Research Strawberry

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 (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
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.
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
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.

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.
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.

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.
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.
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.

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.

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
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
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.
References
1. World Health Organization, Cardiovascular Diseases, 2016. Available from:

http://www.who.int/cardiovascular_diseases/en/
2. Giampieri, F., Forbes-Hernandez, T.Y., Gasparrini, M., Alvarez-Suarez, J.M., Afrin, S.,
Bompadre, S., Quiles, J.L., Mezzetti, B., Battino M. Strawberry as a health promoter: An
evidence based review. Food Funct. 2015, 6, 1386–1398. (47)
3. Giampieri, F., Alvarez-Suarez, J.M., Battino, M. Strawberry and Human Health: Effects
beyondAntioxidant Activity. J. Agric. Food Chem. 2014, 62, 3867–3876. (49)
4. Naemura, A., Mitani T, Ijir Y, Tamura Y, Yamashita T, Okumura M, et. al.. Anti-
thrombotic effect of strawberries. Blood Coagul Fibrinolysis. 2005 Oct; 16(7): 501-9
5. Doaa S Ibrahim and Marwa A E Abd El-Maksoud. Effect of strawberry

(Fragaria × ananassa) leaf extract on diabetic nephropathy in rats. 2015 Feb 3. Available
from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459800/
6. Pinto Mda, S., de Carvalho, J.E., Lajolo, F.M.; Genovese, M.I., Shetty, K. Evaluation of
antiproliferative, anti-type 2 diabetes, and antihypertension potentials of ellagitannins from
strawberries (Fragaria ananassa Duch.) using in vitro models. J. Med. Food 2010, 13, 1–9.
7. Alvarez-Suarez, J.M.; Giampieri, F.; Tulipani, S., Casoli, T., di Stefano, G., González-
Paramás, A.M., et al. One-month strawberry-rich anthocyanin supplementation
ameliorates cardiovascular risk, oxidative stress markers and platelet activation in humans.
J. Nutr. Biochem. 2014, 25, 289–294.
8. Katalinic, V., Milos, M., Kulisic, T., Jukic, M., 2006. Screening of 70 medicinal plant
extracts for antioxidant capacity and total phenols. Food Chem. 94, 550–557
9. Mudnic, D. Modun, I. Brizic, J. Vukovic, I. Generalic, V. Katalinic, T. Bilusic, I.

Ljubenkov, M. Boban. Cardiovascular effects in vitro of aqueous extract of wild strawberry
(Fragaria vesca, L.) leaves. Phytomedicine 16 (2009) 462–469. Available from:
https://www.researchgate.net/publication/23766001_Cardiovascular_effects_in_vitro_of_
aqueous_extract_of_wild_strawberry_Fragaria_vesca_L_leaves
10. Clifford, M.N., & Scalbert, A. (2000). Ellagitannins – Nature, occurrence and dietary
burden. Journal of the Science of Food and Agriculture, 80, 1118–1125.
11. Vaux-Bjerke, A., Kan, F., Barrette, T., Edirisinghe,I., Burton-Freeman, B., Kappagoda, T.
Effect of strawberry powder on blood pressure and flow mediated vasodilation in
individuals with pre-hypertension.
http://www.fasebj.org/content/24/1_Supplement/564.13
12. Duru, Metin. (2012) Effects of Dietary Strawberry (Fragaria x ananassa) Leaf Powder on
Growth Performance, Body Components and Digestive System Broiler Chicks.
International Journal of Agriculture and Biology. Available from: http://world-food.net
13. Seeram, Navindra. (2007). Strawberry Phytochemicals and Human Heatlh: A Review
14. Madhavi Eerike, Ruckmani.Arunachalam, Venkata Raman Yeddula, Venu Gopala Rai
Konda, Prasanth Chary.R. Evaluation of hypolipidemic activity of of ethanolic and
aqueous extracts of Fragaria vesca in high fat diet induced hyperlipidemia in rats [internet].
Int. J. Phar. Sci. Rev. Res., 2014 September-October; Article no. 34: 191-196. Available
from: www.globalresearchonline.net
15. Arpita Basu, Michael Rhone, and Timothy J Lyons. Berries: emerging impact on
cardiovascular health. Nutr Rev. 2010, March. Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068482/
16. Aedíns Cassidy, Kenneth J. Mukamal, Lydia Liu, Mary Franz, A. Heather
Eliassen and Eric B. Rimm. High Anthocyanin Intake Is Associated With a Reduced Risk
of Myocardial Infarction in Young and Middle-Aged Women. 2013 January 14. Available
from: http://circ.ahajournals.org/content/127/2/188
17. Creasy, L.L., Swain, T., 1966. Flavan production in strawberry leaves. Phytochemistry 5,
501–509
18. Jaykaran Charan and ND Kantharia. How to calculate sample size in animal studies?.
Journal of Pharmacology and Pharmacotherapeutics (2013); 4 (4): 303- 306. Available
from www.ncbi.nlm.nih.gov
19. Bekesho Galeta, Egasu Makonnen, Asfaw Debella, Abiy Abebe, Net Sanet Fekadu. In vitro
vasodilatory activity and possible mechanisms of crude extracts and fractions of Moringa
stenopetala (Baker f.) Cufod. leaves in isolated thoracic aorta of guinea pigs [internet].
Journal of experimental pharmacology 2016: 8 35-42. Available from:

https://www.dovepress.com
20. OpenEpi: Open Source Epidemiologic Statistics for Public Health, 2013. Available from:
http://www.openepi.com/Menu/OE_Menu.htm
21. Bradamente, S., Barenghi, L., & Villa, A. (2004). Cardiovascular protective effects of
resveratrol. Cardiovascular Drug Review, 22, 169-188.
22. Corder, R., Wartburton, R., Khan, N., Brown, R., & Wood, E. (2004). The procyanidin-
induced pseudolaminar shear stress response: a new concept for reversal of endothelial
dysfunction. Clin Sci (London), 107, 513-517.
23. Kim, S., Kang, K., Kim, K., & Kim, N. (2000). Procyanidins in crataegus extract evoke
endothelium dependent vasorelaxation in rat aorta. Life Sci, 67, 121-131.
24. Modun, D., Music, I., KAatalinic, V., Salamunic, I., & Boban, M. (2003). Comparison of
protective effects of catechin applied in vitro and in vivo on ischemia-reperfusion injury in
the isolated rat hearts. Croatian Med J, 44, 690-696.
25. Stoclet, J., Chataingneau, T., Ndiaye, M., Oak, M., & El Bedoui, J. (2004). Vascular
protection by dietary polyphenols. Eur. J. Pharmacol, 500, 299-313.
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 5 (Ring A, Ring B) Exposure to 0.2mg/10mL strawberry ethanol
Slide 6 (Ring A, Ring B) extract
- diameter measured immediately, 5 minutes
Slide 5 (Ring A, Ring B) Exposure to 2.0mg/10mL strawberry ethanol
extract
Slide 5 (Ring A, Ring B) Exposure to 5.0/10mL strawberry ethanol
Slide 6 (Ring A, Ring B) extract
Total: 23 slides
46 aortic rings
12 rings per group
2 rings per slide
(Rings A & B)
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
Epinephrine + 5mg/10m 6 14.03 7.48 15.83 8.35
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
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.
Epinephrine 0.2mg/10ml Strawberry Ethanol
Extract
(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
Table 6
Group 3 (Epinephrine + 2mg/10ml Strawberry Ethanol Extract) Raw Data:
Aortic Ring Changes Following Exposure to Epinephrine and 2mg/10ml Strawberry Ethanol
Epinephrine 2mg/10ml Strawberry Ethanol Extract
(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
Table 7
Group 4 (Epinephrine + 5mg/10ml Strawberry Ethanol Extract) Raw Data:
Aortic Ring Changes Following Exposure to Epinephrine and 5mg/10ml Strawberry Ethanol
Epinephrine 5mg/10ml Strawberry Ethanol Extract
(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
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)
Table 9
ANOVA Table of the Four Groups at 10 min

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
variation square
Between Groups 674.97 3 224.99 1.06 0.38
Within Groups 4001.03 19 210.58
Total 4675.99 22
10.08 3 0.017
variance

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

1 6 11.41 8.59
2 5 13.75 6.5
3 6 14.03 7.48
ANOVA Table
Source of Mean
variation square
Between Groups 24.33 2 12.16 0.20 0.81
Within Groups 817.69 14 58.40
Total 842.03 16

0.31 2 0.85
variance

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

1 6 14.64 7.84
2 5 15.75 8.1
3 6 15.83 8.35
ANOVA Table
Source of Mean
variation square
Between Groups 5.18 2 2.59 0.03 0.96
Within Groups 918.38 14 65.59
Total 923.56 16

0.01 2 0.99
variance

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

Research Strawberry

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VASODILATORY EFFECTS OF STRAWBERRY 2

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

studied to improve cardiovascular function in general.

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

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

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

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

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

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

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

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

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

One major pathophysiologic mechanism of hypertension can be attributed to vascular

Institutional Animal Care and Use Committee (IACUC).

developed by the Philippine Association Animal Science.

the deep ground covered with disinfectants.

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

induce stress and aggressive behavior.

contact with other rat groups.

procedure is used for analysis.

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

study would be:

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

animals to be used for the experiment is acceptable:

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

Collection of Plant Material and Preparation of Treatments. Organically grown

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

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

concurrently with Rings B.

A were measured concurrently with Rings B.

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

A were measured concurrently with Rings B.

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

results are included in the appendices.

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

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

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

aortic rat rings.

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

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

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

of the relative size of the aortic rings.

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

capabilities of the strawberry fruit.

1. World Health Organization, Cardiovascular Diseases, 2016. Available from:

5. Doaa S Ibrahim and Marwa A E Abd El-Maksoud. Effect of strawberry

9. Mudnic, D. Modun, I. Brizic, J. Vukovic, I. Generalic, V. Katalinic, T. Bilusic, I.

Journal of experimental pharmacology 2016: 8 35-42. Available from:

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

Analysis of Variance (ANOVA)

Chi square d.f p-value1

95% CI of individual 95% CI assuming equal

1 19.71 -5.11 44.53 5.23 34.18

Analysis of Variance (ANOVA)

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

95% CI of individual 95% CI assuming equal

Analysis of Variance (ANOVA)

Chi square d.f p-value1

95% CI of individual 95% CI assuming equal

1 11.41 2.39 20.42 3.38 19.43

Analysis of Variance (ANOVA)

Chi square d.f p-value1