토끼 안점막자극 대체시험법으로서의 HET-CAM assay 소개

김채욱 ( Chae Wook Kim ),정경미 ( Kyoung Mi Jung ),이지해 ( Ji Hae Lee ),윤준원 ( Jun Won Yun ),서병휘 ( Byung Fhy Suh ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.1

The hen`s egg test-chorioallantoic membrane(HET-CAM) assay, which employs the vascular membrane of a fertile hen`s egg, has been actively studied by several countries as an alternative method to the Draize rabbit eye irritation test. There are two methods to evaluate the eye irritation potential, reaction time method(RTM) and end point assessment(EPA), between which can be chosen as the apt method depending on the characteristics of test material. In this study, we employed the EPA method to compare with the Draize rabbit eye irritation test method and then calculated the Pearson correlation coefficient value. The Result of Pearson correlation was high(0.986, p=0.002) with 5 test materials: 3 raw materials(IPMS 03, VCP-IS-2Na, Seletinoid: Amorepacific R&D center, Korea), 1 commercial cleansing form(Amorepacific R&D center, Korea) and 1 surfactant (Amisoft LS-11, Ajinomoto, Japan). According to these results, we suppose that the HET-CAM assay is a good method to replace the Draize eye irritation test as the tests of 5 materials have proven. Despite our results, additional studies with more materials are necessary to validate this method as an alternative to the Draize rabbit eye irritation test method.

안점막자극시험의 대체시험법으로서의 CAM-TBS assay 소개

김채욱 ( Chae Wook Kim ),정경미 ( Kyoung Mi Jung ),서병휘 ( Byung Fhy Suh ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.2

The hen`s egg test-chorioallantoic membrane (HET-CAM) assay, which employs the vascular membrane of a fertile hen`s egg, has been actively studied by several countries as an alternative method to the Draize rabbit eye irritation test. However, the scoring system of the original HET-CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Moreover, the test required skill when the effects of test substance are to be regarded. To overcome these disadvantages, Hagino et al. developed and objective evaluation technique using the amount of trypan blue absorbed at the site of treatment as an indicator of injury to the CAM (Chorioallantoic membrane-trypan blue staining, CAM-TBS). In this study, we evaluate the CAM-TBS methods and used three materials (1%, 2%, 5% SLS, tween 20 and PBS) to evaluate the correlation of CAMTBS with HET-CAM test. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the HET-CAM test score(r=0.958). According to these results, we suppose that the trypan blue staining assay using CAM-TBS may be a good method to replace the HET-CAM test as an alternative to Draize rabbit eye irritation test. Despite our results, additional studies with more materials are necessary to validate this method.

연구논문 : 토끼 안점막자극 대체시험법으로서의 HET-CAM assay 소개

김채욱 ( Chae Wook Kim ),정경미 ( Kyoung Mi Jung ),이지해 ( Ji Hae Lee ),윤준원 ( Jun Won Yun ),서병휘 ( Byung Fhy Suh ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.1

The hen`s egg test-chorioallantoic membrane(HET-CAM) assay, which employs the vascular membrane of a fertile hen`s egg, has been actively studied by several countries as an alternative method to the Draize rabbit eye irritation test. There are two methods to evaluate the eye irritation potential, reaction time method(RTM) and end point assessment(EPA), between which can be chosen as the apt method depending on the characteristics of test material. In this study, we employed the EPA method to compare with the Draize rabbit eye irritation test method and then calculated the Pearson correlation coefficient value. The Result of Pearson correlation was high(0.986, p=0.002) with 5 test materials: 3 raw materials(IPMS 03, VCP-IS-2Na, Seletinoid: Amorepacific R&D center, Korea), 1 commercial cleansing form(Amorepacific R&D center, Korea) and 1 surfactant (Amisoft LS-11, Ajinomoto, Japan). According to these results, we suppose that the HET-CAM assay is a good method to replace the Draize eye irritation test as the tests of 5 materials have proven. Despite our results, additional studies with more materials are necessary to validate this method as an alternative to the Draize rabbit eye irritation test method.

연구논문 : 안점막자극시험의 대체시험법으로서의 CAM-TBS assay 소개

김채욱 ( Chae Wook Kim ),정경미 ( Kyoung Mi Jung ),서병휘 ( Byung Fhy Suh ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.2

The hen`s egg test-chorioallantoic membrane (HET-CAM) assay, which employs the vascular membrane of a fertile hen`s egg, has been actively studied by several countries as an alternative method to the Draize rabbit eye irritation test. However, the scoring system of the original HET-CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Moreover, the test required skill when the effects of test substance are to be regarded. To overcome these disadvantages, Hagino et al. developed and objective evaluation technique using the amount of trypan blue absorbed at the site of treatment as an indicator of injury to the CAM (Chorioallantoic membrane-trypan blue staining, CAM-TBS). In this study, we evaluate the CAM-TBS methods and used three materials (1%, 2%, 5% SLS, tween 20 and PBS) to evaluate the correlation of CAMTBS with HET-CAM test. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the HET-CAM test score(r=0.958). According to these results, we suppose that the trypan blue staining assay using CAM-TBS may be a good method to replace the HET-CAM test as an alternative to Draize rabbit eye irritation test. Despite our results, additional studies with more materials are necessary to validate this method.

열수 녹차추출물이 생쥐의 체중 및 부고환 지방축적 조절에 미치는 영향

박필준(Pil Joon Park),김채욱(Chae Wook Kim),조시영(Si Young Cho),라찬수(Chan Su Rha),서대방(Dae Bang Seo),이상준(Sang Jun Lee) 한국식품과학회 2010 한국식품과학회지 Vol.42 No.1

본 연구에서는 기존 주정추출 방법에 의하여 인위적으로 카테킨 함량을 높인 녹차추출물의 효능이 아니라 음용시 사용되는 녹차가 지닌 일반적인 카테킨 함량에 의한 항비만 효능을 검토하기 위해, 열수 분무건조 방법을 적용하여 제조된 국내산 제주 열수 녹차추출물(aq-GTE)을 C57BL/6J 생쥐에 고지방 식이를 통한 비만을 유도시키며 동시에 농도를 달리하여 8주간 섭취시킨 뒤, 체중과 체내 지방함량 및 혈중 지질함량에 대한 분석을 실시하여 항비만 효능을 확인하였다. 그 결과, aq-GTE 첨가시 HFD 군간 식이 섭취량에는 차이가 없었으나 체중 증가량에 있어서는 aq-GTE에 농도 의존적으로 체중 증가량이 감소됨을 확인하였다. 또한 부고환 지방의 무게는 aq-GTE의 섭취 농도에 따라 유의하게 감소하였고, 체중 변화에 따른 비교에서도 통계적으로 유의하게 부고환 지방 무게의 감소가 확인되었다. 비만을 식이로 유도한 실험군에 나타나는 steatosis 증가 현상이 aq-GTE를 처리시 농도의존적으로 억제되어 감소하는 현상이 발생함을 관찰할 수 있었으며, 그 외에 혈액 분석을 통하여 확인할 수 있는 이상증상은 없는 것으로 확인되었다. 결론적으로 본 시험에서 사용한 aq-GTE는 C57BL/6J 생쥐에서 체중 증가와 체지방 축적 감소 및 지질대사 개선 효과를 나타내었으며, 이는 기존의 주정추출 방식으로 카테킨 함량을 높인 녹차 추출물뿐만 아니라 일반 녹차의 카테킨 함량도 충분히 그 효과를 볼 수 있다는 결과를 검증한 것이고, 향후 aq-GTE의 인체에 미치는 효과에 대한 연구가 수행되어야 할 것으로 사료된다. To obtain the best yield of the beneficial ingredients in green tea, such as catechins, green tea powder is most often prepared by ethyl alcohol extraction. However, the taste, cost and composition of ethyl alcohol extract is different from aqueous spray-dried green tea extract (aq-GTE). Specifically, aq-GTE has a better flavor, lower production costs and higher purity when compared to ethyl alcohol extract. In this study, we elucidated the effect of aq-GTE on diet-induced obesity in male C57BL/6J mice following dose-dependent oral administration of aq-GTE. After eight weeks, the body weight was reduced by 13-17% in mice fed 200 mg/kg bw aq-GTE (12.468±0.45 g; p<0.05) and 20-25% in mice fed 400 mg/kg bw aq-GTE (11.259±0.61 g; p<0.05) when compared with the high-fat diet (HFD) control group mice (14.714±0.95 g; p<0.05). The correlation between epididymal fat accumulation and body weight also decreased by approximately 26.6% (p<0.05) in mice fed a HFD with aq-GTE 400 mg/kg bw. Finally, serum parameters such as the triglyceride, glucose and cholesterol levels in the HFD groups were reduced by the aq-GTE 400 mg/kg bw diet. Analysis on glutamic-pyruvic transaminase, blood urea nitrogen and development of hepatic steatosis revealed no histologic evidence of hepatotoxicity in HFD mice fed aq-GTE. Overall, our results imply that aq-GTE is able to regulate body weight and fat accumulation in mice.

In vitro 및 in vivo에서 검은콩 추출물의 육모 효과

전희영(Hee Young Jeon),김승훈(Seung Hun Kim),김채욱(Chae Wook Kim),신현정(Hyun Jeong Shin),서대방(Dae Bang Seo),이상준(Sang Jun Lee) 한국식품과학회 2011 한국식품과학회지 Vol.43 No.6

본 연구에서는 검은콩의 육모 촉진 효과를 평가하기 위하여 검은콩 추출물을 이용하여 모유두 세포의 성장 촉진과 모세혈관 확장에 미치는 효과를 평가하였으며, C57BL/6 마우스 모델을 이용하여 검은콩 추출물의 경구 섭취가 모발 성장에 미치는 효과를 2% minoxidil과 비교하여 평가하였다. 그 결과 검은콩 추출물이 모유두 세포의 증식을 촉진하고 모세혈관을 확장 시키는 효과가 있으며, 우수한 성장기 모낭 유도 촉진 효과를 통해 모발의 성장을 촉진하는 것을 확인하였다. 또한, 혈액의 항산화능을 증가시키고 혈중 중성 지질을 감소하는 효과를 가져 혈액을 맑게 하고 혈액 순환에 도움을 주어 육모 및 모발 건강 증진에 기여할 수 있음을 확인하였다. 검은콩 추출물의 우수한 육모 효과를 확신하기 위해서는 사람을 대상으로 6개월 이상의 인체 효능 평가와 검은콩 추출물 내의 유효 성분을 규명하는 연구가 후속으로 진행되어야 할 것이다. 그러나 본 연구는 민간에서 널리 알려진 검은 콩의 육모 촉진 효과를 체계적으로 평가하였다는 것에 큰 의의가 있으며, 이를 통하여 검은콩 추출물이 혈액 순환 개선 및 모발 성장 주기 조절을 통해 모발 건강 개선 및 육모 촉진제로써 이용될 수 있다고 기대할 수 있다. It is commonly believed that black soybean (Glycine max: BB) prevents and alleviates hair loss. However, few studies have directly assessed the effect of BB on hair growth. We presently evaluated the mitosis induction on dermal papillae cell and mitogenic effect on NIH3T3 cells in vitro. To elucidate the hair growth promoting effect in vivo, anagen induction and hair restoration were examined in a shaving model of C57BL/6 mice. We also conducted biochemical analysis of blood plasma. Significant stimulation of dermal papillae and NIH3T3 cell proliferation were observed by treatment of BB in a dose-dependent manner. BB markedly promoted hair growth and significantly improved blood total antioxidant capacity and reduced lipid peroxidation and triglyceride level. These results suggest that BB has hair growth promoting effect and it is a potent candidate for the prevention of hair loss.

인공피부를 이용한 피부자극 대체시험 밸리데이션 연구

정경미 ( Kyoung Mi Jung ),문지영 ( Ji Young Moon ),이수현 ( Su Hyon Lee ),김채욱 ( Chae Wook Kim ),박철범 ( Cheol Beom Park ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.1

Several alternative in vitro methods for identifying skin irritants have been developed recently, the most promising of which use reconstituted human skin model. In this study, the two reconstituted human skin models, KeraskinTM and EPI-200, showed promising results in terms of interlaboratory variability and predictive capacity. The main endpoint measured for both KeraskinTM and EPI-200 was cell viability via MTT. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1α release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. In the interlaboratory validation study for KeraskinTM, sensitivity, specificity and accuracy of the KeraskinTM were 90%, 44% and 68%(MTT assay only), respectively. The KeraskinTM model was overly sensitive, resulting in the prediction of the non-irritant chemicals as irritant. The EPI-200 model had an accuracy of 63%, with a sensitivity rate of 70% and a specificity rate of 56%. Based on these results, although predictive capacity of KeraSkinTM model was considered insufficient, due to a low specificity, we concluded that this KeraSkinTM model is useful potential alternative skin irritation method. Further studies are in progress to improve the test protocols and prediction models for KeraSkinTM.

인공각막모델을 이용한 안점막자극 대체시험법 연구

정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),김채욱 ( Chae Wook Kim ),임경민 ( Kyung Min Lim ),손영숙 ( Young Sook Son ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1

Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In order to improve the sensitivity and specificity of alternative eye irritation test, we developed a three-dimensional human corneal model that uses normal human corneal epithelial cells. Our corneal tissue model consists of normal human corneal epithelial cells on cell culture inserts at the air liquid interface, differentiating to form a stratified, squamous epithelium similar to that found in the human cornea, exhibiting in vivo like morphological and histological characteristics. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to in vivo data as well as previously published data obtained in the other three-dimensional corneal model test. Although an overall accuracy of 85% was obtained (sensitivity=100% and specificity=67%), further experiments are required to confirm and validate these preliminary results.

인공각막모델을 이용한 안점막자극 대체시험법 연구

정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),김채욱 ( Chae Wook Kim ),임경민 ( Kyung Min Lim ),손영숙 ( Young Sook Son ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1

Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In order to improve the sensitivity and specificity of alternative eye irritation test, we developed a three-dimensional human corneal model that uses normal human corneal epithelial cells. Our corneal tissue model consists of normal human corneal epithelial cells on cell culture inserts at the air liquid interface, differentiating to form a stratified, squamous epithelium similar to that found in the human cornea, exhibiting in vivo like morphological and histological characteristics. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to in vivo data as well as previously published data obtained in the other three-dimensional corneal model test. Although an overall accuracy of 85% was obtained (sensitivity=100% and specificity=67%), further experiments are required to confirm and validate these preliminary results.