Introduction

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

world and ranked third in the list of leading causes of death in the country in 2010 [1]. Tumor

growth and systemic metastasis are highly dependent on angiogenesis. 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]. The action of agents with anti-angiogenic

activity can help prevent and treat cancer and one of the agents that have been suspected to

have anti-angiogenic property is wine. Some wines possess naturally occurring substances

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

clinical outcomes [7]. Recent studies have shown that red wine polyphenolic compounds

(RWCP) have anti-angiogenic properties preventing the formation of new blood vessels which

explains their beneficial 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. The Cordillera Region, is most popular for

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

produced 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]. Rice wine or tapuy, is a traditional

alcoholic rice drink originated from Banaue and Mountain Province [18]. Strawberry 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 apoptotic

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

carcinogenic agents [23]. Lastly, yacon is rich in phenolic components with strong antioxidant

effects used for medicinal infusion [24]. In studying the anti-angiogenic potential of these

wines, chorioallantoic membrane (CAM) assay can be used [27][28].

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

wines versus red wine using CAM assay in duck embryo. The specific objectives of this study

were to determine the number of new blood vessels formed in CAM and determine if there is

a significant difference between the controls and the local wines.

Materials and Methods

The researchers utilized duck (Anas platyrhynchos) embryos, divided into four experimental

groups and two control groups. The negative 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. 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.

Results

Representative area of the CAM was used and photo-documented using Samsung S6 Edge

1440x2560 pixels (~577 ppi pixel density) and were enhanced by Adobe Photoshop to further
visualize the vascularity of the CAM. Manual counting of the blood vessels were done to

quantify the blood vessels formed.

A B

C D

E F

Figure 1. Blood Vessel formed. (A) Negative Control (B) Positive Control (C) Bugnay Wine
(D) Rice Wine (E) Strawberry Wine (F) Yacon Wine

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

vessels, averaging 27.9. 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, 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

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.

Table 1. 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
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.

Table 2. 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 (Mann-
Treatment p-value
Whitney U 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*

Conclusions and Recommendations

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

demonstrates an anti-angiogenic activity in CAM assay. 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 mechanisms by which bugnay wine and

strawberry wine elicit their anti-angiogenic properties. We recommend that a phytochemical

analysis be added, therapeutic and toxic dose of bugnay wine and strawberry wine shall be

established, and to compare and contrast the manufacturing processes specific for each wines

in future studies. A larger sample size should also be used and should involve a more accurate

way of quantifying the blood vessels, to yield more reliable results.