Research Plan

Lowering Low-Density Lipoprotein Cholesterol in Dyslipidemia Using Sunflower (Helianthus

annuus) Seeds Ethanolic Extract in Zebrafish (Danio rerio) Model of Atherosclerosis

Leader:

Francene Hosillos

Members:

Agie Mae Roxas

Arielle Mae Castro

Aubrey Alfonso

Claire Gem Bajacan

Clark Kent Mateo

Emmanuel Nicolas

Katrina Mae Pascual

Chryztle Khate Hernandez

Paulene Mae San Pedro

Ronald Bong Villareal

 Rationale:

An estimated 3.8 million men and 3.4 million women die each year from Coronary Heart

Disease and its primary cause is atherosclerosis, the hardening and narrowing of the arteries due to

the build-up of fatty material and plaque, that reduces blood flow through the coronary arteries to

the heart muscle (Health Knowledge Organization United Kingdom, 2017). Dyslipidemia (high

cholesterol), the commonest cause of cardiovascular diseases, leads to lipid deposits on the arterial

wall, thereby aggravating the process of atherosclerosis (Garg, Aggarwal, & Sharma, 2015). An

accumulation of Low-Density Lipoproteins (LDL) in the arterial intima is an early step in

atherosclerosis, however despite the proposed strengths of the associations between cholesterol,

lipoproteins, and atherosclerosis, underlying mechanisms have not been completely clarified

(Sigurdsson, 2016). One in two Filipino adults faces borderline to high-cholesterol levels, with

46.9 percent Filipinos aged 20 and above at increased risk of cardiovascular diseases (Food and

Nutrition Research Institute, 2016).

Despite the existence of statins such as Atorvastatin (Lipitor), Pravastatin (Lipostat),

Simvastatin (Zocor), and Fluvastatin (Lescol) that generally help lower cholesterol levels, the

intake likely needs to remain for the long term because once the maintenance is halted, cholesterol

levels will go back up (Mayo Foundation for Medical Education and Research, 2018). Side-effects

of statins include muscle pain, liver damage, increased blood sugar levels which may eventually

lead to type 2 diabetes, and on some occasions, memory loss and confusion (Mayo Foundation for

Medical Education and Research, 2016). Surgical procedures such as stent placement, bypass
surgery, and balloon angioplasty not only are costly, ranging approximately from Php 60,000 –

Php 520,000 (Philippine Heart Center, 2017).

Sunflower (Helianthus annuus) seeds contains: good fats that may help protect the

arteries including monounsaturated and polyunsaturated fatty acids, B complex vitamins that are

helpful for a healthy nervous system, it is also a good source of phosphorus, magnesium, iron,

calcium, potassium, protein and vitamin E, as well as trace minerals, zinc, manganese, copper,

chromium and carotene (Green 2018). Sunflower seeds and pistachios were richest in

phytosterols (270-289 mg/100 g) which reduce blood levels of cholesterol, enhance the immune

response and decrease risk of certain cancers. Also, dried unhulled sunflower seeds are an

excellent source of vitamin E, the body's primary fat-soluble antioxidant that reduce blood levels

of cholesterol, and enhance the immune response (The George Mateljan Foundation, 2019).

Containing 35.17mg of Vitamin E (alpha-tocopherol), monosaturated fats (18.528g),

polyunsaturated fats (23.137g), magnesium (325mg), phosphorus (660mg), and potassium

(645mg) (United States Department of Agriculture - Agricultural Research Service, 2018).

Zebrafish shares 70 percent of genes with humans due to its comparable genetic structure

and 84 percent of genes are known to be related to human disease where embryos are nearly

transparent, allowing researchers to easily examine the development of internal structures (Your

Genome Organization, 2014). Through a combination of genetic, developmental, and

physiological advantages, the zebrafish has emerged as a major mechanistic discovery platform

for studying dyslipidemia and atherosclerosis from dietary interventions, exploration of various
aspects of lipoprotein production and processing, and genetic models for dyslipidemia and early

atherosclerosis (Schlegel, 2016).

This study aims to assess the potency of Sunflower (Helianthus annuus) seeds ethanolic

extract in lowering LDL cholesterol levels in dyslipidemic zebrafishes – fed with high

cholesterol diet (HCD) – as a model for atherosclerosis to act as a potential treatment and

contribute to the field of cardiology.

Research Questions:

This study will be conducted to evaluate the effect of Helianthus annuus seeds Ethanolic

Extract in reducing the risk of developing dyslipidemia using Danio Rerio model of

atherosclerosis.

1. What will be the effect of the varying concentrations (2, 3.25, 6.25, 17.5, 25, 50, 100

µg/ml) of HASEE on the mortality of Danio rerio in terms of:

A. Mortality rate after 24 hours post-treatment application (%)

B. Mortality rate after 48 hours post-treatment application (%)

C. Mortality rate after 72 hours post-treatment application (%)

2. Do the varied concentrations (2, 3.25, 6.25, 17.5, 25, 50, 100 µg/ml) of HASEE affect the

cholesterol level of Danio rerio?

 3. Do the varied concentrations (2, 3.25, 6.25, 17.5, 25, 50, 100 µg/ml) of HASEE affect the

heart pumping rate of Danio rerio?

A. Heart rate (beats per minute)

4. How do the varied concentrations of HASEE affect the cholesterol level of Danio rerio

fed with high cholesterol diet (HCD)?

5. What will be the effect of the varied concentrations of HASEE to lipid production present

in Danio Rerio?

6. Does the effect of the varied concentrations affect the appetite of Danio Rerio?

A. Lipids metabolized after 0.5 hours post-feeding

B. Lipids metabolized after 1 hour post-feeding

C. Lipids metabolized after 2 hours post-feeding

D. Lipids metabolized after 4 hours post-feeding

E. Lipids metabolized after 8 hours post-feeding

F. Lipids metabolized after 24 hours post-feeding

7. What will be the effect of the varied concentrations of sunflower (Helianthus annuus) seeds

ethanolic extract to development of zebrafish (Danio Rerio) in terms of?

A. Blood Vascular Development

B. Lymphatic Vascular Development

C. Dorsal Migration of Endothelial Cells

8. What will be the effect of the varied concentrations of sunflower (Helianthus annuus) seeds

ethanolic extract on the patterns of movement of zebrafish (Danio Rerio) in terms of:

A. Braking Movement

B. Maneuvering Movement