INTRODUCTION

Background of the Study

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 cases and decrease such presented numbers. 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]. 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].

Statement of the Problem

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

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. Two hundred microliters (200 L) of each

wines were introduced to the CAM on Day 5 and then left to incubate for 24 hours.

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 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
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 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 (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 should 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.