The drug discovery research of clinical-use antioxidants, which may control various vascular
disorders caused by the oxidative stress, is extremely important. We present the development of an
in vivo evaluation system of antioxidants for their vascular protective activities using the chick
embryonic chorioallantoic membrane (CAM). In the case of 12-days chick embryos, the topical
administration of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) induced their
vascular injuries against the CAM veins and venous capillaries but without substantial fatal damage.
Artepillin C, a potent natural antioxidant, did not show the chick embryo’s venous injury, and
pre-treatment with artepillin C would tend to protect the CAM veins injuries induced by the
post-administration of AAPH. These results suggest that artepillin C might be able to protect the
AAPH-induced vascular injury in in vivo. In conclusion, we showed the possibility of in vivo
evaluation system of antioxidants for their vascular protective activities using the chick embryo

Abe, Chiaki

Hori, Hitoshi

Nakata, Eiji

Uto, Yoshihiro

Tokushima University Institutional Repository

Development of an in vivo Evaluation System  of Antioxidants for their Vascular Protective Activities  using the Chick Embryonic Chorioallantoic Membrane  by  Chiaki ABE1, Yoshihiro UTO2*, Eiji NAKATA2, Hitoshi HORI2   Abstract The drug discovery research of clinical-use antioxidants, which may control various vascular disorders caused by the oxidative stress, is extremely important.  We present the development of an in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryonic chorioallantoic membrane (CAM).  In the case of 12-days chick embryos, the topical administration of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) induced their vascular injuries against the CAM veins and venous capillaries but without substantial fatal damage.  Artepillin C, a potent natural antioxidant, did not show the chick embryo’s venous injury, and pre-treatment with artepillin C would tend to protect the CAM veins injuries induced by the post-administration of AAPH.  These results suggest that artepillin C might be able to protect the AAPH-induced vascular injury in in vivo.  In conclusion, we showed the possibility of in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryo.    Key words: Antioxidant, Artepillin C, Chick embryo chorioallantoic membrane, vascular injury   1. Introduction      Agents with anti-inflammatory and antioxidant actions have been demonstrated to exert beneficial effects on cardiovascular diseases (1).  In fact, the in vitro evaluation system of antioxidants is being mainly used now.  It, however, has a problem of no appreciable in vivo redox status of the antioxidants.  Preclinical assays generally using mammalian models such as mouse and rat are time-consuming, expensive, and require special facilities and limitation from ethical and legal points of view.  Therefore, the developments of an alternative method of the mammalian models are necessary.        The chick embryo is frequently used as an alternative experimental animal for angiogenic investigations (2).  Current laws regulating animal experimentation in Japan allows us to use chick embryo without authorization from our animal experimentation committee.  The development of an in vivo model using the chick embryo has more advantages, such as, handy-size, quickness, less expensiveness, and possible building in  1 Graduate School of Advanced Technology and Science, The University of Tokushima 2 Division of Bioinformatics Engineering,   Department of Life System,   Institute of Technology and Science,   Graduate School of The University of Tokushima * The University of Tokushima, 2-1 Minamijosanjima,    Tokushima, 770-8506, Japan???????ソシオテクノサイエンス??????? BULLETIN OF INSTITUTE OF TECHNOLOGY AND SCIENCE THE UNIVERSITY OF TOKUSHIMA- 70 -high-throughput screening than a general animal model.        We adopted artepillin C as an antioxidant to evaluate the utility of our designed in vivo model using the chick embryo.  Artepillin C is a diprenyl-p-hydroxycinnamic acid first isolated from Baccharis species as a major constituent (>5%) in Brazilian propolis (3).  Artepillin C showed a potent antioxidative activity for lipid peroxidation (4).  We succeeded in the first total synthesis of artepillin C (5), and then we termed “isoprenomics” for medicinal chemistry of isoprenoids involved in their structural analyses, biosyntheses, biological functions, and chemotypes (6).        In this paper, we present the development of an in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryo.    2. Materials and Methods  2.1. In vivo model for the evaluation of antioxidants using the 6- or 12-days chick embryo      Fertilized chicken eggs were purchased from Goto Hatchery, Inc. (Gifu, Japan).  The pro- or anti-oxidative effects of AAPH and artepillin C on the chick embryonic chorioallantoic membrane (CAM) vascular were evaluated according to the CAM method (7).  CAM vascular injury was judged by ratio to total number of egg of inhibited one; digestion (Figure 1B) or disappearance (Figure 1C) of CAM blood vessel within the silicon ring.  Figure 1A shows the control (no injury).        Fertilized chicken eggs (Ishii co-ltd, Inc., Tokushima, Japan) were incubated in a humidified incubator at 37.6 °C for 9 days.  At day 10, air cell region and a part of the CAM blood vessel region (2 x 2 cm) on the egg shell were cut off with a grinder.  A shell membrane was peeled off from CAM and the shell membrane was removed.  Opened shell window was sealed with opsite and then the egg was incubated at 37.6°C in a humidified atmosphere until day 12.  At day 12, a silicon ring was placed on the CAM blood vessel and various concentrations of antioxidant solution including 1% methyl cellulose/0.9% NaCl (5% DMSO) were dropped into the silicon ring.  Opened shell window was sealed again with tegaderm and the egg was incubated at 37.6°C in a humidified atmosphere for 24 hours.  CAM veins (size: ca. 0.5 mm) and venous capillaries (size: < 0.1 mm) injury were judged by according to the above-mentioned method.  The significance of difference between different groups was analyzed by the multiple numbers of eggs.    2.2. Protection effect of artepillin C for AAPH-induced oxidative vascular injury      At day 12, a silicon ring was placed on the CAM blood vessel, and various concentrations of artepillin C including 1% methyl cellulose/0.9% NaCl (5% DMSO) were dropped into the silicon ring.  Opened shell window was sealed again with tegaderm and the egg was incubated at 37.6°C in a humidified atmosphere for 1 hour.  The topical administration of AAPH (3 mg) into the silicon ring and egg was incubated at 37.6°C in a humidified atmosphere for 24 hours.  CAM veins and venous capillaries injury were judged according to the description of 2.1.    3. Results  3.1. CAM vascular toxicity of AAPH or artepillin C for 6- and 12-days chick embryo      The topical administration of AAPH (a dose from 100 to 300 g), a water-soluble prooxidant, to the CAM of 6-days chick embryos, resulted in their vascular injuries of percentages from 0 to 100%  dose dependently, which were correlated with their increased fatal rate of percentages from 0 to 71% (Table 1).  Artepillin C (a dose from 0.1 to 10 g), a potent natural antioxidant, tends to show a weak vascular injury and fatal damage (each 33% at 1.0 g) (Table 2).        In the case of 12-days chick embryos, AAPH (doses from 0.3 to 7.0 mg) induced their vascular injuries up to 80% against the CAM veins and venous capillaries without substantial fatal damage (Table 3).  Artepillin C, with its dose of up to 30 g, did not show the CAM veins injury and fatality of chick embryo (Table 4).    ???????ソシオテクノサイエンス??????? BULLETIN OF INSTITUTE OF TECHNOLOGY AND SCIENCE THE UNIVERSITY OF TOKUSHIMA- 71 -3.2. Protection effect of artepillin C for AAPH-induced oxidative vascular injury      In the case of 12-days chick embryos, pre-treatment with artepillin C (a dose of 1 and 10 g) tends to protect the CAM veins injuries, and the injury rate has de-creased up to 20%, induced by the post-administration of 3 mg of AAPH (Table 5).     Figure 1. CAM images of no injury (A), digestion (B), and disappearance (C) of CAM blood vessel  Table 1. Embryo (6-days) and CAM vascular toxicity of AAPH AAPH (g) CAM vascular injury rate (%) Embryo fatal rate (%) Number of eggs   0  14 14 7 100   0  0 5 150  33 11 9 200  86 43 7 250  86 71 7 300 100 71 7  Table 2. Embryo (6-days) and CAM vascular toxicity of artepillin C Artepillin C (g) CAM vascular injury rate (%) Embryo fatal rate (%) Number of eggs  0  0  0 7    0.1 17  0 6    1.0 33 33 6 10 17  0 6  Table 3. Embryo (12-days) and CAM vascular toxicity of AAPH AAPH (mg) Veins injury rate (%) Venous capillary injury rate (%) Embryo fatal rate (%) Number of eggs  0  0 20  0 5 0.3  0 20 20 5 1.0  0 20  0 5 3.0 40 60  0 5 7.0 80 80  0 5 ???????ソシオテクノサイエンス??????? BULLETIN OF INSTITUTE OF TECHNOLOGY AND SCIENCE THE UNIVERSITY OF TOKUSHIMA- 72 -Table 4. Embryo (12-days) and CAM vascular toxicity of artepillin C Artepillin C (g) Veins injury rate (%) Venous capillary injury rate (%) Embryo fatal rate (%) Number of eggs  0  0  0 0 5    0.1 20 60 0 5    1.0  0 20 0 5 10  0 25 0 4 30  0  0 0 2  Table 5. Protection effect of artepillin C for AAPH-induced vascular injury Artepillin C (g) AAPH (mg) Veins injury rate (%) Venous capillary injury  rate (%) Embryo fatal rate (%) Number of eggs  0 0  0  20  0 5  0 3 80 100 40 5    0.1 3 40 100  0 5  1 3 20  80  0 5 10 3 20  60  0 5  4. Discussion      In this paper, we presented the development of the new in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryo.  In the case of 6-days chick embryo, not only AAPH but also artepillin C showed the CAM vascular injury and embryo’s fatality.  These results suggest that immature developing CAM vasculature might have low tolerance to oxidative injury. Besides, artepillin C did not show the CAM veins injury without their fatal damage while AAPH had shown vascular injuries as observed in the case of 12-days chick embryo.  The CAM blood vessels of chick embryo formed during the day 4 or 5 and its rapid capillary proliferation continued until day 11 (8), therefore we thought that this different result depends on the maturity of the CAM blood vessels.  We presumed that artepillin C would tend to protect the CAM veins injuries induced by AAPH because of the amphiphilic property of artepillin C having both vascular penetrativeness and still water solubility.  Our developed in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryo might be useful for the drug discovery research of clinical-use antioxidants.    5. Conclusion      In our developed in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryo, we demonstrated that artepillin C would tend to protect the AAPH-induced vascular injury of 12-days chick embryonic CAM.    Acknowledgments      This work was supported by the 2007 Research Project of Faculty and School of Engineering, The University of Tokushima.    References 1. D. L. Bhatt : Anti-inflammatory agents and antioxidants as a possible "third great wave" in cardiovascular secondary prevention, Am. J. Cardiol., 101(10A), 4D-13D (2008). 2. R. Auerbach, L. Kubai, D. Knighton and J. Folkman : A simple procedure for the long-term cultivation of chicken embryos, Dev. Biol., 41(2), 391-394 (1974). 3. H. Aga, T. Shibuya, T. Sugimoto, M. Kurimoto and S. Nakajima : Isolation and identification of antimicrobial compounds in Brazilian propolis, Biosci. Biotech. Biochem., 58, 945-946 (1994). 4. K. Hayashi, S. Komura, N. Isaji, N. Ohishi and K. ???????ソシオテクノサイエンス??????? BULLETIN OF INSTITUTE OF TECHNOLOGY AND SCIENCE THE UNIVERSITY OF TOKUSHIMA- 73 -Yagi : Isolation of antioxidative compounds from Brazilian propolis: 3,4-Dihydroxy-5-prenylcynnamic acid, a novel potent antioxidant, Chem. Pharm. Bull., 47(11), 1521-1524 (1999). 5. Y. Uto, A. Hirata, T. Fujita, S. Takubo, H. Nagasawa and H. Hori : First total synthesis of artepillin C established by o,o'-diprenylation of p-halophenols in water, J. Org. Chem., 67(7), 2355-2357 (2002). 6. Y. Uto, S. Ae, D. Koyama, M. Sakakibara, N. Otomo, M. Otsuki, H. Nagasawa, K. L. Kirk and H. Hori : Artepillin C isoprenomics: design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity, Bioorg. Med. Chem., 14(16), 5721-5728 (2006). 7. S. Nakayama, Y. Uto, K. Tanimoto, Y. Okuno, Y. Sasaki, H. Nagasawa, E. Nakata, K. Arai, K. Momose, T. Fujita, T. Hashimoto, Y. Okamoto, Y. Asakawa, S. Goto and H. Hori : TX-2152: a conformationally rigid and electron-rich diyne analogue of FTY720 with in vivo antiangiogenic activity, Bioorg. Med. Chem., 16(16), 7705-7714 (2008). 8. D. Ribatti, B. Nico, A. Vacca, L. Roncali, P. H. Burri and V. Djonov : Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti-angiogenesis in vivo, Anat. Rec., 264, 317-324 (2001). ???????ソシオテクノサイエンス??????? BULLETIN OF INSTITUTE OF TECHNOLOGY AND SCIENCE THE UNIVERSITY OF TOKUSHIMA- 74 -

ジェネティック制御下にある発育鶏卵を用いた工学的in vivo 薬剤評価系の開発

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ジェネティック制御下にある発育鶏卵を用いた工学的in vivo 薬剤評価系の開発

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