Toxicology in Vitro 24 (2010) 16551661

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Toxicology in Vitro
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In vitro and in vivo anti-angiogenesis effect of shallot (Allium ascalonicum): A heat-stable and avonoid-rich fraction of shallot extract potently inhibits angiogenesis
Parivash Sey a, Ali Mostafaie b,*, Kamran Mansouri b, Delnia Arshadi a, Hamid-Reza Mohammadi-Motlagh b, Amir Kiani b
a b

School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

a r t i c l e

i n f o

a b s t r a c t
This study has been undertaken to elucidate the anti-angiogenic properties of shallot extract in vitro and in vivo and also to dene the responsible fraction and its stability. After preparation of the extract of shallot bulbs with 50% ethanol, the extract was successively fractionated into n-hexane, ethyl acetate, n-butanol and aqueous fractions. The ethyl acetate fraction was further fractionated to three subfractions using thin layer chromatography. Anti-angiogenic activity of fractions and subfractions were examined on human umbilical vein endothelial cells (HUVECs) in collagen matrix and chicken chorioallantoic membrane (CAM) models. Among the fractions, ethyl acetate fraction and one of its subfractions potently inhibited angiogenesis in vitro and in vivo. Furthermore, ethyl acetate fraction sustained its inhibitory effect signicantly even after treatment in high thermal and low pH conditions. These ndings provided a useful basis for further investigations on shallot as a useful herb with therapeutic or preventive activity against angiogenesis related disorders. 2010 Published by Elsevier Ltd.

Article history: Received 27 January 2010 Accepted 30 May 2010 Available online 4 June 2010 Keywords: Allium ascalonicum Anti-angiogenic Human umbilical vein endothelial cells Chicken chorioallantoic membrane Flavonoid

1. Introduction Angiogenesis is the process of generating new capillaries from pre-existing vessels. In adults, the proliferation rate of endothelial cells is very low compared with many other cell types. Physiological exceptions in which angiogenesis occurs under tight regulation are found in the female reproductive system and during wound healing (Hyder and Stancel, 1999). Unregulated angiogenesis may result in different pathologies (Folkman, 1995), such as rheumatoid arthritis (Koch, 1998), diabetic retinopathy (Ferrara and Alitalo, 1999), psoriasis and juvenile hemangiomas (Powell, 1999). Finally, tumor growth and metastasis are angiogenesis-dependent (Hanahan, 1998). Angiogenesis is regulated by a balance between proangiogenic factors including vascular endothelial cell growth factor (VEGF) (Cao et al., 2008; OReilly et al., 2007) and anti-angiogenic factors including transforming growth factor-b and endostatin (Roberts, 2008; Nyberg et al., 2005). Because deregulated angiogenesis is associated with disease progression, especially tumor development, inhibition of neovessel growth has become a target

* Corresponding author. Address: Medical Biology Research Center, P.O. Box 6714869914, Sorkheh Lizheh, Kermanshah, Iran. Tel.: +98 831 4276473; fax: +98 831 4276471. E-mail address: amostafaie@kums.ac.ir (A. Mostafaie). 0887-2333/$ - see front matter 2010 Published by Elsevier Ltd. doi:10.1016/j.tiv.2010.05.022

in drug development. Natural compounds from medicinal plants display diverse pharmacological activities and have advantages over synthetic drugs, such as smoother action and better tolerance (Nagy et al., 2003). For example, plant derived natural products such as genistein (Fotsis et al., 1993), isoliquitrin (Kobayashi et al., 1995), ginsenoside (Sato et al., 1994) and torilin (Kim et al., 2000) have potent effects on endothelial cell proliferation or tube formation. Allium a genus of more than 500 species belongs to the family of Liliaceae. However, only a few of them are important as food plants, notably onion, garlic, shallot, chive, leek, and rakkyo. Such plants have been used for many centuries for their pungency and avoring value, for their medicinal properties, and in some parts of the world, their use also has religious connotations (Fenwic and Hanley, 1985; Cavalito and Bailey, 1944; Ariga and Seki, 2006; Lawson, 1996; Eidi et al., 2006). To date, there are few clinical reports about pharmacological properties of shallot. These studies include: in vitro antioxidant and anti-bacterial activities of shallot (Yin and Cheng, 1998), hypoglycemic effect of shallot and garlic aqueous extract in rats with fructose induced insulin resistance (Jalal et al., 2007), isolation of antifungal peptide with human immunodeciency virus type 1 reverse transcriptase inhibiting activity (Wang and Ng, 2002) and the antiproliferative effect of shallot chloroformic extract on two tumor cell lines, MCF-7 and HeLa (Ghodrati Azadi et al., 2008). Analysis of shallot extract has

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conrmed the presence of avone and polyphenolic derivatives such as quercetin, quercetin 40 -glucoside, quercetin 7-glucoside, quercetin 40 ,3-diglucoside, and quercetin mono-D-glucose at high concentrations (Fattorusso et al., 2002). Previously, we indicated that aqueous extract of Allium ascalonicum bulbs has noticeable anti-angiogenic activity on rat aorta ring model without toxic effect on endothelial cells at 50800 lg/ml (Mohammadi Motlagh et al., 2008). The present study was undertaken to fractionate shallot extract and reveal the responsible fraction that inhibits angiogenesis in vitro and in vivo.

2.6. Preparative thin layer chromatography (TLC) Ethyl acetate fraction was applied to aluminum-backed TLC plates coated with 0.2 mm layers of silica gel 60 F254. Pure quercetin was used as a control for comparison. The fraction and quercetin were spotted and placed in TLC tank. The mobile phase was ethyl acetatemethanolwater (6.5:1:1.5). The plates were airdried and observed under UV light (254 and 366 nm) and Rf values were calculated. After detection, the spots were scraped and dissolved in solvent and centrifuged for 10 min at 10,000g. The supernatant was collected and solvent evaporated under reduced pressure. The powder was dissolved in ethanol and used for antiangiogenic assay (nal concentration of ethanol in test and control wells was kept below 1% for the assay).

2. Materials and methods 2.1. Chemicals Quercetin, potassium acetate, aluminum chloride, ethanol, methanol, ethyl acetate, butanol, hexane, LDH cytotoxicity assay kit (Sigma Chemical Co., USA). Coated cytodex microcarriers (Amersham Pharmacia Biotech, USA), Fetal bovine serum (FBS), Trypan blue 0.4% (Gibco, NY, USA), Silica gel 60 F254 (Merck, Darmstadt, Germany). Human umbilical vein endothelial cells (HUVECs) obtained from the National Cell Bank, Pasteur Institute of Iran. 2.2. Plant material Shallot bulbs (A. ascalonicum L.) were purchased from the local vegetable market at Kermanshah (Iran). 2.3. Preparation of ethanolic extract and fractions Preparation of ethanolic extract and fractions was performed using successive fractionation (Park et al., 2003; Zhi-Heng et al., 2009). The extract was prepared by blending shallot bulbs in a blender. The homogenate was extracted with 50% (v/v) ethanol by stirring for 24 h. The extract was ltered and centrifuged at 12,000g for 20 min at 4 C and then evaporated under reduced pressure to dryness. For solvent fractionation, the extract was resuspended in distilled water, and then partitioned successively with n-hexane (Hex), ethyl acetate (EA) and n-butanol (BuOH), leaving a residual aqueous fraction (Aq). Each fraction was evaporated under reduced pressure to yield Hex, EA, BuOH and Aq fractions, respectively. 2.4. Cyanidin test

2.7. Cell culture Human umbilical vein endothelial cells (HUVECs) were taken out from nitrogen tank and after melting at 37 C, a complete culture medium was added and the mixture was centrifuged for 5 min at 1000g. The pellet was washed for an additional time and re-suspended in MCDB131 containing 10% fetal bovine serum (FBS). After cell count and determining cell viability, the suspension was transferred into appropriate cell culture asks and then the culture medium was added. The cultured cells were transferred to 37 C incubator with 5% CO2.

2.8. Cytotoxicity and proliferation assays Cell viability was determined by the Trypan blue exclusion and LDH assay. In brief, HUVECs were seeded at a density of 1 104 cells per well into 96-well plates. After 24 h, shallot fractions and quercetin at different concentrations were added and the cells were cultured for additional 48 h. The experimental procedure for LDH assay was done according to Linfords method (Linford and Dorsa, 2002). A group of wells was treated for 45 min with only culture medium (negative control) and culture medium containing 1% Triton X-100 (maximum LDH release). Plates were centrifuged at 200g, and 100 ll of LDH assay mixture was added and incubated at 37 C for 30 min and the LDH release was estimated at 495 nm. For proliferation assay, the cells were counted after adding different doses of the fractions and quercetin against control wells by a coulter counter (KX-21 Sysmex Co). All these experiments were performed at triplicates.

2.9. HUVEC capillary tube formation in three-dimensional collagen gel The assay was based on the method of Shinoda (Khandelwal, 2007). To one ml of each fraction (2 mg/ml in 80% ethanol), few drops of concentrated HCl and 2 cm magnesium ribbon was added. After appearing red color, addition of increasing amount of sodium hydroxide to the residue shows yellow coloration which decolorizes after addition of the acid. 2.5. Total avonoids determination Aluminum chloride colorimetric method was used for avonoids determination (Chang et al., 2002). Each fraction (0.5 ml) was mixed with 1.5 ml of methanol, 0.1 ml of aluminum chloride (10% w/v), 0.1 ml of potassium acetate (1 M) and 2.8 ml distilled water. The mixture was remained at room temperature for 30 min, and then its absorbance measured at 415 nm. The calibration curve was prepared using quercetin solutions at concentrations 15.62125 lg/ml. 2.9.1. Preparation of cytodex-3-microcarrier beads The cytodex-3-microcarrier beads were pre-swelled in phosphate buffer, and then rinsed with MCDB131 under sterile hood (Nehls and Drekhahn, 1995).

2.9.2. Development of in vitro angiogenesis model HUVECs were mixed with cytodex-3-microcarriers at an appropriate ratio in MCDB131 medium supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 lg/ml streptomycin. Then, cell-coated beads were cultured in collagen matrix and the culture medium was added. In order to monitor anti-angiogenic effect of shallot fractions and quercetin, the cells were treated with different concentrations of such compounds against controls, and the results analyzed microscopically after 4872 h using a specialized software package (AE-31; Motic) according to an standard method (Grifth et al., 2005).

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2.10. Activity of the EA fraction after heat and acid treatments To determine the stability of anti-angiogenic effect of EA fraction, this fraction was subjected to different thermal conditions (40, 60, 80, 100 C) for 20 min and even autoclaved at 121 C. In separate experiments, pH of the fraction decreased to 2 with 2 N HCl, and after several hrs in this pH, it was neutralized with 2 N NaOH. 2.11. Chick chorioallantoic membrane (CAM) model For in vivo anti-angiogenesis activity of EA fraction, chicken chorioallantoic membrane (CAM) was used according to method of Kirchenr et al. (1996) with some modications. Briey, fertilized chicken eggs were incubated at 37 C at constant humidity. On incubation day 3, a square window was opened in the shell and 0.51 ml albumin was removed by an 18-gauge hypodermic needle through a small hole drilled at the narrow end of the eggs, to allow detachment of the developing CAM, and the window was then sealed with glass. On day 7 of development, blank lter discs and discs containing different concentrations of EA fractions (310 ng/egg) and 10 ll distilled water as negative control, were placed on the top of growing CAMs under sterile conditions. The zones around the discs were observed macroscopically 48 h after disc placement, and blood vessel analysis was based on evaluation of angiogenesis by three independent expert observers within a 100-mm2 area surrounding the applied disc. 2.12. Statistical analysis Statistical analysis was calculated based on Students t-test, and all data were presented as mean SD. P < 0.05 was considered to be statistically signicant.

3.3. Flavonoid contents of ethanolic extracts and its fractions Flavonoid contents of the fractions based on quercetin calibration curve (y = 0.008x 0.016, r2 = 0.998) have been shown at Table 1. Ethyl acetate fraction had the highest avonoid content (38% of total avonoid) comparing with other fractions, followed by Hex, BuOH and Aq fractions. 3.4. Separation of ethyl acetate subfractions by TLC Quercetin was identied by observing the characteristic yellow zone. The Rf of quercetin was determined 0.8 based on TLC experiment in 365 nm UV light. The ethyl acetate fraction was separated to three subfractions (namely A, B, C) using TLC. These subfractions were appeared as three dark zones against a green background when exposed to UV light (254 nm) and their Rf values were estimated as: A: 0.25, B: 0.58 and C: 0.67. 3.5. Effect of shallot fractions and quercetin on growth and viability of HUVECs The viability of endothelial cells was evaluated by the Trypan blue and LDH assays. The results of the tests have been shown in Table 2 and Fig. 1. As the results indicated, HUVEC cells had viability of more than 90% even at up to 2 lg/ml of EA fraction (Fig. 1). LD50 of EA fraction was estimated 3.2 lg/ml (Table 2). We examined the effects of shallot fractions and quercetin (as positive control) on HUVECs proliferation. As the results showed (Table 3), the EA fraction at 100 ng/ml did not signicantly decrease the number of cells, but at 300 ng/ml and higher concentrations inhibited proliferation of HUVECs signicantly. Finally, the 50% proliferation inhibition (IC50) of EA fraction on HUVECs was estimated at 1 lg/ ml (Fig. 2 and Table 3). 3.6. Anti-angiogenesis activity of shallot fractions on HUVEC model

3. Results 3.1. Ethanolic fractions of shallot The bulbs of shallot (200 g) was extracted with 50% ethanol and concentrated to dryness under reduced pressure to give an ethanol extract (80 g). The ethanol extract was successively fractionated into n-hexane (0.25%), ethyl acetate (0.65%), n-butanol (8.5%) and aqueous (90.6%) fractions. 3.2. Cyaniding test All fractions showed positive reactions on magnesium hydrochloric acid interaction, a characteristic of avonoid compound. Among the fractions, ethyl acetate fraction showed greater intensity color (red), violence color depended to concentration of avonoid. Three-dimensional culture of HUVECs was applied as an in vitro model to screen the inhibitory effect of shallot fractions, quercetin and isolated subfractions A, B and C. After 3 days of treatment, untreated control wells gave branching pattern of tube like vessels. Among the shallot fractions, the EA fraction showed the highest

Table 2 Survival-inhibitory effects of shallot fractions and quercetin on HUVECs. Sample Hexan fraction Ethyl acetate fraction Butanol fraction Aqueous fraction Quercetin LDso (lg/ml) 16 3.2 40 700 300

Table 1 Flavonoid contents in the shallot fractions. Sample Ethanolic extract Hexan fraction Ethyl acetate fraction Butanol fraction Aqueous fraction Volume of sample (ml) 20 2 3 8 4 Concentration of avonoid (lg/ml) 20.75 23.25 52.29 3.91 31.875 Amount of avonoid (lg) 415 5.4 46.5 2 156.87 5 31.25 1 127.5 3.2 Percentage of avonoid in sample/ethanolic extract 100 11 38 7.5 31

Each value was obtained by calculating the average of three experiments.

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ited in wells which treated with 8 lg/ml of Hex, 25 lg/ml of BuOH, 100 lg/ml of Aq fractions and 25 lg/ml of quercetin. Among the EA subfractions, the subfraction C completely inhibited vessel formation in three-dimensional collagen-cytodex model. The subfraction B showed activity of about 80% and subfraction A had very weak inhibitory effect on angiogenesis in vitro (Fig. 4). 3.7. Effect of heat and low pH on anti-angiogenic properties of EA fraction We examined the stability of EA fraction treated in different conditions including high thermal and low pH conditions. The results showed that the heat and acid treated EA fraction of shallot still retained its anti-angiogenic activity signicantly (Fig. 3). 3.8. Anti-angiogenic effect of EA fraction on CAM model To determine the anti-angiogenic effect of EA fraction of shallot in vivo, we also examined the fraction on chicken chorioallantoic membrane (CAM) model. In this assay, after day 7, the anti-angiogenic response of test and control samples were judged. The fraction started to inhibit the developing angiogenesis at a low dose as 3 ng/egg (Fig. 5 B) and completely abolished it at the dose of 10 ng/egg (Fig. 5 C), whereas the negative control group treated with only water did not show anti-angiogenic effects. 4. Discussion Plants are complex chemical cocktails with medicinal properties acting on multiple pathways that initiate and maintain tumor angiogenesis (Fan et al., 2006). Shallot is widely consumed as a component of the diet of many populations, particularly in Asian diets. It is widely believed to be benecial to health and even curative potential against a range of debilitating conditions and diseases (Azeez et al., 1990). Although numerous scientic studies have been undertaken on other members of Allium family such as garlic to test the basis and validity of these beliefs, little parallel work has been carried out on shallot (Leelarungrayub et al., 2006). The results of the present investigation clearly showed that some fractions from shallot bulbs are effective inhibitors of angiogenesis in vitro and in vivo. At rst, anti-angiogenic activity of all fractions of shallot extract was carried out on HUVECs to nd the fraction with the highest activity. To examine further if endothelial cells viability is affected by shallot fractions and quercetin, the safety of all fractions were evaluated. The fractions inhibited endothelial cell growth and survival in a concentration-dependent manner. As shown (Tables 2 and 3), the lowest LD50 and IC50 for HUVECs were exhibited by the ethyl acetate fraction. The anti-angiogenic potential of ethyl acetate fraction on cytodex-3-microcarrier beads model showed that this fraction from 500 ng/ml could inhibit angiogenesis completely. Therefore, EA fractions in a dose-dependent manner had potent inhibition of sprouting and capillary tube formation on HUVEC, without considerable toxic effect on the cells up to 2000 ng/ml. We also evaluated anti-angiogenic effect of EA fraction on CAM neovascularization as a reliable in vivo model for study of angiogenesis. The EA fraction prevented embryonic angiogenesis in a dose-depended manner; which indicated that A. ascalonicum also has anti-angiogenesis potential in vivo. One of the richest sources of avonoids in human diet is common onion (Allium cepa) and shallot ( A. ascalonicum). Leighton et al. (1992) reported that shallot contains the highest level of total avonols among the onion varieties. Furthermore, the ndings of Fattorusso et al. (2002) showed that, bulbs of shallot had high concentrations of quercetin, isorhamnetin, and their glycosides. Yang et al. (2004) reported that acetone extract of shallot have high a-

Fig. 1. Effect of ethyl acetate fraction on viability of human umbilical vein endothelial cells. Values are expressed as mean SD from at least three independent experiments. *P < 0.05 vs. control, n = 3.

Table 3 Growth-inhibitory effects of shallot fractions and quercetin on HUVECs. Sample Hexan fraction Ethyl acetate fraction Butanol fraction Aqueous fraction Quercetin IC50 (lg/ml) 9 1 16 150 10

The cells were incubated with various concentrations of shallot tractions and quercetin in MCDB131 medium with FBS 10% for 48 h cell proliferation was measured by cell counter assay. The IC50 value was the estimated concentration that resulted in 50% inhibition of cell proliferation under the specied experimental conditions.

Fig. 2. Inhibition rate of ethyl acetate fraction on growth of human umbilical vein endothelial cells. Values are expressed as mean SD from at least three independent experiments. *P < 0.05 vs. control, n = 3.

inhibitory activity on three-dimensional culture of HUVECs, and this inhibition was dose-dependent. This fraction at 100 ng/ml could not inhibit the beads from sprouting and no difference was observed between the results of treated and control wells. The EA fraction at 300 ng/ml, caused partial inhibition, and eventually at 5001000 ng/ml inhibited angiogenesis perfectly (Fig. 3). Therefore, the EA fraction of shallot in a dose-dependent manner inhibited the sprouting of the cultured HUVECs on 3-microcarrier beads, with no cytotoxic effects on the cells at the mentioned concentrations. In addition, capillary tube formation was completely inhib-

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Fig. 3. Effect of the EA fraction on in vitro angiogenesis. HUVECs were cultured on 3-microcarrier beads and seeded in 3-dimensional cytodex-3-microcarrier model. Sprouting at control was induced by adding medium containing growth supplements (A). Angiogenesis of the endothelial cells treated with ethyl acetate at 500 ng/ml (B). Panels (C) and (D) show the inhibition of sprouting by EA fraction (800 ng/ml) treated by heat (121 C), and acid, respectively.

Fig. 4. Effect of the separated subfractions of EA fraction by TLC on angiogenesis at 10 ll of sample per each well of microplate (1 ml). Part A shows angiogenesis of the untreated endothelial cells (control). (B) 42%, (C) 79% and (D) 100% abolish of angiogenesis by A, B and C subfractions, respectively. Size bar is 92 lm.

Fig. 5. Anti-angiogenic effect of ethyl acetate fraction on CAM model of angiogenesis (arrows show neovasculars forming in the chicken chorioallantoic membrane); (A) control, (B) treatment with EA fraction at 3 ng/egg, (C) treatment with ethyl acetate fraction at 10 ng/egg.

vonoid content (34.4 2.1 mg of catechin quiv/100 g of sample). In comparison, we found that total avonoid content in ethanolic extract was 41.5 5.4 (mg of quercetin quiv/100 g of sample). Ethyl acetate fraction of A. ascalonicum, which exhibited the greatest

anti-angiogenic activity, contained the highest amount of avonoid compounds, followed by Hex, BuOH and Aq fractions (Table 1). Quercetin is an important constituent of the avonoid family and is found in high concentrations in shallot. The anti-angiogenic

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activity of quercetin was also documented (Tan et al., 2003; Igura et al., 2001). Herein, we found that quercetin (as positive control) inhibited in vitro angiogenesis completely at 25 lg/ml. We also demonstrated that the anti-angiogenic effect of EA fraction and quercetin can be related to the effect on proliferation of HUVECs. Tan et al. (2003) reported that quercetin inhibited several important steps of angiogenesis including proliferation, migration, and tube formation of endothelial cells in vitro and exerted anti-angiogenic activity in vivo (CAM) and all these effects were concentration-dependent. Our study conrms the recently published nding of Tan et al. Furthermore, our results of preparative TLC indicated that EA fraction can be isolated to at least three subfractions. Among the isolated compounds, subfractions B and C showed anti-angiogenic activities. Flavonoids in silica gels appear as dark spots (at 245 nm) and depending on their structures appear as dark yellow, green, or blue (at 365 nm) (Andersen and Markham, 2006). Because of abundance of organosulfur compounds of Allium plants, these compounds may be responsible for some benecial properties of these plants. However, evidences have shown that organosulfur compounds lost their most effects at high thermal conditions. For example, Agarwal (1996) studied the therapeutic actions of garlic constitutes and demonstrated that the antithrombotic properties of Allicin (one of the organosulfur important compounds) are destroyed at high pH conditions or at temperatures above 56 C. More recently, it has been found that the anti-bacterial effect of shallot was stable at the temperatures of 121 C (Amin and Kapadnis, 2005). Our results revealed that heat and acid treated EA fraction retained its anti-angiogenic effect signicantly. Based on the recent evidences and our ndings, it seems that there is a close relationship between the anti-bacterial and anti-angiogenic activities of shallot EA fraction. Furthermore, Lombard and co-workers demonstrated that quercetin in onion remains stable after treatment at high temperatures (Lombard et al., 2005). Finally, it is concluded that anti-angiogenic activity of shallot ethanolic extract can depend on different chemical compositions including sulphur-containing compounds like allicin and polyphenolic compounds like avonoids. However, our results supports the possibility that anti-angiogenic activity of shallot EA fraction and its isolated subfractions can be due to the presence of heat and low pH stable constituents, mainly avonoid compounds such as quercetin and its glycoside forms. Because of these properties, shallot fractions can be used as specic angiogenesis inhibitors. 5. Conclusions This study apparently for the rst time reported the inhibitory effects of shallot extract and its fractions on angiogenesis in vitro and in vivo. Furthermore, thermal and low pH stability of EA fraction, beside non-cytotoxic effect at pharmacological doses, reinforces useful properties of shallot and considers this medicinal plant as an interesting choice. However, more quantitative studies need to provide accurate data about nutritional impact of this medicinal plant for treatment of angiogenesis related diseases. Acknowledgments We acknowledge Dr. Yadollah Shakiba, Dr. Reza Khodarahmi, Mr. Shahram Parvaneh and Miss. Maryam Keshavarz (Medical Biology Research Center, Kermanshah University of Medical Sciences) for their valuable advises and helpful assistances. This work is supported partly by a grant from Kermanshah University of Medical Sciences.

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