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VASODILATORY EFFECTS OF STRAWBERRY 2

Abstract

Introduction. A number of research studies have provided substantial evidence on strawberries’

preventive and therapeutic health benefits. With its rich source of phytochemicals and vitamins,

strawberries have been highly ranked among dietary sources of antioxidants and have been

studied to improve cardiovascular function in general.

Methodology. This study specifically evaluated the vasodilatory effects of the strawberry fruit

by measuring the changes in the diameter of the aortic rings harvested from the descending aorta

of twenty three 8 weeks old male Sprague-dawley spp. rats, exposed to strawberry (Fragaria x

ananassa “Sweet Charlie”) ethanol extract, at different concentrations specifically 0.2 mg/10mL

distilled water, 2mg/10mL and 5mg/10mL. Aortic ring diameter measurement was done using

light microscope (40x) with an installed micrometer immediately, 5 minutes and 10 minutes after

exposure to treatments.

Results and Discussion: Control group, exposed to 20 uL hydralazine (20mg/mL), displayed an

average of 20-30% dilatation over the course of 10 minutes. Groups exposed to strawberry

ethanol extracts at 0.2mg/10mL, 2mg/10mL & 5mg/10mL demonstrated a percentage dilatation

of about 10-15% dilatation. In general, the groups exposed to strawberry ethanol extracts only

achieved half the percent dilatation that occurred with hydralazine. Nevertheless, the extracts at

different concentrations revealed a significant percentage dilatation in rat aortic rings.

Conclusion: Strawberry ethanol extract appears to be an effective vasodilator and can potentially

be used to lower blood pressure in the management of hypertension.

Keywords: Strawberry (Fragaria x ananassa “Sweet Charlie”), Ethanol Extract,

Vasodilation, Hypertension.
VASODILATORY EFFECTS OF STRAWBERRY 3

Vasodilatory Effects of Strawberry (Fragaria x ananassa “Sweet Charlie”) Ethanol Extracts in

Sprague-Dawley Rats

Cardiovascular diseases have been the leading cause of morbidity and mortality in the

Philippines for the past decades. Major form of this ailment includes hypertension, a chronic

disorder characterized by a persistently elevated blood pressure above 140/90 mmHg or greater.

Often referred to as the “silent killer”, its course often remains asymptomatic and if left

undiagnosed, it can result to damage to target organs including the kidneys, eyes, heart and brain.

[1]

Fresh strawberries contain nutrients including ascorbic acid [2] and are considered in folk

medicine as a potential remedy to various conditions due to their astringent and diuretic

properties [3]. It is also studied to have antithrombogenic [4] and antidiabetic [5] properties. In

the form of fruit paste, they are used to heal skin diseases, wounds and the juice for inflammation

of the nerves and lungs. [5][6][7] Antioxidants in strawberries also help to lessen the risk of

cardiovascular incidents by inhibition of LDL-cholesterol oxidation or improved vascular

endothelial function [8]. These beneficial effects have been mostly attributed to the phenolic

compounds that are found in strawberry [9]. The bioactive properties of different strawberry

parts (fruits, leaves and roots) have been related to the presence of various phenolic compounds,

such as hydroxycinnamic acid and ellagic acid derivatives (e.g., ellagitannins), and flavonols

[10].

Apart from all the documented therapeutic effects, properties of strawberry fruits on

vasodilation have not been extensively measured and studied. A previous study on the

vasodilatory effects of strawberry was conducted and it revealed that powderized California
VASODILATORY EFFECTS OF STRAWBERRY 4

strawberry, taken daily, can potentially benefit individuals with prehypertension. It is

recommended that more research, however, is needed to strengthen this association [11].

Strawberries (family: Rosaceae; genus: Fragaria; species: ananassa) are widely grown

and consumed in the Cordillera Region where this study is conducted. They are popular due to

their desirable sweet taste and attractive aroma. After thorough review of available literatures,

the researchers were not able to find studies utilizing Fragaria x ananassa “Sweet Charlie” in

blood vessel vasodilation determination. This specific species of strawberry is opted for the study

since strawberries grown in the region is of this species. A study revealed that phytochemical

compounds in Fragaria x ananassa responsible for its beneficial effects are significantly higher in

leaves than fruits [12]. However, fruits were used in this study considering that the fruit is the

one popularly consumed in the human diet [13] and it is widely distributed and is more

accessible. Most studies on strawberry’s therapeutic properties used aqueous extracts. After

exhaustion of all possible resources, the researchers were not able to find experiments utilizing

ethanol extracts of the strawberry fruit in determining vasodilatation. This study thereby utilizes

ethanol extraction of strawberry as this method may enhance its antioxidant properties, which

then may potentially contribute to its vascular regulatory effects [14].

One major pathophysiologic mechanism of hypertension can be attributed to vascular

endothelial dysfunction which lead to a decrease in the diameter of the vessel lumen [15]. This

pathophysiologic pathway can be targeted by induced vasodilation from which how many

antihypertensive agents work including potentially, strawberry fruit ethanol extracts, if proven.

In this study, we will examine the direct, dose-dependent effects of strawberry (Fragaria x

ananassa “Sweet Charlie”) in Sprague Dawley rats as experimental models. Effects on the

harvested rat aortic rings will be measured to evaluate its vasodilatory effects.
VASODILATORY EFFECTS OF STRAWBERRY 5

Method

Animal Welfare

The experiments were performed following all the guidelines established and approved

by the Ethics Committee of Saint Louis University. The researchers based their methodology

from the ethical guidelines in the use of experimental animals for research, outlined by

Institutional Animal Care and Use Committee (IACUC).

Furthermore, the researchers have abided to the proper care and use of animals in

laboratory experiments as stated in the R.A. No. 8485 – Animal Welfare Act of 1198 and its

Implementing Rules and Regulations under DA Administration no. 40 series of 1998 and Code

of Practice for the Care and Use of Laboratory Animals in the Philippines, 2nd edition, 2002,

developed by the Philippine Association Animal Science.

After conducting the experiments, the rats were packed in polythene bags and buried in

the deep ground covered with disinfectants.

Study Design

This study utilized controlled experimental research design in which an in vitro analysis

of the effects of strawberry (Fragaria x ananassa “Sweet Charlie”) ethanol extracts in the aortic

ring diameter of rats (Sprague-dawley spp.) was performed.

Experimental Animal

Twenty (20) 8-week old male Sprague-dawley spp. rats weighing 150-250 g were

procured from a local pet shop and were housed in the SLU Animal Housing Unit, Baguio City.

The rats were randomly grouped and separated into five rats per cage (5 cages) as they

already belong to the same criteria such as sex, age and weight. They were grouped into five rats
VASODILATORY EFFECTS OF STRAWBERRY 6

per cage considering that they prefer group living but not with a very large group as this can

induce stress and aggressive behavior.

For rats weighing 150-300g, a cage height requirement of 22 cm (9 inches) and length of

60cm (24 inches) should be provided. The researchers used a rectangular 24 x 12 in cage to

allow upright standing behavior, stretch upright for interaction with other rats, allow access of

food and water, and to provide enough space to move around to avoid bony and cartilaginous

damage. The rat cages were made from plastic containers covered with the original plastic cover

and one of the walls was replaced by wire mesh for ventilation and to allow visual and olfactory

contact with other rat groups.

The rats were acclimatized for 9 days (March 25 to April 3) in the SLU Animal House

Laboratory. This facility is maintained at 21-27 degrees Celsius and is well-ventilated. They

were given pellets with 20-21 CP (Crude Protein) level, and purified drinking water.

Study Sample

Sample size is determined using the “resource equation” method. This method is used

when it is not possible to assume about effect size and to estimate standard deviation as no

previous findings are available, or when multiple endpoints are measured or complex statistical

procedure is used for analysis.

In this method, a value “E” is measured. “E” is the degree of freedom of analysis of

variance (ANOVA). The sample size will be dependent on the value of E which should only be

between 10 and 20, keeping in mind that a value less than 10 will entail increasing the number of

study animals to increase the chance of producing a significant result. Whereas a result of 20 or

more would not strengthen the chance of getting a significant result.


VASODILATORY EFFECTS OF STRAWBERRY 7

The working formula that is advised in some of the previous literatures related to the

study would be:

E= total number of study animals – total number of groups

Shown below is the computation used in the study to determine whether the number of study

animals to be used for the experiment is acceptable:

E = (5 study animals per group x 4 groups) – 4 groups


E = 20 – 4
E = 16

The considered acceptable sample size in this study is, therefore, 16. The reduction in the

sample size will not affect the results significantly as it is within the acceptable range of sample

size.

Experimental Procedure

Collection of Plant Material and Preparation of Treatments. Organically grown

strawberry (Fragaria x anananassa “Sweet Charlie”) was purchased from Benguet State

University and was specifically named by a licensed agriculturist. Exactly 1 kg of fresh

strawberry was oven-dried at Saint Louis University Natural Science Laboratory that yielded

400g of dried specimens. Dried strawberry fruits were soaked in 95% ethanol for 3 days. They

were filtered using cotton-plugged funnel and the filtrate was subjected to rotary evaporation.

Extraction via Rotary evaporation of the strawberry was performed at Don Mariano Marcos

Memorial State University Laboratory, March 28, 2017, 6 days prior to the experiment proper.

Formulations of specific dosages for treatment were carried out the same day of the

experiment proper.

Laboratory experiment was performed at the SLU Animal Research facility on April 3,

2017.
VASODILATORY EFFECTS OF STRAWBERRY 8

Isolation and Preparation of Aortic Rings. Rats were put to sleep using 90% Ether, and

euthanized via dislocation of the atlanto-occipital bone. Sternotomy was done to gain access to

the rat’s thoracic cavity. The rat’s heart and surrounding vessels were harvested and were

immediately washed using .9% NaCl solution. Descending thoracic aorta was carefully detached

from the heart and cleared of adjacent tissues including fats, leaving the layers intact particularly

the tunica media and intima. 2 aortic rings measuring 1mm thick were isolated from each of the

descending aorta and were placed in a clean and dry microscope slide.

Evaluation of the Vasodilatory Effects of Treatments. The diameter of all rings was

measured prior to any interventions. Light microscope (40x) installed with a micrometer was

used to carry out the measurements. Experimental aortic rings were initially exposed to 1 drop

(20 μL) of epinephrine (1mg/mL) for pre-contraction for a period of 5 minutes to achieve the

peak effects of epinephrine. Accurate measurement of the treatment quantity was achieved using

a calibrated micropipette. After peak, sustained contraction, the aortic rings were exposed to

various treatments that are divided in to Group 1 (Positive Control Group), Group 2 (Treatment

Group 1), Group 3 (Treatment Group 2) and Group 4 (Treatment Group 3) accordingly.

Group 1 (Positive Control Group). 12 aortic rings, 2 rings from each of the 5 different

mice were used for the control group, 2 rings placed on 1 clean and dry slide. The aortic ring on

the left side of the slide, designated as ring “A”, did not receive any form of treatment whereas

the one on the right side, designated as ring “B”, was exposed to 1 drop (20 μL) of Hydralazine

(20mg/mL). Measurements were done immediately after exposure. Further measurements were

performed at 5, and 10 minutes after exposure to treatments. Rings A were measured

concurrently with Rings B.


VASODILATORY EFFECTS OF STRAWBERRY 9

Group 2 (Treatment Group 1). 12 aortic rings, 2 rings from each of the 5 different mice

were used for the treatment group 1. All of the rings were initially measured prior to treatments.

Rings B were pre-contracted and were exposed to 0.2 mg/10mL dose of strawberry ethanol

extract. Measurements were done immediately, 5 minutes and 10 minutes after exposure. Rings

A were measured concurrently with Rings B.

Group 3 (Treatment Group 2). 10 aortic rings, 2 rings from each of the 5 different mice

were used for the treatment group 2. Two more rings(1 slide) are supposed to be included in this

group, however, precontraction result turned out to be paradoxical, as Ring B dilated with

epinephrine. It is presumed that the isolated structure must have been trachea rather than the

aorta. The rest of Rings B were precontracted and were exposed to 2.0 mg/10mL dose of

strawberry ethanol extract. Measurements were done immediately, 5 minutes and 10 minutes

after exposure. Rings A were measured concurrently with Rings B.

Group 4 (Treatment Group 3). 12 aortic rings, 2 rings from each of the 5 different mice

were used for the treatment group 2. All of the rings were initially measured prior to treatments.

Rings B were precontracted and were exposed to 5.0 mg/10mL dose of strawberry ethanol

extract. Measurements were done immediately, 5 minutes and 10 minutes after exposure. Rings

A were measured concurrently with Rings B.

A summary of groups with the corresponding treatments can be found in table 1 of the

appendix.

Overall, relaxation, which is a measure of inhibition of contraction in whole, spirally cut

aortic strip pre-contracted with contracting agent, were determined using a measurement before

and after treatment administration and were individually recorded and calculated. All tabulated

results are included in the appendices.


VASODILATORY EFFECTS OF STRAWBERRY 10

Results

Results revealed that the comparison of pre-treated rings to the effects of hydralazine (n=6),

0.2 mg/10ml strawberry extract (n=6), 2 mg/10ml strawberry extract (n=5) and 5 mg/10 ml of

strawberry extract (n=6), shown in Table 2 and reflected in Figure 1, exhibited a statistically

non-significant difference in percentage vasodilation, when comparing the control and the

treatment groups, after 5 min (p value = 0.76) and 10 min (p value = 0.38) of exposure. The control,

Hydralazine group, revealed an average percent dilatation of 19.71 (sd = 23.66) percent change

and 27.62 (sd = 24.83) percent at 5 and 10 min exposure respectively. Whereas, the different doses

of extracts showed a similar vasodilatory trend nearly half the capability of hydralazine at 11.41

(sd = 8.59), 13.75 (sd = 6.50), 14.03 (sd = 7.48) at 5 min respectively; and 14.64 (sd = 7.84), 15.75

(sd= 8.10), 15.83 (sd= 8.35) at 10 min, correspondingly. The aortic rings revealed a similar

statistically non-significant difference in percentage vasodilatation when the pre-contracted rings

were exposed at intervals of 5 minutes (p value = 0.81) and 10 minutes (p value = 0.96) to the

different dosages of extracts. Inferential statistical results were computed using OpenEpi -

ANOVA (Analysis of Variance) with a confidence interval of 95%.

Discussion

Hypertension remains as one of the top 10 causes of mortality and morbidity worldwide.

Proper treatment relies on targeting the factors affecting Blood Pressure, namely Cardiac Output

and Total Peripheral Resistance. Escalating total or systemic peripheral resistance coincides with

the increase in cases of hypertension and management relies on vasodilatation. Hydralazine

remains as one of the drugs given for hypertension, similar vasodilatory effects were observed in

aortic rat rings.


VASODILATORY EFFECTS OF STRAWBERRY 11

Strawberries (family: Rosaceae; genus: Fragaria; species: anasa) are widely grown and

consumed in the Cordillera Region. Previous studies have revealed its potential to increase the

lumen of blood vessels due to the presence of polyphenols, which has been shown to produce

endothelial-dependent vasodilatation [25]. Additional researches attributed the vasodilatory

effects to the presence of procyanidin B1, present in wild strawberries [22, 23]. Furthermore,

Resveratrol, one of the most examined polyphenolic compound with proven beneficial biological

effects includes vasomodulatory capability [21]. As previously stated by past researches, the

vasodilatory effect of strawberries is most likely not the result of one specific polyphenol

compound but of a collection of different chemical compounds exhibiting vasodilatory effects.

In our study, we examined the direct vascular changes in rat aortic rings resulting from its

exposure to hydralazine and the different dosages of the strawberry ethanol extract. In terms of

the overall vasodilatory capability, the drug compared – hydralazine, displayed an unrivaled

percentage dilatation over all of the extracts, with an average of 20 - 30% percentage dilatation

over the course of 10 min. The different strawberry extracts, namely 0.2mg/10ml, 2mg/10ml and

5mg/10ml of extracts, only achieved half the percent dilatation capability of hydralazine.

Nevertheless, all of the extracts revealed a significant percentage dilatation in rat aortic rings.

The discrepancy of the results could have stemmed from the fact that hydralazine,

composed only of the active vasodilatory component, has a higher capability to increase the

lumen of the aortic ring: as compared to an extract, composed of different phytochemicals and

polyphenols which exhibits only a proven vasodilatory capability from previous protocols [24].

The effect on the mechanical dilatation resulting from the impact of the fluid dropped on the

aortic ring, as a confounder, was excluded in the research because there was no immediate

vasodilation observed in the aortic rings upon initial exposure. Rather, the vasodilatory effects of
VASODILATORY EFFECTS OF STRAWBERRY 12

the treatments were observed at least 5 minutes after exposure coinciding with the expected peak

action of the treatments. In addition, Ring A per slide (no treatment) did not exhibit any changes

along the course of the experiment, signifying that direct exposure to the environment did not

induce any change in the parameter being measured. Further analysis of the results revealed that

the dosages namely 0.2mg/10ml, 2 mg/10ml and the max dose of 5mg/10ml revealed a

seemingly minute linear increase in vasodilation. The study revealed that the least dose and the

highest dose exhibited statistically the same vasodilatory capabilities, which was opposite from

the previous dose-dependent capabilities of strawberry extracts seen in the study done by Modun

[21]. This effect could be attributed to the achievement of the maximal dose for the maximum

attributable vasodilatory effect. Further reiterating that a smaller dose than 0.2 mg/10ml of

extract could still be tested to exhibit a dose-response vasodilatory effect.

Although the present study yielded some preliminary findings, its design is not without

flaws. A number of caveats needs to be noted regarding the present study. The limitations are as

follows: In the harvesting of the rings, the researchers formed three dissecting teams to gather the

aortic rings from the Sprague Dawley rats. Although, the procedures in rat dissection were

almost the same, minute differences in the techniques and location of dissection may have

altered the specimen. In addition, the size of rats significantly hindered the surgical team because

of the relative size of the aortic rings.

Based on the discussion above, future researches should be cautious of the limitations

stated. The recommendations of t are as follows: Conduction of the procedure should be

performed by a single individual for a uniform technique. Larger subjects would have yielded an

easier dissection as well as data interpretation. In addition, a smaller dose for the extract,

including further chemical extraction of the main active component found in strawberries
VASODILATORY EFFECTS OF STRAWBERRY 13

correlating to its vasodilatory property, should be done to fully isolate the vasodilatory

capabilities of the strawberry fruit.

In conclusion, the ethanol extract of Fragaria ananasa “Sweet Charlie” strawberry fruit

appears to be an effective direct aortic ring vasodilator with half the capability of hydralazine, a

potent vasodilating drug. The study shows that strawberry ethanol-extracts have the capability to

vasodilate rat aortic rings which can have further implications in the management of blood vessel

resistance.

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Appendix A

Table 1
Summary of the control and treatment groups
GROUP AORTIC RINGS TREATMENT

Positive Slide 1 (Ring A, Ring B) Ring A: no treatment


Control Slide 2 (Ring A, Ring B) Ring B:
Group Slide 3 (Ring A, Ring B) Precontraction w/ epinephrine
Slide 4 (Ring A, Ring B) - diameter measured after 5 minutes
Slide 5 (Ring A, Ring B) Exposure to hydralazine
Slide 6 (Ring A, Ring B) - diameter measured immediately, 5 minutes
and 10 minutes after exposure
Treatment Slide 1 (Ring A, Ring B) Ring A: no treatment
Group 1 Slide 2 (Ring A, Ring B) Ring B:
Slide 3 (Ring A, Ring B) Precontraction w/ epinephrine
Slide 4 (Ring A, Ring B) - diameter measured after 5 minutes
Slide 5 (Ring A, Ring B) Exposure to 0.2mg/10mL strawberry ethanol
Slide 6 (Ring A, Ring B) extract
- diameter measured immediately, 5 minutes
and 10 minutes after exposure
Treatment Slide 1 (Ring A, Ring B) Ring A: no treatment
Group 2 Slide 2 (Ring A, Ring B) Ring B:
Slide 3 (Ring A, Ring B) Precontraction w/ epinephrine
Slide 4 (Ring A, Ring B) - diameter measured after 5 minutes
Slide 5 (Ring A, Ring B) Exposure to 2.0mg/10mL strawberry ethanol
extract
- diameter measured immediately, 5 minutes
and 10 minutes after exposure
Treatment Slide 1 (Ring A, Ring B) Ring A: no treatment
Group 3 Slide 2 (Ring A, Ring B) Ring B:
Slide 3 (Ring A, Ring B) Precontraction w/ epinephrine
Slide 4 (Ring A, Ring B) - diameter measured after 5 minutes
Slide 5 (Ring A, Ring B) Exposure to 5.0/10mL strawberry ethanol
Slide 6 (Ring A, Ring B) extract
- diameter measured immediately, 5 minutes
and 10 minutes after exposure
Total: 23 slides
46 aortic rings
12 rings per group
2 rings per slide
(Rings A & B)
VASODILATORY EFFECTS OF STRAWBERRY 17

Table 2
Summary Table for the Mean Percent Dilatation and Standard Deviation of all groups after 5 min
and 10 min of exposure.
After 5 min Exposure After 10 min Exposure
Group n Mean Percent Standard Mean Standard
Dilatation Deviation Percent Deviation
Dilatation
Epinephrine and 6 19.71 23.66 27.62 24.83
Hydralazine
Epinephrine + 0.2mg/10m 6 11.41 8.59 14.64 7.84
Strawberry Ethanol Extract
Epinephrine + 2mg/10m 5 13.75 6.50 15.75 8.10
Strawberry Ethanol Extract
Epinephrine + 5mg/10m 6 14.03 7.48 15.83 8.35
Strawberry Ethanol Extract

Table 3
Summary Table for the ANOVA results at 5 min and at 10 min.
Group P value (5 min) P value (10 min)
All Groups 0.76 0.38
Strawberry Ethanol Extracts only 0.81 0.96
Note: Confidence Interval = 95%

Table 4
Group 1 (Epinephrine + Hydralazine) Raw Data:
Aortic Ring Changes Following Exposure to Epinephrine and Hydralazine after 5 min and
10min.
Aortic Ring Diameter (μm)
Epinephrine Hydralazine
Rat No Initial Immediate 5 min Immediate 5min 10min
Diameter Diameter (μm) Diameter (μm) (μm)
(μm) (μm) (μm)
Rat 1 37 37 35 35 58 60
Rat 2 35 35 32 32 34 32
Rat 3 41 41 40 40 42 46
Rat 4 44 44 42 42 52 53
Rat 5 36 36 32 32 36 37
Rat 6 42 42 40 40 42 55
Note: Initial Diameter – Initial Diameter of the aortic ring after placement
Immediate Diameter – Diameter of the aortic ring after immediate exposure to the treating agent
5min – Diameter of the aortic ring after 5 minutes of exposure to the treating agent
10min – Diameter of the aortic rings after 10 minutes of exposure to the treating agent
VASODILATORY EFFECTS OF STRAWBERRY 18

Table 5
Group 2 (Epinephrine + 0.2mg/10ml Strawberry Ethanol Extract) Raw Data:
Aortic Ring Changes Following Exposure to Epinephrine and 0.2mg/10ml Strawberry Ethanol
Extract after 5 min and 10min.
Aortic Ring Diameter (μm)
Epinephrine 0.2mg/10ml Strawberry Ethanol
Extract
Rat No Initial Immediate 5 min Immediate 5min 10min
Diameter Diameter (μm) Diameter (μm) (μm)
(μm) (μm) (μm)
Rat 1 37 37 36 36 42 44
Rat 2 31 31 29 29 35 35
Rat 3 45 45 41 41 46 47
Rat 4 59 59 55 55 56 57
Rat 5 36 36 35 35 41 42
Rat 6 34 34 30 30 30 32
Note: Initial Diameter – Initial Diameter of the aortic ring after placement
Immediate Diameter – Diameter of the aortic ring after immediate exposure to the treating agent
5min – Diameter of the aortic ring after 5 minutes of exposure to the treating agent
10min – Diameter of the aortic rings after 10 minutes of exposure to the treating agent

Table 6
Group 3 (Epinephrine + 2mg/10ml Strawberry Ethanol Extract) Raw Data:
Aortic Ring Changes Following Exposure to Epinephrine and 2mg/10ml Strawberry Ethanol
Extract after 5 min and 10min.
Aortic Ring Diameter (μm)
Epinephrine 2mg/10ml Strawberry Ethanol Extract
Rat No Initial Immediate 5 min Immediate 5min 10min
Diameter Diameter (μm) Diameter (μm) (μm)
(μm) (μm) (μm)
Rat 1 33 33 30 30 35 37
Rat 2 37 37 30 30 35 35
Rat 3 48 48 47 47 48 48
Rat 4 37 37 30 30 35 36
Rat 5 33 33 30 30 35 35
Note: Initial Diameter – Initial Diameter of the aortic ring after placement
Immediate Diameter – Diameter of the aortic ring after immediate exposure to the treating agent
5min – Diameter of the aortic ring after 5 minutes of exposure to the treating agent
10min – Diameter of the aortic rings after 10 minutes of exposure to the treating agent
VASODILATORY EFFECTS OF STRAWBERRY 19

Table 7
Group 4 (Epinephrine + 5mg/10ml Strawberry Ethanol Extract) Raw Data:
Aortic Ring Changes Following Exposure to Epinephrine and 5mg/10ml Strawberry Ethanol
Extract after 5 min and 10min.
Aortic Ring Diameter (μm)
Epinephrine 5mg/10ml Strawberry Ethanol Extract
Rat No Initial Immediate 5 min Immediate 5min 10min
Diameter Diameter (μm) Diameter (μm) (μm)
(μm) (μm) (μm)
Rat 1 41 41 39 39 46 46
Rat 2 46 46 40 40 50 52
Rat 3 47 47 41 41 47 47
Rat 4 48 48 37 37 41 41
Rat 5 47 47 45 45 51 52
Rat 6 41 41 40 40 41 42
Note: Initial Diameter – Initial Diameter of the aortic ring after placement
Immediate Diameter – Diameter of the aortic ring after immediate exposure to the treating agent
5min – Diameter of the aortic ring after 5 minutes of exposure to the treating agent
10min – Diameter of the aortic rings after 10 minutes of exposure to the treating agent
VASODILATORY EFFECTS OF STRAWBERRY 20

Table 8
ANOVA Table of the Four Groups at 5 min

Analysis of Variance (ANOVA)


Group N (count) Mean Std. Dev. Std. error

1 6 19.71 23.66
2 6 11.41 8.59
3 5 13.75 6.5
4 6 14.03 7.48

ANOVA Table

Source of Mean
Sum of squares d.f F statistics p-value1
variation square
Between Groups 222.64 3 74.21 0.389 0.76
Within Groups 3616.67 19 190.35
Total 3839.32 22

Chi square d.f p-value1


Test for equality of
10.55 3 0.01
variance

95% CI of individual 95% CI assuming equal


sample mean variance

Group Mean Lower Limit Upper Limit Lower Limit Upper Limit

1 19.71 -5.11 44.53 5.23 34.18


2 11.41 2.39 20.42 -3.06 25.88
3 13.75 5.67 21.82 -3.38 30.88
4 14.03 6.18 21.87 -0.44 28.50
1
p-value (two-tailed)
VASODILATORY EFFECTS OF STRAWBERRY 21

Table 9
ANOVA Table of the Four Groups at 10 min

Analysis of Variance (ANOVA)

Group N (count) Mean Std. Dev. Std. error


1 6 27.62 24.83
2 6 14.64 7.84
3 5 15.75 8.1
4 6 15.83 8.35

ANOVA Table
Source of Mean
Sum of squares d.f F statistics p-value1
variation square
Between Groups 674.97 3 224.99 1.06 0.38
Within Groups 4001.03 19 210.58
Total 4675.99 22
Chi square d.f p-value1
Test for equality of
10.08 3 0.017
variance

95% CI of individual 95% CI assuming equal


sample mean variance
Group Mean Lower Limit Upper Limit Lower Limit Upper Limit
1 27.62 1.56 53.67 12.39 42.84
2 14.64 6.41 22.86 -0.58 29.86
3 15.75 5.691 25.80 -2.26 33.76
4 15.83 6.88 24.41 0.42 30.87
1
p-value (two-tailed)
VASODILATORY EFFECTS OF STRAWBERRY 22

Table 10
ANOVA Table of the Different Strawberry Extracts at 5 min

Analysis of Variance (ANOVA)


Group N (count) Mean Std. Dev. Std. error

1 6 11.41 8.59
2 5 13.75 6.5
3 6 14.03 7.48

ANOVA Table
Source of Mean
Sum of squares d.f F statistics p-value1
variation square
Between Groups 24.33 2 12.16 0.20 0.81
Within Groups 817.69 14 58.40
Total 842.03 16

Chi square d.f p-value1


Test for equality of
0.31 2 0.85
variance

95% CI of individual 95% CI assuming equal


sample mean variance

Group Mean Lower Limit Upper Limit Lower Limit Upper Limit

1 11.41 2.39 20.42 3.38 19.43


2 13.75 5.67 21.82 4.26 23.23
3 14.03 6.18 21.87 6.00 22.05
1
p-value (two-tailed)
VASODILATORY EFFECTS OF STRAWBERRY 23

Table 11
ANOVA Table of the Different Strawberry Extracts at 10 min

Analysis of Variance (ANOVA)


Group N (count) Mean Std. Dev. Std. error

1 6 14.64 7.84
2 5 15.75 8.1
3 6 15.83 8.35

ANOVA Table

Source of Mean
Sum of squares d.f F statistics p-value1
variation square
Between Groups 5.18 2 2.59 0.03 0.96
Within Groups 918.38 14 65.59
Total 923.56 16

Chi square d.f p-value1


Test for equality of
0.01 2 0.99
variance

95% CI of individual 95% CI assuming equal


sample mean variance

Group Mean Lower Limit Upper Limit Lower Limit Upper Limit

1 14.64 6.41 22.86 6.14 23.13


2 15.75 5.69 25.80 5.69 25.80
3 15.83 7.06 24.59 7.33 24.32
1
p-value (two-tailed)
VASODILATORY EFFECTS OF STRAWBERRY 24

Figure 1:
Vasodilatory Effects of Hydralazine and the Different doses of the Extract on rat Aortic Rings

30

25
Percentage Vasodilatation

20

15

10

0
Epinephrine + Epinephrine + Epinephrine + 2mg/10 ml Epinephrine + 5mg/10ml
Hydralazine 0.2mg/10ml Extract Extract Extract
Control and Treatment Groups at 5 min and 10 min

5min 10min
VASODILATORY EFFECTS OF STRAWBERRY 25

Appendix B

ANIMAL CARE AND USE STATEMENT


(Protocol Review Form)

I. PROCEDURE(S) OR TITLE OF RESEARCH/STUDY:


Vasodilatory Effects of Strawberry (Fragaria ananassa x Sweet Charlie) Ethanol Extracts
on Sprague Dawley Rats
II. PURPOSE / OBJECTIVE
General Object:
To determine the vasodilatory effects of strawberry fruit (Fragariavescalinn.) ethanol
extracts on Sprague Dawley rats
Specific Objective
1. To determine the changes in rat aortic ring diameter after exposure to strawberry
fruit ethanol extracts.
2. To determine the significant difference among dose-dependent vasodilatory effects
of strawberry fruit extracts using the following dosages:
a) 0.2 mg extract /10 ml distilled H2O
b) 2 mg extract /10 ml distilled H2O
c) 5 mg extract /10 ml distilled H2O
3. To determine vasodilatory effects of the extracts to endothelium-denuded aortic
rings.
III. DURATION OR TIME FRAME:
The experiment is expected to be performed for at least 3-4 weeks from the
extraction process to the determination of vasodilatory effect until the last day.
Experimentation is scheduled on February 20, 2017 until March 07, 2017
IV. RESPONSIBLE PERSON OR PRINCIPAL INVESTIGATOR:
A. NAME: Dr. Leo Emmanuel Buñag
QUALIFICATION: Research Adviser
B. NAME: Adrian F. Bugayong
QUALIFICATION: Group leader
V. BACKGROUND AND SIGNIFICANCE OF THE PROCEDURE OR RESEARCH:

Cardiovascular diseases have been the leading cause of morbidity and mortality in
the Philippines for the past decades. Major form of this ailment includes hypertension, a
chronic disorder characterized by a persistently elevated blood pressure above 140/90
mmHg or greater. Often referred to as the “silent killer”, its course often remains
asymptomatic and if left undiagnosed, it can result to damage to target organs including
the kidneys, eyes, heart and brain. [1]
Fresh strawberries contain nutrients including ascorbic acid [2] and are considered
in folk medicine as a potential remedy to various conditions due to their astringent and
diuretic properties [3]. It is also studied to have antithrombogenic [4] and antidiabetic [5]
VASODILATORY EFFECTS OF STRAWBERRY 26

properties. In the form of fruit paste, they are used to heal skin diseases, wounds and the
juice for inflammation of the nerves and lungs. [5][6][7] Antioxidants in strawberries also
help to lessen the risk of cardiovascular incidents by inhibition of LDL-cholesterol
oxidation or improved vascular endothelial function [8]. These beneficial effects have been
mostly attributed to the phenolic compounds that are found in strawberry [9]. The bioactive
properties of different strawberry parts (fruits, leaves and roots) have been related to the
presence of various phenolic compounds, such as hydroxycinnamic acid and ellagic acid
derivatives (e.g., ellagitannins), and flavonols [10].
Apart from all the documented therapeutic effects, properties of strawberry fruits
on vasodilation have not been extensively measured and studied. A previous study on the
vasodilatory effects of strawberry was conducted and it revealed that powderized
California strawberry, taken daily, can potentially benefit individuals with
prehypertension. It is recommended that more research, however, is needed to strengthen
this association [11].
Strawberries (family: Rosaceae; genus: Fragaria; species: ananassa) are widely
grown and consumed in the Cordillera Region where this study is conducted. They are
popular due to their desirable sweet taste and attractive aroma. After thorough review of
available literatures, the researchers were not able to find studies utilizing Fragaria x
ananassa “Sweet Charlie” in blood vessel vasodilation determination. A study revealed
that phytochemical compounds in Fragaria x ananassa responsible in its beneficial effects
are significantly higher in leaves than fruits [12]. However, fruits were used in this study
considering that the fruit is the one popularly consumed in human diet [13] and it is widely
distributed and is more accessible. Most studies on strawberry’s therapeutic properties used
aqueous extracts. After exhaustion of all possible resources, the researchers were not able
to find experiments utilizing ethanol extracts of the strawberry fruit in determining
vasodilatation. This study thereby utilizes ethanol extraction of strawberry as this method
may enhance its antioxidant properties, which then may potentially contribute to its
vascular regulatory effects [14].
One major pathophysiologic mechanism of hypertension can be attributed to
vascular endothelial dysfunction which lead to decreased in the diameter of the vessel
lumen [15]. This pathophysiologic pathway can be targeted by induced vasodilation from
which how many antihypertensive agents work including potentially, strawberry fruit
ethanol extracts, if proven. In this study, we will examine the direct, dose-dependent effects
of strawberry (Fragaria x ananassa “Sweet Charlie”) in Sprague Dawley rats as
experimental models. Effects on the harvested rat aortic rings will be measured to evaluate
its vasodilatory effects

VI. DESCRIPTION OF METHODOLOGIES/ EXPERIMENTAL DESIGN


A. Type of animal to be used (species): Sprague Dawlery Rats
B. Source of the animals: VMU College of Veterinary Medicine
C. Reason? Basis for selecting the animal species: Closest genome to humans
D. Sex and number of animals: 30 Males
E. Quarantine and/or acclimation or conditioning process: five animals will be
placed per cage (6). Room will be conditioned with a temperature of 21-27C,
humidity of40-70 %. The room is provided with well-ventilation as deemed
VASODILATORY EFFECTS OF STRAWBERRY 27

appropriate. Lighting provided for 12 hours light and 12 hours dark exposure
pattern.
F. Animal care procedures
1.Cage type: Metal Rat Cages
2.Number of animals per cage: 5
3.Cage cleaning method: minimum once a week or as often as needed
4.Room temperature. Humidity, ventilation and lighting
a) Room temperature: 21-27C
b) Humidity: 40-70%
c) Ventilation: well ventilation as deemed appropriate
d) Lighting: 12 hours light and 12 hours dark exposure pattern
5.Animal Diet and feeding and watering methods
a) Diet: pellets with 20-21 CP (Crude Protein level)
b) Water: ad libitum
G. Experimental or animal manipulation methods
1.General description of animal manipulation methods (including method of
conditioning):
Thirty male Sprague-dawley spp. mice weighing 250-300 g will be
procured and housed at Saint Louis University - Animal Laboratory,
Benguet, Cordillera Administrative Region. The Sprague-dawley spp. rats
will be placed in 6 different cages (5 rats in each cage) and acclimatized for
1 week. The animals will be receiving a balanced commercially available
pelleted rat feed and will be provided with clean drinking water. Based from
“resource equation” method[18], 25 rats (5 rats in each group) will be used
to determine the vasodilatory effect of FragariaVesca Linn. (strawberry)
ethanol extracts. The remaining 5 rats will serve as backup in case death is
encountered.
2.Dosing method (including frequency, volume, route, method of restraint
and expected outcome or effects):
No dosing will be done at this stage of experiment.
3.Specimen or biological agent (blood, urine etc.) collection method
(including frequency, volume, route and method of restraint)
No specimen or biological agent is to be collected at this stage.
4.Animal examination procedure and frequency of examinations (including
retraining method)
none
5.Use of anesthetics ( including drug, dosage , frequency)
Ether will be used to induce general anesthesia to the rats prior to
dissection.
6.Surgical procedure (type and purpose):
VASODILATORY EFFECTS OF STRAWBERRY 28

Dislocation of the atlanto-occipital bone will be performed to


prepare the rats for aortic harvesting. Surgical procedures will be assisted
by a veterinarian. Descending thoracic aorta will be dissected and cleared
of adjacent tissues including fats, leaving the layers intact particularly the
tunica media and intima. Specimen will be soaked in Krebs Henseleit
solution.
7.If euthanasia of animals will be done, indicate/describe the method
selected.
Dislocation of the atlanto-occipital bone will be performed. All
animal carcasses will be packed in polythene bags and will be buried deep
in the ground covered with lime and disinfectants.
H. Is there a none-animal model applicable for the procedure/study?
None
I. Indicate the names and qualification of all personnel who will be responsible for
conducting the procedure
a) Bugayong, Adrian F. (Leader)
b) Pascua, Arbel Joy L. (Asst. Leader)
c) Bai, Chalatorn S.
d) Cancino, Ryan S.
e) Chinayog, Sean Paulo G.
f) Ko, Philip T.
g) Langpuyas, Ingrid Mariz V.
h) Movilla, Thessalonica Anne M.
i) Padiernos, Rosemarie Christine F.
j) Smith, Yvonnie E.
k) Terrado, Reo Famela P.
l) Valentin, Jemimah G.
m) Veradero, Ryan Louis M.
J. Post-experimental procedure (if any) especially, if physical manipulation like
surgical procedure will be done or if the animal will be euthanized. Please indicate
the method of carcass disposal:
Dislocation of the atlanto-occipital bone will be performed to prepare the
rats for aortic harvesting. Descending thoracic aorta will be dissected and cleared
of adjacent tissues including fats, leaving the layers intact particularly the tunica
media and intima. Specimen will be soaked in Krebs Henseleit solution.
All animal carcasses will be packed in polythene bags and will be buried
deep in the ground covered with lime and disinfectants.

VII. DECLARATION BY THE RESPONSIBLE PERSON:

I ACCEPT RESPONSIBILITY FOR ASSURING THAT THE PROCEDURE/ STUDY WILL


BE CONDUCTED IN ACCORDANCE WITH THE APPROVED PROTOCOL.
VASODILATORY EFFECTS OF STRAWBERRY 29

I ASSURE THAT ALL PERSON WHO USE THIS PROTOCOL AND WORK WITH
ANIMALS HAVE RECEIVED APPROPRIATE TRAINING/INSTRUCTIONS IN
PROCEDURAL AND HANDLING TECHNIQUES, AND ON ANIMAL WELFARE
CONSIDERATIONS.
I AGREE TO OBTAIN WRITTEN APPROVAL FROM THE INSTITUTIONAL ANIMAL
CARE AND USE COMMITTEE PRIOR TO MAKING ANY CHANGES AFFECTING MY
PROTOCOL. I ALSO AGREE TO PROMPTLY NOTIFY THE IACUC IN WRITING OF ANY
EMERGENT PROBLEMS THAT MAY ARISE IN THE COURSE OF THIS STUDY
INCLUDING THE OCCURRENCE OF ADVERSE SIDE EFFECTS.
Signature of Responsible Person:

ADRIAN F. BUGAYONG, RN Date:_____________


Leader
ARBEL JOY L. PASCUA, RMT Date:_____________
Asst. Leader
VASODILATORY EFFECTS OF STRAWBERRY 30

Appendix C
Letter for the use of the Animal Laboratory

February 10, 2017

Evelyn E. Oda, PhD


Dean, School of Natural Sciences
4th floor, Dr. Jose P. Rizal building, Saint Louis University
Bonifacio Street 2600 Baguio City Philippines

Dear Madam:

We, 2nd year students of SLU-School of Medicine, are currently conducting a research entitled
“Vasodilatory Effects of Strawberry (Fragaria ananassa x Sweet Charlie) Ethanol Extracts
on Sprague Dawley Rats.” Our research aims to determine (1) the changes in rat aortic ring
diameter after exposure to strawberry fruit ethanol extracts (2) the significant difference between
dose-dependent vasodilatory effects of strawberry fruit extracts on Sprague Dawley Aortic Rings,
using the following dosages: a. 0.2 mg extract /100ml distilled H2O, b. 2 mg extract /100 ml
distilled H2O, c. 5 mg extract /100 ml distilled H2O and (3) the vasodilatory effects of the extracts
to endothelium-denuded vessel.

In line with this, we would like to request your good office for the use of the Animal Laboratory
from February 20, 2017 to March 07, 2017. Attached to this letter will be our daily routine and
plans for the dates given hereof with a table of chemicals needed for the experiment. Also, we will
be responsible for providing care, food and rat cages for our experimental animals once they arrive.

Thank you very much and we hope for your kind consideration.
God bless!

Respectfully yours,

BUGAYONG, Adrian F.
PASCUA, Arbel Joy L.
BAI, Chalatorn S
CANCINO, Ryan S.
CHINAYOG, Sean Paulo G.
KO, Philip T.
LANGPUYAS, Ingrid Mariz B.
MOVILLA, Thesalonica Anne M.
PADIERNOS, Rosemarie Christine F.
SMITH, Yvonnie E.
TERRADO, Reo Famela P.
VALENTIN, Jemimah G.
VERDADERO, Ryan Louis
VASODILATORY EFFECTS OF STRAWBERRY 2

Endorsed by:
Dr. Leo Emmanuel Buñag Dr. John Anthony Domantay
Department of Pharmacology Dean of School of Medicine