Running head: ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND

YACON WINES 1

Anti-angiogenic Property of Bugnay Wine,

Philippine Rice Wine (Tapuy), Strawberry Wine and Yacon Wine, versus Red Wine

using Chorioallantoic Membrane Assay in Duck (Anas platyrhynchos) Embryo

Matthew Apollo M. Acop, Mikhail Isaiah B. Geloso, Christian Jake N. Llano, Krystel J. Batino,

Jerleen Jill Q. Epler, Genelyza Theresa F. Garcia, Carizza Joy B. Nuega,

Tatiana Wiezell C. Orcilla, Ashley Joan B. Pinas, Eileen Mae L. Sagorsor,

Manjot Singh, Joan D. Solomon

Saint Louis University - School of Medicine

Dr. John Anthony Domantay, Ph.D. Ed. Mgmt., FPSP, FPMASP

Dr. Leo Emmanuel R. Bunag, FPSECP

ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 2

Abstract

Introduction: Epidemiological studies have proven that regular consumption of red wine reduces

the risk for cancer. This is attributed to its polyphenols which are naturally occurring compounds

that inhibit angiogenesis, the abnormal growth of new blood vessels needed for tumor growth. The

Philippines, particularly the Cordillera Region, is popular for producing its own wines which have

a myriad of health benefits. Their anti-angiogenic properties, however, remain to be further

elucidated.

Objectives: This research aims to determine the anti-angiogenic property of locally made wines,

namely, bugnay wine, Philippine rice wine (tapuy), strawberry wine, and yacon wine in

comparison to red wine.

Methods: Chorioallantoic membrane assay in duck (Anas platyrhynchos) embryo was performed

to determine the anti-angiogenic effect of the wine samples.

Results: Using Mann-Whitney U test, the significant difference (p<0.05) of each wines were

determined. Quantitative analysis shows that bugnay wine (p=0.00397) and strawberry wine

(p=0.00794) significantly inhibit the proliferation of blood vessels in a normal CAM. However,

the statistical comparison of the negative control with yacon wine (p=0.34286) and rice wine

(p=0.44386) showed no significant inhibition of vascular growth. Between bugnay wine (p=0.006)

and strawberry wine (p=0.019), bugnay wine showed greater inhibition on blood vessel formation.

Conclusion: Comparing all the results of the tested wines, bugnay wine showed the greatest inhibitory

effect to angiogenesis, and rice wine, the least.

Keywords: Anti-angiogenesis, bugnay wine, strawberry wine, Philippine rice wine, yacon wine,

CAM assay

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 3

Background

Cancer is one of the severe human diseases which cause increasing mortality every year in

the world. The World Health Organization predicted that the number of cancer deaths worldwide

would increase from 7.6 million to 17 million deaths in 2030. In the Philippines, cancer ranked

third in the list of leading causes of death in the country in 2010 [1]. Based on 96.5 million

populations of the Philippines as of 2012, the newly diagnosed cancer cases per year is 98,200, the

incidence per 100,000 people per year is 140, and the risk of getting cancer before age 75 is 14.8%

[2]. Tumor growth and systemic metastasis are highly dependent on angiogenesis. Though it is a

normal multistep process by which new capillaries are formed from pre-existing capillaries, it is

also significant in the development and repair of damaged tissues, tumor growth, invasion and

metastasis [3,4]. It is, therefore, particularly unwanted, as it is a key event that feeds tumor growth

and cancer metastases. Angiogenesis inhibition offers an opportunity to interrupt an early, rate-

limiting step in tumorigenesis and prevent the progression of an early tumor to a malignant type

[5]. Because of this, anti-angiogenic approaches to prevent and treat cancer represent a priority

area in investigative tumor biology [6]. Thus, the action of agents with anti-angiogenic activity

can help prevent cases and decrease such presented numbers. One of the agents that have been

suspected to have anti-angiogenic property is wine. Wine has been regarded by many societies as

an essential and healthful beverage. Some wines possess naturally occurring substances that would

be responsible for their antioxidant, anti-inflammatory and anti-clotting roles which improved

clinical outcomes [7]. Studies showed that beneficial properties of wine, independent from the

presence of alcohol, have been attributed to the polyphenol content of wine [8] [9]. Recent studies

have shown that red wine polyphenolic compounds (RWCP) have anti-angiogenic properties

preventing the formation of new blood vessels which explains, at least in part to, their beneficial

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 4

effect on heart diseases and cancer [10]. The expression of growth factor-induced vascular

endothelin growth factors (VEGF), which plays an important role in the formation of pro-

inflammatory and pro-thrombotic molecules in atherosclerotic plaque, is strongly inhibited by

RWCP at concentrations that are likely to be achieved in the blood after moderate consumption of

red wine [11].

Wines are widely produced in the Philippines, being identified as one of the principal and

important countries for wine in Southeast Asia and in the world [12]. Having a growing wine

industry, Philippine wineries produce several types of wine that use plants, such as tropical fruit,

rice, sugar cane, bugnay, yacon and coffee [13] [14]. The Cordillera Region, in particular, is most

popular for tapuy, a fermented rice wine, and strawberry wine [15]. Since these wines are

popularly produced in the Philippines, especially in the Cordillera region, the researchers would

like to study four locally made wines regarding their anti-angiogenic property. Bugnay is native to

the Philippines and contains phenolics flavonoids, anthocyanins and carotenoids [16]. It was

reported that the ripe fruit was non-genotoxic and has anti-mutagenic properties [17]. Philippine

rice wine or tapuy, is a traditional alcoholic rice drink that originated from Banaue and Mountain

Province [18]. It is an excellent source of carbohydrates and good source of vitamins and minerals

[19]. Strawberry is a very good source of dietary fiber, vitamins, minerals and omega-3 fatty acids

and they provide an outstanding variety of phytonutrients, flavonols, hydroxybenzoic acids,

stilbenes and resveratrol [20]. Resveratrol was found to inhibit nitric oxide, an important pro-

tumoral substance playing part in the initiation, growth, and metastasis of various cancers.

[21][22]. Strawberry extracts and their metabolites such as hydroxyphenylacetic acid showed

apopototic effects in HT-29 colon cancer cells and may suggest their possible contribution as anti-

carcinogenic agents [23]. Yacon is rich in phenolic components with strong antioxidant effects

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 5

used for medicinal infusion [24]. A study of the crude extracts of dried leaves and tubers of yacon

yielded phenolic acids contributing to the radical scavenging activity detected [25][26]. In studying

the anti-angiogenic potential of these wines, chorioallantoic membrane (CAM) assay can be used.

According to Seow and his colleagues, CAM has become a widely used tool for the determination

of both angiogenic and anti-angiogenic activities of many drugs including herbal extracts [27].

Moreover, Wan stated that CAM is a highly vascular lining in the inner surface of the egg shell

and it is as well easily accessible [28].

At the time being, the anti-angiogenic property of the four locally manufactured wines

selected for the study has not yet been broadly elucidated. In this research, new understanding of

ways to reduce cancer growths was sought to be achieved. This study may also contribute to the

promotion and improvement of the winery industry of the country.

The researchers primarily attempted to determine the anti-angiogenic property of bugnay

wine, Philippine rice wine (tapuy), strawberry wine, and yacon wine versus red wine using

chorioallantoic membrane (CAM) assay in duck embryo. The specific objectives of this study

were: (1) to determine the number of new blood vessels formed in CAM treated with bugnay wine,

Philippine rice wine (tapuy), strawberry wine, yacon wine, and red wine; and (2) to determine if

there is a significant difference in the number of new blood vessels formed in CAM between the

controls and (a) bugnay wine, (b) Philippine rice wine (tapuy), (c) strawberry wine, and (d) yacon

wine.

Methods

General

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 6

The study was conducted using an experimental design. The researchers utilized duck

(Anas platyrhynchos) embryos, divided into four experimental groups and two control groups.

Thirty zero-day old duck eggs were collected from a local hatchery and were incubated at 37.5C

at the Natural Sciences Research Unit of Saint Louis University. The samples were acclimatized

for four days prior to the experiment proper and were routinely manually rotated. The viability of

the eggs was checked through candling.

There were four treatment groups and two control groups. The control groups,

specifically the negative control group and positive control group, received distilled water and red

wine respectively. The four treatment groups are as follows: bugnay wine, strawberry wine, rice

wine and yacon wine. Five duck eggs were assigned to each group, following the protocol

published by Dohle et al, and were labelled accordingly

The duck eggs were randomly selected for sampling treatment using the OpenEpi

website. The numbers are assigned accordingly: (a) negative control group: 23, 5, 25, 7, 12; (b)

positive control group, red wine: 21, 24, 29, 22, 3; (c) bugnay wine: 26, 11, 18, 16, 9; (d) strawberry

wine:28, 30, 20, 27, 17; (e) rice wine: 6, 15, 1, 19, 10and (f) yacon wine: 4, 13, 14, 8, 2. Five eggs

were designated for each group.

Preparation of Wine Material

The four (4) locally manufactured wines are bugnay wine, strawberry wine, tapuy and

yacon wine. All were acquired from Jasons Homemade Products (Ques finest) at Longlong,

Puguis, La Trinidad, the only BFAD registered firm in the area. The red wine (Novellino Merlot)

was purchased from the SM Baguio City Supermart.

Since the anti-angiogenic property of red wine polyphenols has already been established

in a study conducted by Oak and colleagues, red wine was chosen for the positive control group.

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 7

A similar study conducted in University of Santo Tomas, Manila, that employed CAM assay, also

used red wine as their positive control.

The wines were properly stored in room temperature. The four wines had 12% alcohol

content per 750 mL net volume. Equally, the red wine had 12% alcohol content per 750 mL net

volume.

Preparation of Duck Embryos

The freshly laid duck eggs were purchased and delivered from a local hatchery in

Santiago, Tarlac and were transported to Baguio City and incubated at the Natural Sciences

Research Unit. Thirty (30) duck eggs were purchased for each of the three (3) trials.

Chorioallantoic Membrane (CAM) Assay

Anti-angiogenic effect of the wine samples was investigated in vivo using CAM assay.

The chorioallantoic membrane (CAM) assay for screening the effect of test samples on

angiogenesis was performed according to the method given by Chen and co-workers (2013). At

the seventh day of incubation, the eggs were candled and inspected for egg viability and position

of embryo. All the eggs were wiped with 70% ethanol to reduce contamination from egg surface

and were air dried. A small window opening was made in the blunt end (air space) of the shell

using an inoculating needle. Adapted from the research of Uzma and Khan, using a micropipette,

200l of wine was directly applied onto the CAM according to their corresponding treatment

group. The inoculated CAMs were sealed using sterile Parafilm and returned to the humidified

atmosphere until 24 hours after administration of the test samples. The CAMs were harvested by

removing the shell of the egg then extraction of the embryos onto a petri dish. Representative area

of the CAM was used and photo-documented using Samsung S6 Edge 1440x2560 pixels (~577

ppi pixel density). The blood vessels in each CAM were then quantified.

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 8

Statistical Analysis

All analyses were performed using Real Statistics Using Excel for Windows and

Macintosh. The Mann-Whitney U-test, a nonparametric alternative test to the independent sample

t-test, was used to compare the individual treatment group against the negative and positive control

groups. The group rank difference obtained was used to determine the statistical significance of

the anti-angiogenesis properties of each wine. Statistical significance was accepted at p < 0.05.

Results

Chorioallantoic Membrane Blood Vessel Counting

This study was designed to determine the anti-angiogenic property of local wines, namely, bugnay

wine, rice wine, strawberry wine, and yacon wine, in terms of blood vessels formed in CAM. The said

wines were also compared to red wine, the positive control, as to their anti-angiogenic property. Mann-

Whitney U Test was used to analyse the relationship between the negative control and positive control to

the wine samples.

Two hundred microliters (200 L) of each wines were introduced to the CAM on Day 5 and were

then left to incubate for 24 hours. After which, the blood vessels formed were photodocumented and were

enhanced by Adobe Photoshop to further visualize the vascularity of the CAM (see Figure 1 in Appendix

2). Manual counting of the blood vessels were used to quantify the blood vessels formed.
A B

Table 1 (Appendix 2) presents an overview Fof the counted blood vessels of each egg after
E
incubation and treatment of wines. Table 2 (Appendix 2) shows the mean number of blood vessels

formed per trial. Graph 1 represents the mean total of each wines. In the graph, the wine closest to

the average or mean number of red wine (positive control) is bugnay wine, followed by strawberry

wine, rice wine and lastly, yacon wine.

Anti-Angiogenic Effects of Sample Wines on CAM

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 9

Using Mann-Whitney U test, the significant difference of each wine was determined, as

seen on Table 3. Comparing the wines and the negative control, bugnay wine and strawberry wine

were significant (p<0.05) at values of p= 0.004 and p= 0.008, respectively. However, rice wine (p

= 0.444) and yacon wine (p = 0.343), were not significant. The quantitative analysis indicate that

among the four wines, only bugnay wine (p=0.006) and strawberry wine (p=0.019) significantly

inhibited the proliferation of blood vessels in a normal CAM. Between bugnay wine (p=0.006)

and strawberry wine (p=0.019), bugnay wine showed greater inhibition on blood vessel formation

than the latter. Comparing all the results of the tested wines, bugnay wine showed the greatest

inhibitory effect to angiogenesis, and rice wine, the least.

Discussion

This study examined the possible anti-angiogenic activity of bugnay, tapuy, strawberry,

and yacon wines. To the best of our knowledge, little evidence was found in the literature of the

anti-angiogenic properties of these wines. However, these wines may contain properties that can

possibly play a significant role in anti-angiogenesis.

An initial objective of the study is to count the number of blood vessels formed in each

treatment. Table 1 presents the average number of blood vessels formed for each treatment per

trial, and it can give us a slight inkling as to which of the wines demonstrated an anti-angiogenic

effect. The results show that CAM assay treated with red wine produced the least amount of blood

vessels, averaging 27.9 (Table 2). This is consistent with other literature, and reflective of its anti-

angiogenic property. Next to red wine were the CAMs treated with bugnay wine and strawberry

wine followed, producing an average of 45.8 and 49.13 blood vessels, respectively. These numbers

may be suggestive of their possible significant anti-angiogenic property. On the other hand, the

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 10

average blood vessels formed in CAMs treated with rice wine and yacon wine were close to the

control, suggesting that their anti-angiogenic activity is not significant.

To determine if these wines contain a significant level of anti-angiogenic activity, we

statistically compared them individually to the negative control first, and then to red wine, which

is the positive control. Table 3 compares the individual wines with the negative control. The data

shows that bugnay wine and strawberry wine contain a significant anti-angiogenic property when

compared to the negative control. Further analysis also reveals that both bugnay wine and

strawberry wine have significant levels of difference with that of the positive control, the red wine.

Although demonstrable anti-angiogenic effects are observed with both wines, these results do not

denote their degree of anti-angiogenic activity as to potency.

The anti-angiogenic effects of strawberry wine could be attributable to its component

called resveratrol, an anti-angiogenic polyphenol also present in red wine [20] [21]. On the other

hand, bugnay wine, which is less studied, contains phenolic flavonoids, anthocyanins, and

carotenoids that could explain its inhibitory effect on angiogenesis [15]. In previous studies,

flavonoids were found to be capable of regulating the expression of vascular endothelial growth

factor (VEGF), matrix metalloproteinases (MMPs), and epidermal growth factor receptors

(EGFRs), which would ultimately result to an anti-angiogenic effect [28]. Similarly, anthocyanins

were also found to reduce VEGF and VEGF receptors in endothelial cells, which would also

prevent angiogenesis [29]. Finally, carotenoids present in bugnay were also associated with

inhibition of angiogenesis by suppressing fibroblast growth factor 2 (FGF-2) and its receptor [30].

Although the exact mechanism of anti-angiogenesis exerted by bugnay wine and strawberry wine

were not explored in this study, the findings of previous studies provide explanation to such

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 11

inquiry. This suggests that the result of this study is congruent with the findings of the previews

studies.

In contrast, the yacon wine and rice wine did not demonstrate a significant anti-angiogenic

effect. The result in rice wine is different from a similar study we have found. In a study involving

magkeolli (traditional Korean rice wine), they evaluated its anti-angiogenic activity, and concluded

that it prevented tube formation in vitro [31]. However, it is worth mentioning that aside from

variety in the rice used, the said study used human umbilical vein endothelial cells (HUVECs)

while we used CAM assay. Secondly, they explored on how the magkeolli demonstrates its anti-

angiogenic activity, while we measured if rice wine demonstrates a significant anti-angiogenic

effect in contrast to other wines. Basically, it is still possible that rice wine, in general, actually

contains anti-angiogenic properties, but further studies should be conducted to support such claim.

On the other hand, various literatures about yacon also noted phenolic compounds as its component

which has an anti-angiogenesis effect. Hence, just like that of the rice wine, a deeper and more

technical study might be more suitable to explore such inferences about yacon wine [24] [25].

Recommendations

This research has thrown-up many questions in need of further investigation. As to the

methods performed, the viability of the eggs is very crucial in CAM assay. Future researchers,

who wish to perform such procedure, should make sure that the hatchery be able to provide the

eggs with the accurate age requirement. As to the results, extensive work is required to be able to

isolate the exact properties and mechanisms by which bugnay wine and strawberry wine elicit their

anti-angiogenic properties. We also recommend that a phytochemical analysis be added in future

methodologies. Furthermore, it is an important issue for future research to establish the therapeutic

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 12

and toxic dose of bugnay wine and strawberry wine for consumers. The researchers also suggest

that further studies compare and contrast the manufacturing processes specific for each wines. This

may have varying effects not only on the content, but also to their beneficial effects, as well.

Finally, we recommend that future studies on the current topic should involve a larger sample size,

and should involve a more accurate way of quantifying the blood vessels, in order to yield more

reliable results.

Limitation

The research was limited in several ways. First, the study used a small sample size due to

practical constraints in maintaining the viability of the eggs during transportation. This affected

the samples and, consequently, the results as well. A larger sample size couldve made the results

more reliable. Furthermore it is beyond the scope of this study to determine the exact mechanism

and chemicals involved in the anti-angiogenic effects of the wines. Thirdly, the manual counting

of blood vessels is prone to human error which could have affected the data. Finally, although the

study has successfully demonstrated the anti-angiogenic property of bugnay wine and strawberry

wine; it is beyond our capability to determine the exact degree or potency of their anti-angiogenic

property.

Application

The results of this study would encourage further studies on the anti-angiogenic property

of bugnay wine and other local wines. This would also add to the beneficial effects of such

beverages and could encourage use of locally manufactured wines, which could hopefully improve

further the local economy.

Conclusion

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 13

In conclusion, this study revealed that locally made bugnay wine and strawberry wine

demonstrates an anti-angiogenic activity in CAM assay. We suspect that compounds inherent to

bugnay wine and strawberry, specifically resveratrol for strawberry wine and flavonoids,

anthocynanins, and carotenoid for bugnay wine, are responsible for their anti-angiogenic action.

On the other hand, yacon wine and rice wine did not have a significant anti-angiogenic effect in

CAM assay. Further research is needed to understand the exact mechanism behind these results.

Funding

This research project was self-financed.

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Appendices

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 19

Appendix 1: Acquisition, processing of materials, and CAM assay

ACQUISITION OF MATERIALS
WINES
The red wine was
purchased at the SM City
Baguio Supermart.
The Bugnay wine, rice
wine, strawberry wine, and
yacon wine were all
acquired from Jasons
Homemade Products (Ques
finest) at Longlong, Puguis,
La Trinidad.

PROCESSING OF MATERIALS

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 20

The fertilized eggs

incubated at 37.5 C.
The samples were
acclimatized for seven
days prior to the
experiment proper.

Viability checking

Preparation of wines

CHORIOALLANTOIC MEMBRANE ASSAY

20
ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 21

CAM
assay
proper

RESULTS
Negative Control

Red wine

Bugnay wine

21
ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 22

Rice wine

Strawberry wine

Yacon wine

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 23

Figure 1. Blood Vessel formed. (A) Negative Control (B) Positive Control (C) Bugnay Wine (D)

Rice Wine (E) Strawberry Wine (F) Yacon Wine

A B

Table 1 (Appendix 2) presents an overview of Fthe counted blood vessels of each egg after
incubation and treatment of wines. Table 2 (Appendix 2) shows the mean number of blood vessels
formed per trial, andE this is better presented in Graph 1. Graph 2 represents the mean total of each
wines. In these two graphs, the wine closest to the average or mean number of red wine (positive
control) is bugnay wine, followed by strawberry wine, rice wine and lastly, yacon wine.

Appendix 2

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 24

Computations for branch point frequency and CAM Score

Table 1. Average Number of Blood Vessel Counted per Trial Counted

Negative Red Wine (Positive Bugnay Strawberry Yacon

Rice Wine
Control Control) Wine Wine Wine
Trial 1 62 28.4 46.6 45.8 44.4 51
Trial 2 60.8 27.8 45.6 55.4 49.8 63.8
Trial 3 59.8 27.4 45.2 67 53.2 58.6

Table 2. Mean of Blood Vessel Counted

Negative Red Wine Bugnay Rice Wine Strawberry Yacon

Control (Positive Wine Wine Wine
Control)
Total 60.9 27.9 45.8 56.1 49.13 57.8
Average

Table 3. Analysis of the Anti-angiogenic Property of Wines (Bugnay, Rice wine,

Strawberry wine and Yacon wine) as compared to Negative Control and Positive Control

Test Statistics
Treatment (Mann-Whitney U p-value
Test)
Bugnay wine 2.507 0.004*
Rice wine 0.144 0.444
Negative Control
Strawberry wine 2.207 0.008*
Yacon wine 0.433 0.343
Positive Control Bugnay wine 2.507 0.006*
(Red Wine) Strawberry wine 2.207 0.019*

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ANTI-ANGIOGENESIS: BUGNAY, RICE, STRAWBERRY AND YACON WINES 25

Graph 1. Mean of blood vessel formed

Graph 2. Mean total of blood vessel formed.

Number of Blood Vessels 60

50

40

30

20

10

0
Negative Red Wine Bugnay Wine Rice Wine Strawberry Yacon Wine
Control (Positive Wine
Control)

25