seal the cover slip.18 The cells

will be observed under a binocular microscope with 400x magnification in the laboratory of

Natural Science Research Institute (NSRI) – UP Diliman Campus to take a closer observation to

the cells, and the stages of mitosis. The mitotic index and mitotic phase index will be computed

using the data obtained.

The mitotic index (MI) will be computed by counting the numbers of cells under the

mitotic phase in 1000 cells using the formula below:

total number of dividing cells

Mitotic Index (MI) = x 100
1000

In addition, Mitotic Phase Index (MPI) will be computed by counting the number of cells

in the prophase, metaphase, anaphase, and telophase using the formula below:

total number of dividing cells (P/M/A/T)

Mitotic Phase Index (MI) = x 100
total number of dividing cells

Will bete Disposal

After the conduct of the study, the chemicals, reagents, and solvents used will be

disposed into their organic and inorganic containers. Used and unused Allium cepa will be put in

a black plastic bag to be brought in the municipal facility recovery area.

18
O. A. El-Shahaby, HM Abdel Migid, M. I. Soliman, and I. A. Mashaly. "Genotoxicity Screening of
Industrial Will betewater Using the Allium сера Chromosome Aberration Assay." Pak. J. Biol Sci 6, no. 1 (2003):
23-28.
 The microorganisms and agar used in the antimicrobial test will be autoclaved. The brine

shrimps will be bleached while zebra fish embryos will be put into ice and disposed according to

the regulated research institution’s guidelines.

Data analysis. To determine the effect of various concentrations of X. hongkongensis

extract, Analysis on Variance (ANOVA) will be used. Trend Line Fit Regression Analysis in

Microsoft Excel 2016 will be used to determine the half-maximal effective concentration (EC50)

on Onion Root Tip Chromosomal Aberration Assay and brine shrimp lethality assays.
 References

Alvin Ming Chak Tang, Regent Yau Ching Lam & Mike Wing Keung Leung “Xylaria

hongkongensis sp. nov. from an urban tree” Mycotaxon Volume 128, pp. 37–40 April–

June 2014

Alizza Yvette B. Memita, Sheena D. Macalinao, Lea Esperanza G. Reyes, Ernesto E. Damian Jr.

And Rich Milton R. Dulay “Toxicity And Teratogenicity OF Dieffenbachia amoena Leaf

Extract” IJBPAS, August 2018, 7(8): 1591-1600

Ananya Mandal, “What is staphylococcus aureus?”, (2018) News Medical Life Sciences,

Accessed from: https://www.news-medical.net/health/What-is-Staphylococcus-

Aureus.aspx

Cresencio C. Cabuga Jr., Julene Joy Z. Abelada , Rene Rose Q. Apostado , Brent Joy H.

Hernando, John Erick C. Lador, Owen Lloyd P. Obenza, Christian James R. Presilda,

Honelyn C. Havana “Allium cepa test: An evaluation of genotoxicity” Proceedings of the

International Academy of Ecology and Environmental Sciences, 2017, 7(1): 12-19

Dulay, Rich Milton R., Sofronio P. Kalaw, Renato G. Reyes, and Esperanza C. Cabrera.

"Embryo-toxic and teratogenic effects of Philippine strain of Lentinus tigrinus (tiger

sawgill basidiomycetes) extract on zebrafish (Danio rerio) embryos." Annals of

Biological Research 5, no. 6 (2014): 9-14.

El-Fadaly, H. A., Sherif Mohamed El-Kadi, Mostafa Maher El-Moghazy, Ahmed Ali Soliman,

and Mahmoud Salama Mahmoud El-Haysha. "Correlation Between Active Components

of Rocket (Eruca sativa) as Cytotoxicity (Brine Shrimp Lethality Assay)." American

Journal of Biomedical Science and Engineering 3, no. 2 (2017): 20-24

El-Shahaby, O. A., HM Abdel Migid, M. I. Soliman, and I. A. Mashaly. "Genotoxicity Screening

of Industrial Will betewater Using the Allium сера Chromosome Aberration Assay." Pak.

J. Biol Sci 6, no. 1 (2003): 23-28.

Geirid Fiskesjo, “The allium test-as a standard in environmental monitoring. Hereditas” 102:99-

112. (1985)

Geirid Fiskesjo, “The allium test-an alternative in environmental studies: the relative toxicity of

metal ions. Mutation Research” 197: 243-260. (1985)

Healthdirect. “Antioxidants. Healthdirect”. (2018), Accessed from

https://www.healthdirect.gov.au/antioxidants

Hudzicki, Jan. "Kirby-Bauer disk diffusion susceptibility test protocol." (2009).

Jayanth A, Muralidhar S.Talkad, “Neuroprotective Activity Of Stachytrapheta Indica On

Rotenone Induced Parkinson’s Disease In Zebra Fish” World Journal Of Pharmacy And

Pharmaceutical Sciences Volume 3, Issue 7, 940-955, 2014

Jiao-Jiao Zhang , Ya Li , Tong Zhou , Dong-Ping Xu , Pei Zhang , Sha Li and Hua-Bin Li

“Bioactivities and Health Benefits of Mushrooms Mainly from China” Molecules 2016

Jill Seladi-Schulman. “E. coli infection”, (2017), Healthline, Accessed from

https://www.healthline.com/health/e-coli-infection

Linda Crampton “Penicillin, Lovastatin, and Cyclosporine: Medicines From Fungi” Owlcation

August 30, 2019

Lilybeth F. Olowa and Olga M. Nuñeza “Brine Shrimp Lethality Assay of the Ethanolic Extracts

of Three Selected Species of Medicinal Plants from Iligan City, Philippines”

International Research Journal of Biological Sciences, November 2013 Vol. 2(11), 74-

77
NCCIH, “Antioxidants: In Depth. National Center for Complimentary and Integrative Health”,

(2013), Accessed from https://nccih.nih.gov/health/antioxidants/introduction.htm

Peter Firbas & Tomaz Amon, “Chromosome damage studies in the onion plant Allium cepa L..

Caryologia. International Journal of Cytology, Cystosystematics and Cytogenetics.”2014

67:1, 25-35

Sarah, Quazi Sahely, Fatema Chowdhury Anny, and Mir Misbahuddin. "Brine shrimp lethality

assay." Bangladesh Journal of Pharmacology 12, no. 2 (2017): Online-Jun.