KR20020006132A - Counteracting composition against heavy metal containing chatechin isolated green tea - Google Patents

Counteracting composition against heavy metal containing chatechin isolated green tea Download PDF

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KR20020006132A
KR20020006132A KR1020000039618A KR20000039618A KR20020006132A KR 20020006132 A KR20020006132 A KR 20020006132A KR 1020000039618 A KR1020000039618 A KR 1020000039618A KR 20000039618 A KR20000039618 A KR 20000039618A KR 20020006132 A KR20020006132 A KR 20020006132A
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이순재
최정화
박근용
이인구
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A23V2250/214—Tea

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Abstract

PURPOSE: A composition for detoxification of heavy metal containing catechin present in green tea as an effective component is provided. Whereby, the composition has excellent effects on suppressing the accumulation of cadmium in the body, stimulating bone metabolism, improving kidney function and blood circulation. CONSTITUTION: The composition for detoxication of heavy metal contains green tea catechin as an effective component. The assay of detoxication effect of the composition toward heavy metal comprises: measuring the suppression of accumulation of cadmium in the body; measuring the improvement in physiological function of bone; measuring the improvement in bone metabolism; measuring an effect on reduction in bone density and content of bone organic materials; measuring an effect on antithrombus and anti-inflammation; measuring an effect on kidney function and blood circulation; and measuring an effect on cardiac function.

Description

녹차 카테킨을 유효성분으로 함유하는 중금속 해독용 조성물{Counteracting composition against heavy metal containing chatechin isolated green tea}Counteracting composition against heavy metal containing chatechin isolated green tea}

본 발명은 카드뮴의 체내 축적을 억제시키고, 골조직 대사를 원활히 하며, 카드뮴 중독으로 인한 신기능 장애를 개선시키고 혈액순환 장애 개선 작용이 있는 녹차 카테킨을 유효성분으로 함유하는 중금속 해독용 조성물에 관한 것이다.The present invention relates to a composition for detoxifying heavy metals containing green tea catechin as an active ingredient, which inhibits accumulation of cadmium in the body, facilitates bone tissue metabolism, improves renal dysfunction due to cadmium intoxication, and improves blood circulation disorders.

최근 산업의 발달로 납, 카드뮴, 수은 등의 중금속 오염이 급증하고 그로 인한 공해병이 날로 심각하여 국내외적으로 큰 사회문제가 되고 있다. 그 중에서도 카드뮴(cadmium : Cd)의 공업적 이용 증가로 인해 토양, 공기, 물이 오염되고 또한 인산비료와 하수, 카드뮴 함유 살균제의 이용 등을 통해 상당량이 토양에 축적되고있다.Recently, due to the development of industry, heavy metal pollution such as lead, cadmium, and mercury has increased rapidly, and the pollution caused by it has become serious. Among them, the increase of industrial use of cadmium (Cd) is contaminated with soil, air and water, and considerable amount is accumulated in soil through the use of phosphate fertilizer, sewage, and cadmium-containing fungicides.

우리나라의 전국적인 카드뮴 섭취량에 대한 보고는 아직 없으나 1980∼1990년 사이의 지역별 카드뮴 섭취 실태 조사에 의하면 일인당 평균 섭취량이 55∼84㎍/day로 Joint FAO/WHO Expert Committee on Food Additives에서 허용한 카드뮴에 대한 식이 섭취 잠정 기준인 57∼71㎍/day를 상회하고 있어 경계해야 할 단계에 진입했다고 볼 수 있다.Although there is no report on the nationwide cadmium intake in Korea, according to the survey of cadmium intake by region between 1980 and 1990, the average intake per person is 55 ~ 84㎍ / day, which is based on the cadmium allowed by the Joint FAO / WHO Expert Committee on Food Additives. Korea's dietary intake was over the preliminary standard of 57-71 ㎍ / day, indicating that it has entered the phase of alertness.

카드뮴의 인체중독 현상으로는 간, 위장, 중추신경의 장애 등의 급성중독 현상과 신장기능의 장애, 칼슘 흡수 장애와 골다공증 등의 골장해를 일으키는 이따이 이따이(itai iati) 병과 같은 만성중독증이 있다. 또한 심장이나 혈액순환에도 영향을 주어 고혈압, 동맥경화증과 같은 심혈관계 질환과 신장장해를 유발할 수 있으며, 동물이나 사람에서 카드뮴이 고혈압을 유발하고 병상이 사람의 본태성 고혈압과 극히 유사하다. 또 체내 카드뮴의 축적량이 많아지게 되면 신장기능 부전과 조직손상이 오고 그 첫 신호로서 단백뇨와 함께 β2-마이크로글로블린 (microglobulin), 레티놀-바인딩 프로틴(retinol-binding protein)과 이뮤노글로블린(immunoglobulin) 등 저분자 단백질의 배설이 증가하며 저농도로 장기간 카드뮴에 노출되었을 때 단백뇨와 같은 신세뇨관 기능장애를 유발한다.Cadmium human poisoning includes acute poisoning such as liver, gastrointestinal, and central nervous system disorders, and chronic poisoning such as itai iati, which causes bone disorders such as disorders of kidney function, calcium absorption, and osteoporosis. It also affects the heart and blood circulation, which can cause cardiovascular diseases such as hypertension and atherosclerosis and kidney failure. Cadmium causes hypertension in animals and humans, and the condition is very similar to essential hypertension in humans. In addition, as the amount of cadmium in the body increases, renal failure and tissue damage occur, and proteinuria as the first signal is β 2 -microglobulin, retinol-binding protein and immunoglobulin. The excretion of low molecular weight protein is increased, and low concentrations of prolonged exposure to cadmium cause renal tubular dysfunction such as proteinuria.

한편 카드뮴의 임상적 중독증상이 동물 체내 영양상태와 관련이 깊다는 여러 연구 보고가 있다. 실험동물을 대상으로 한 카드뮴 독성에 대한 해독과 영양물질과의 관련 연구로는 카드뮴 중독이 완화되거나 체내 카드뮴 축적량을 낮출 수 있는식이인자로는 식이 중에 칼슘수준을 높였을 때(Masanori Ando, Yasuyoshi Sayato, and Toshiaki Osawa : Studies on the Disposition of Calcium in Bones of Rats afrer Continuous Oral Administration of Cadmium.Toxicollogy and applied pharmacology 46:625∼632, 1978), 식이섬유를 공급하였을 때(김미경, 백주은 : 쌀과 보리의 식이 섬유가 흰쥐의 지방 및 Cadmium 대사에 미치는 영향.한국영양학회지 30(3):252∼265, 1997), 구리와 철분을 적정수준으로 섭취하였을 때(김애정, 승정자: 장기간 카드뮴에 중독된 흰쥐에서 카드뮴 축적에 대한 식이 구리와 철분의 효과.한국영양학회지 29(1):70∼76, 1996), 칼슘과 단백질 수준을 높인 경우(권오란: 식이 단백질과 Calcium 수준이 흰쥐의 Cadmium 중독 및 해독에 미치는 영향. 이화여대 박사학위청구논문 1991) 등의 여러 연구가 있다. 그 외에도 칼슘, 단백질, 시스테인 등이 카드뮴 중독시 신장기능 장애를 완화시킨다는 보고도 있다(김미경, 서명숙: 식이내 단백질의 수준과 종류가 흰쥐의 Cadmium 중독에 미치는 영향.한국영양학회지 29(6):578∼589, 1996 ; 이혜영, 김미경: 식이내 Cadmium과 단백질 수준이 흰쥐의 체내 단백질 대사 및 Cadmium 중독에 미치는 영향.한국영양학회지 21(6):410∼420, 1998 ; 류정미, 김미경: 식이내 Cysteine 수준이 흰쥐의 카드뮴과 납주독에 미치는 영향.한국영양학회지 29(6):597∼607, 1996).Several studies have reported that clinical symptoms of cadmium are closely related to nutritional status in animals. Studies on the detoxification and nutritional substances of cadmium toxicity in experimental animals have shown that dietary factors that can alleviate cadmium poisoning or reduce cadmium accumulation in the body are high calcium levels in the diet (Masanori Ando, Yasuyoshi Sayato). , and Toshiaki Osawa: Studies on the Disposition of Calcium in Bones of Rats afrer Continuous Oral Administration of Cadmium.Toxicollogy and applied pharmacology 46 : 625-632, 1978). Effects of Dietary Fiber on Fat and Cadmium Metabolism in Rats Korean Journal of Nutrition 30 (3) : 252 ~ 265, 1997) When Ingestion of Copper and Iron in Proper Levels effects of dietary copper and iron for cadmium accumulates 29 (1) Journal of Korea nutrition: When enhanced 70-76, 1996), calcium and protein levels (gwonoh is: dietary protein and calcium level white Impact of Cadmium poisoning and detoxification effects. There are several studies, such as Ewha Womans University doctoral dissertation in 1991). In addition, calcium, protein, cysteine, etc. is also reported to relieve the city of cadmium poisoning impaired renal function (Kim, Mi - Kyung, seomyeongsuk: Journal of Korea Nutrition 29 Effect of level and type of protein in diets on Cadmium poisoning in rats (6): Effects of Dietary Cadmium and Protein Levels on Body Metabolism and Cadmium Intoxication in Rats Korean Journal of Nutrition 21 (6) : 410 ~ 420, 1998; Ryu Jung-mi, Mi-Kyung Kim: Intracellular Cysteine Effects of Levels on Cadmium and Lead Poisoning in Rats, Journal of the Korean Nutrition Society 29 (6) : 597 ~ 607, 1996).

또한 최근에는 동물체내의 중금속 축적과 중독증상을 완화시키기 위해 천연물질을 이용하여 중금속을 체외로 배설시킬 수 있는 연구가 활발히 진행되고 있다. 그 중에서도 특히 일상생활에서 손쉽게 이용할 수 있는 차에 대한 관심이 증가되고 있고, 뽕잎차 및 녹차가 중금속 제거작용에 쓰인다고 한다.In recent years, research has been actively conducted to excrete heavy metals in vitro using natural materials to alleviate heavy metal accumulation and poisoning symptoms in animals. Among them, in particular, the interest in tea that can be easily used in everyday life is increasing, mulberry leaf tea and green tea is said to be used for heavy metal removal.

녹차는 다양한 약리효과가 있는 기능성 식품으로 잘 알려져 있고 이러한 기능성은 다엽중에 함유되어 있는 여러 가지 유효성분 중 녹차에 특별히 많이 존재하는 카테킨류에 기인한다고 볼 수 있다. 카테킨은 폴리페놀계 화합물로서 녹차 중에 다량 존재하며, 이 성분은 강한 항산화 작용을 가질 뿐 아니라, 금속이온과 착염을 형성하여 중금속을 제거함으로써 중금속의 독성을 해독하는 것으로 알려지고 있다.Green tea is well known as a functional food with various pharmacological effects, and this functionality can be attributed to catechins, which are particularly present in green tea among various active ingredients contained in the multileaf. Catechin is a polyphenol-based compound, which is present in large amounts in green tea, and it is known to detoxify the toxicity of heavy metals by removing complex metals by forming complex salts with metal ions.

체내에 흡수된 카드뮴의 체내분포, 이동 및 독성 발현 등과의 관련성을 검토하기 위하여 간장과 신장이외의 다른 장기에서 카드뮴의 축적량을 알아보거나 장기간동안 경시적 변화를 검토한 연구는 본 발명자가 한국영양학회지에 발표한 '한국산 녹차, 우롱차 및 홍차가 카드뮴에 중독된 흰쥐 간조직의 항산화적 해독작용에 미치는 영향'[윤연희, 이순재,한국영양학회지 27(10):1007∼1017,1994]정도에 불과하다. 특히 카드뮴의 독성으로 인한 뼈조직이나 신장의 병변, 혈소판 응집과 혈관수축 등의 생리적, 생화학적 관찰이나 순환장애 등의 임상적 증상에 미치는 구체적인 기전이나 종합적인 연구가 아직까지 미비할 뿐 아니라 천연물을 이용한 해독기전에 관한 연구는 매우 부족한 실정이다.In order to examine the association between cadmium absorbed in the body, the distribution, migration, and toxic expression, the study was conducted to investigate the accumulation of cadmium in organs other than liver and kidney or to examine changes over time for a long time. The effect of Korean green tea, oolong tea and black tea on the antioxidant detoxification effect of cadmium-addicted rat liver tissues was published in [Yon Yeon-hee, Lee Soon-jae, Korean Journal of Nutrition and Nutrition 27 (10) : 1007 ~ 1017,1994]. Do. In particular, specific mechanisms and comprehensive studies on physiological and biochemical observations such as bone tissue and kidney lesions due to the toxicity of cadmium, and physiological and biochemical observations such as platelet aggregation and vasoconstriction or circulatory disorders have not been completed yet. Research on the detoxification mechanism used is very insufficient.

한편 녹차의 중금속에 대한 연구는 전술한 바와 같이 다소 보고된 바 있고 또 이 등(이서래, 이정희, 최성인 : 녹차 음료의 중금속 제거효과. 제2회 국제 녹차 심포지움 발표논문집, 77∼78, 1993)이 카드뮴과 같은 중금속이 체내에 축적될 때 녹차는 중금속의 체내 흡수를 억제하고 배설을 촉진함으로써 해독작용을 갖는 것으로 보고한 바 있으나, 지금까지 국내외적으로 카테킨 성분 자체만을 추출하여생체실험을 통한 카드뮴 해독작용에 대한 보고는 없었다.On the other hand, studies on heavy metals of green tea have been reported as described above, and Lee et al. (Lee Seo-rae, Lee Jung-hee, Choi Sung-in: Effect of removing heavy metals from green tea beverages. 2nd International Green Paper Symposium, 77 ~ 78, 1993) When heavy metals such as cadmium accumulate in the body, green tea has been reported to have a detoxifying effect by inhibiting the absorption of heavy metals in the body and promoting excretion, but until now only domestic and foreign extracts of the catechin component itself, cadmium through biological experiments There were no reports of detoxification.

본 발명자들은 상기와 같은 점에 착안하여 만성 카드뮴 중독시 가장 문제가 심각한 질병인 골대사 장애 및 심혈관 질환 유발에 대한 병리현상과 또 이를 완화시킬 수 있는 방안을 모색하고자 흰쥐에 만성적으로 카드뮴을 중독시킨 후 녹차 카테킨의 골대사장애 완화 및 혈액순환 개선 기전을 관찰하여 녹차 카테킨이 카드뮴의 체내 축적을 억제시키고, 골조직 대사를 원활히 하며, 카드뮴 중독으로 인한 신기능 장애를 개선시키고 혈액순환 장애 개선에 효과가 있음을 확인하였다.In view of the above, the present inventors have chronically poisoned cadmium in rats in order to explore pathologies for bone metabolism disorders and cardiovascular disease, which are the most serious diseases in chronic cadmium poisoning, and ways to alleviate them. By observing green tea catechins to reduce bone metabolic disorders and improve blood circulation, green tea catechins have been shown to inhibit cadmium accumulation in the body, facilitate bone tissue metabolism, improve renal dysfunction due to cadmium poisoning, and improve blood circulation disorders. It was.

따라서, 본 발명의 목적은 카드뮴의 체내 축적을 억제하고, 골조직 대사를 원활히 하며, 카드뮴 중독으로 인한 신기능 장애를 개선시키고 혈액순환 장애 개선에 효과를 갖는 녹차 카테킨을 유효성분으로 포함하는 조성물을 제공하는데 있다.Accordingly, an object of the present invention is to provide a composition comprising green tea catechin as an active ingredient that inhibits the accumulation of cadmium in the body, facilitates bone tissue metabolism, improves renal dysfunction due to cadmium intoxication and improves blood circulation disorders. have.

본 발명의 상기 목적은 100 g 내외의 sprague-Dawly종 흰쥐를 대상으로 카드뮴을 투여하지 않은 정상군과 카드뮴 투여군으로 나누어 5, 10, 15 및 20주간씩 자유로이 식이와 50ppm Cd2+농도의 식수를 공급하여 사육한 후, 체내 카드뮴 축적 및 배설, 골밀도, 골무기질 함량 및 아라키도닉산 케스케이드(arachidonic acid cascade)계와 혈압변동을 관찰하여 만성 카드뮴 중독으로 인한 골대사 장애 및 혈전생성에 미치는 녹차 카테킨의 효과를 확인, 평가함으로써 달성하였다.The object of the present invention is to divide free diet and drinking water of 50ppm Cd 2+ concentration for 5, 10, 15 and 20 weeks divided into the normal group and the cadmium-administered group in sprague-Dawly species rats of about 100 g Effect of green tea catechin on bone metabolism disorder and thrombus formation due to chronic cadmium poisoning by observing cadmium accumulation and excretion, bone density, bone mineral content, and arachidonic acid cascade system and blood pressure fluctuation after feeding It was achieved by confirming and evaluating.

이하, 본 발명의 구성 및 작용을 설명한다.Hereinafter, the configuration and operation of the present invention.

도 1은 카드뮴 식수 및 식이 공급동안 실험용 쥐의 체중 변화를 나타낸 결과이다.Figure 1 shows the results of body weight change of the experimental rat during cadmium drinking water and dietary supply.

도 2는 만성 카드뮴 중독 쥐의 혈중 카드뮴 농도에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.Figure 2 is a result showing the effect of green tea catechin on blood cadmium concentration of chronic cadmium poisoning rats.

도 3은 20주동안 카드뮴을 공급한 쥐의 적혈구와 백혈구값에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.Figure 3 shows the effect of green tea catechin on the red blood cells and white blood cells of rats fed cadmium for 20 weeks.

도 4는 20주동안 카드뮴을 공급한 쥐의 헤모글로빈농도와 헤마토크리트치에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.Figure 4 shows the effect of green tea catechin on hemoglobin concentration and hematocrit levels of rats fed cadmium for 20 weeks.

도 5는 20주동안 카드뮴을 공급한 쥐의 혈액 중 오스테오칼신(osteocalcin) 수치에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.5 is a result showing the effect of green tea catechin on the level of osteocalcin (osteocalcin) in the blood of rats fed cadmium for 20 weeks.

도 6은 20주동안 카드뮴을 공급한 쥐의 골조직을 광학현미경으로 관찰한 사진도로 A는 대퇴골 정상군의 골조직을 나타내고, B,C 및 D는 카드뮴 투여군의 골조직을 나타내고, C와 D는 녹차 카테킨 공급군의 골조직을 나타내는 사진도이다.6 is an optical microscope photograph of bone tissue of rats fed with cadmium for 20 weeks. A represents bone tissue of a normal femoral group, B, C and D represent bone tissue of a cadmium-administered group, and C and D represent green tea catechins. It is a photograph showing the bone tissue of a supply group.

도 7은 20주동안 카드뮴을 공급한 쥐의 골조직을 전자현미경으로 관찰한 사진도로 A는 대퇴골 정상군의 골조직을 나타내고, B,C 및 D는 카드뮴 투여군의 골조직을 나타내고, C와 D는 녹차 카테킨 공급군의 골조직을 나타내는 사진도이다.FIG. 7 is a photograph showing electron microscopic observation of bone tissue of rats fed with cadmium for 20 weeks. A represents bone tissue of a normal femoral group, B, C and D represent bone tissue of a cadmium-administered group, and C and D represent green tea catechins. It is a photograph showing the bone tissue of a supply group.

도 8은 만성 카드뮴 중독 쥐의 전체 골밀도에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.8 is a result showing the effect of green tea catechin on the total bone density of chronic cadmium poisoning mice.

도 9a와 9b는 각각 만성 카드뮴 중독 쥐의 척추(a)과 골반(b)의 골밀도에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.9a and 9b show the effect of green tea catechin on the bone mineral density of the spine (a) and pelvis (b) of chronic cadmium poisoning mice, respectively.

도 10a와 10b는 만성 카드뮴 중독 쥐의 경골(a)과 대퇴골(b)의 골밀도에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.10a and 10b show the effect of green tea catechin on the bone density of tibia (a) and femur (b) of chronic cadmium poisoning mice.

도 11은 만성 카드뮴 중독 쥐의 전체 골무기질 함량에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.11 is a result showing the effect of green tea catechin on the total bone mineral content of chronic cadmium poisoning rats.

도 12a와 12b는 만성 카드뮴 중독 쥐의 척추(a)와 골반(b)의 골무기질 함량에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.12a and 12b show the effect of green tea catechin on the bone mineral content of the spine (a) and pelvis (b) of chronic cadmium poisoning mice.

도 13은 경골(A)과 대퇴골(B)의 골무기질 함량에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.Figure 13 is a result showing the effect of green tea catechin on the bone mineral content of the tibia (A) and femur (B).

도 14는 만성 카드뮴 중독 쥐의 골칼슘 함량에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.14 is a result showing the effect of green tea catechin on the bone calcium content of chronic cadmium poisoning rats.

도 15는 20주동안 카드뮴을 공급한 쥐의 포스포리파아제 A2(phospholipase A2)활성에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.15 is a result showing the effect of green tea catechins on the Phospholipase A 2 (phospholipase A 2) activity in rats fed a cadmium for 20 weeks.

도 16은 20주동안 카드뮴을 공급한 쥐의 시클로옥시게나아제(cyclooxygenase) 활성에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.16 shows the effect of green tea catechin on the cyclooxygenase activity of rats fed with cadmium for 20 weeks.

도 17은 20주동안 카드뮴을 공급한 쥐의 PGI2/TXA2비에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.17 shows the effect of green tea catechin on the PGI 2 / TXA 2 ratio of rats fed cadmium for 20 weeks.

도 18은 20주동안 카드뮴을 공급한 쥐의 다형핵성 백혈구(polymophonuclear leukocyte)에서의 5'-리폭시게나아제(lipoxygenase) 활성에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.FIG. 18 shows the effect of green tea catechin on 5'-lipoxygenase activity in polymophonuclear leukocytes of rats fed with cadmium for 20 weeks.

도 19는 20주동안 카드뮴을 공급한 쥐의 다형핵성 백혈구에서 루코트리엔 B4(leukotriene B4)활성에 녹차 카테킨이 미치는 영향을 나타낸 결과이다.19 is a result showing the effect of green tea catechins on the base coat Li ¥ B 4 (leukotriene B 4) active in nucleophilic polymorphonuclear white blood cells in rats fed a cadmium for 20 weeks.

본 발명은 녹차 카테킨의 카드뮴 체내 축적 억제 효과를 측정하는 단계; 녹차 카테킨의 골의 생리적 기능 향상 효과를 측정하는 단계; 녹차 카테킨의 골대사 지표 효과를 측정하는 단계; 녹차 카테킨이 골밀도 감소와 골무기질 함량에 미치는 효과를 측정하는 단계; 녹차 카테킨이 항혈전 및 항염증에 미치는 효과를 측정하는 단계; 녹차 카테킨이 신장 기능에 미치는 효과를 측정하는 단계; 녹차 카테킨이 심장기능에 미치는 효과를 측정하는 단계로 구성된다.The present invention comprises the steps of measuring the inhibitory effect of cadmium accumulation in the green tea catechins; Measuring the effect of improving the physiological function of bone of green tea catechin; Measuring the bone metabolic index effect of green tea catechin; Measuring the effect of green tea catechin on bone mineral density and bone mineral content; Measuring the effect of green tea catechins on antithrombosis and anti-inflammatory; Measuring the effect of green tea catechins on kidney function; It consists of measuring the effects of green tea catechins on heart function.

본 발명의 상기 구성에 따르면 녹차 카테킨은 뇨와 변중으로의 카드뮴 배설량을 증가시키고 카드뮴 해독기구인 간장과 신장조직에서의 메탈로사이오닌 (metallothionein:MT) 합성을 촉진시켜 체내 각조직의 카드뮴 축적을 감소시키고, 카드뮴 체내 흡수율과 보유율을 현저하게 감소시키는 효과가 있었다.According to the above configuration of the present invention, the green tea catechin increases the amount of cadmium excreted in urine and stool, and promotes the synthesis of metallothionein (MT) in liver and kidney tissue, which is a cadmium detoxifying device, thereby reducing cadmium accumulation in each tissue in the body. And significantly reduced the cadmium absorption rate and retention rate.

또, 녹차 카테킨은 카드뮴 중독으로 인해 감소된 혈중 적혈구, 백혈구, 헤모글로빈 및 헤마토크릿치를 증가시켰으며, 대퇴골과 혈중에서의 칼슘과 인 등의 무기질 함량을 증가시키고 뇨와 변중 칼슘 배설량은 감소시켜 만성카드뮴 중독 쥐에서의 칼슘대사를 개선키는 효과가 있었다.In addition, green tea catechins increased blood red blood cells, white blood cells, hemoglobin and hematocrit, which were reduced by cadmium poisoning. It was effective in improving calcium metabolism in rats.

또, 녹차 카테킨은 골흡수지표인 뇨중 데옥시피리디노라인 (deoxypyridinoline)과 크로스링크값(crosslinks value) 및 골형성 지표인 혈중 오스테오칼신(osteocalcin) 함량을 감소시켜 골대사 지표를 정상화하였고, 만성카드뮴 중독으로 인한 전체 골밀도, 척추, 골반, 경골 및 대퇴골의 골밀도 감소와 골무기질 함량 손실을 완화하는 효과가 있었다.In addition, green tea catechins normalized bone metabolic index by reducing deoxypyridinoline and crosslinks value and bone osteocalcin, which are bone resorption index, and chronic cadmium poisoning. It was effective in alleviating the loss of bone mineral content and bone mineral density of the total bone density, spine, pelvis, tibia and femur.

또, 녹차 카테킨은 혈소판 포스포리파아제 A2(phospholipase A2), 시크로옥시게나아제(cyclooxygenase) 및 혈청 tolal-TBARS, LDL-TBARS 값을 완화시키고, 혈소판 응집 물질인 트롬복산 A2(thromboxane A2)을 감소시키고 혈소판 응집 억제 물질인 대동맥 프로스타시크린(prostacyclin)을 증가시켜 PGI2/TXA2비 및 PGD2/TXA2비 불균형을 개선시키는 효과가 있었다.The green tea catechins Platelet Phospholipase A 2 (phospholipase A 2), a seek to jade cyclooxygenase (cyclooxygenase), and serum tolal-TBARS, relieve LDL-TBARS value and platelet aggregation substance of thromboxane A 2 (thromboxane A 2 ) decreased the PGI 2 / TXA 2 ratio and PGD 2 / TXA 2 ratio imbalance by reducing the platelet aggregation inhibitor aortic prostacyclin (a).

또, 녹차 카테킨은 다형핵성 백혈구에서 리폭시게나아제(lipoxygenase) 활성과 루코트리엔 B4(leukotriene B4) 생성량을 감소시키는 항염증 작용과 세뇨관 손상지표인 β2-마이크로글로불린을 감소하고, 사구체 여과율의 감소를 막는 신장의 기능의 기능적 손상 억제 효과가 있었다.The green tea catechins Lipoxygenase (lipoxygenase) activity and base coat Li ¥ B 4 (leukotriene B 4) anti-inflammatory and tubular injury indicator of β 2 to reduce the generation amount in polymorphonuclear nucleophilic leukocyte-reducing microglobulin, and glomerular There was an inhibitory effect on the functional impairment of kidney function which prevented the decrease of filtration rate.

또, 녹차 카테킨은 안지오텐신 전환효소(angiotensin converting enzyme: ACE) 활성과 혈압을 정상 수준으로 유지하는 효과가 있었다.In addition, green tea catechins were effective in maintaining angiotensin converting enzyme (ACE) activity and blood pressure at normal levels.

따라서 상기와 같은 효과가 있는 녹차 카테킨을 유효성분으로 하여 통상의 약제 부형제와 혼합하여 캡슐, 정제, 환제 등과 같은 약학적 조성물을 제조하여 제공할 수 있고 또, 녹차 카테킨을 음료를 포함한 각종 가공식품에 일부 함유시켜 기능성 식품을 제조하여 제공할 수 있다.Therefore, the green tea catechin having the above effects as an active ingredient can be mixed with a conventional pharmaceutical excipient to prepare and provide a pharmaceutical composition such as capsules, tablets, pills, etc. In addition, the green tea catechin to various processed foods including beverages Partial inclusion can produce and provide functional foods.

본 발명의 실험에서 사용된 재료와 시약 및 기기는 RIA kit인 6-케토-프로스타그란딘 F1α(prostacyclin), 트롬복산 B2(thromboxane A2), 프로스타그라딘 D2(prostaglandin D2)등은 모두 Amersham 사(buckinghamshire, UK) 제품을 사용하였으며, 분석용 시약인 인도메타친(indomethacin), 아라키돈산(arachidonic acid), 트롬빈(thrombin), 구연산나트륨(sodium citrate), n-핵산(n-hexane), EDTA 등은 sigma사(St.Louis, USA) 제품을 사용하였으며 그외 모든 시약은 특급을 사용하였다. 그리고 식이용 카테킨은 태평양화학 중앙연구소(한국)에 의뢰하여 Ikegaya 등의 방법으로 표 1과 같이 조카테킨(crude catechin) 분말을 조제하여 사용하였다.The materials and reagents and equipment used in the experiment of the present invention, RIA kit of 6-keto-prostaglandin F 1α (prostacyclin), thromboxane B 2 (thromboxane A 2), prostasin Gras Dean D 2 (prostaglandin D 2), etc. All products were manufactured by Amersham (buckinghamshire, UK) and used as analytical reagents, indomethacin, arachidonic acid, thrombin, sodium citrate, and n-hexane. ), EDTA, etc. were used by sigma (St. Louis, USA), and all other reagents were used express. In addition, dietary catechin was used to prepare a crude catechin powder as shown in Table 1 by the method of Ikegaya, etc. commissioned by the Pacific Research Institute (Korea).

본 발명에서 사용한 식이용 녹차 카테킨은 상기 분말 카테킨 외에 녹차로부터 직접 추출분리하여 사용할 수도 있다.The dietary green tea catechin used in the present invention may be used by directly extracting and separating from green tea in addition to the powder catechin.

본 발명 실험에 사용한 녹차 카테킨 분석과 카테킨류의 조성 (Total 및 에피갈로카테킨: EGC, 에피카테킨: EC, 에피갈로카테킨 갈레이트: EGCg, 에피카테킨 갈레이트: ECG...등의 카테킨종류)은 HPLC를 이용한 분석법으로 하였다.Green tea catechin analysis and composition of catechins (total and epigallocatechin: EGC, epicatechin: EC, epigallocatechin gallate: EGCg, epicatechin gallate: ECG ...) It was set as the analysis method using HPLC.

녹차의 crude 카테킨 분말 조성Crude Catechin Powder Composition of Green Tea 카테킨(㎍)/100㎍ powderCatechin (μg) / 100μg powder EGCEGC ECEC EGCgEGCg ECGECG TotalTotal 4.56 ±0.0244.56 ± 0.024 4.52 ±0.0134.52 ± 0.013 38.56 ±0.0638.56 ± 0.06 20.76 ±0.0620.76 ± 0.06 68.40㎍68.40 μg [주] (-) EGC : 에피갈로카테킨 (-) EC : 에피카테킨(-) EGCg : 에피갈로카테킨 갈레이트 (-) ECG : 에피카테킨 갈레이트[Note] (-) EGC: Epigallocatechin (-) EC: Epicatechin (-) EGCg: Epigallocatechin gallate (-) ECG: Epicatechin gallate

이하, 본 발명의 구체적 구성 및 작용을 실시예를 들어 상세히 설명하지만, 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific configuration and operation of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

실시예 1 :녹차 카테킨의 카드뮴 체내 축적 억제 효과Example 1 Inhibition of Cadmium Accumulation in Green Tea Catechin

제 1단계 :실험동물의 사육Step 1: Breeding of Experimental Animals

본 실험에 사용된 실험동물은 무게가 100 g정도 되는 Sprague-Dawley 종 숫컷을 구입하여 실험 시작하기 전 7일간 일정한 환경에서 적응시킨 후 표 2와 같이 식수로 증류수를 공급한 정상군(Normal group)과 카드뮴 식수를 공급한 카드뮴군으로 나누고, 카드뮴 투여군은 다시 식이내 카테킨의 공급 함량에 따라 카테킨을 넣지 않은 Cd-0C군, 카테킨을 0.5% 공급한 Cd-0.5C군 및 1% 공급한 Cd-1.0C군으로 나누었다.The experimental animals used in this experiment were purchased from male Sprague-Dawley species weighing about 100 g and adapted in a constant environment for 7 days before starting the experiment, and then supplied distilled water with drinking water as shown in Table 2 (Normal group). The cadmium-administered group was divided into the Cd-0C group without catechin, the Cd-0.5C group with 0.5% catechin and the Cd- supplied 1%, according to the content of catechin in the diet. Divided into 1.0C group.

카드뮴은 일상생활에서 식수를 통해서 오염될 가능성이 높기 때문에 만성중독시키기 위해 저농도인 50 ppm의 카드뮴을 함유하게 하였다. 카드뮴식수 및 식이의 기본구성은 표 2 및 3과 같다. 식수와 식이는 자유로이 섭취케하며 각각 10주 및 20주간 사육하였다. 체내 카드뮴 흡수율 및 보유율을 관찰하기 위해 5주에 한번씩 700ppm의 카드뮴을 1mL 경구투여하였다(50 ppm 카드뮴함유 식수를 하루 동안의 섭취한 양에 해당함). 또 10주와 20주에는 각각 실험종료 5일전에 메타볼릭케이지(metabolic cage)에 넣고 700 ppm의 카드뮴을 1일 1 mL씩 투여하였으며, 카드뮴 비공급군에는 동량의 증류수를 경구투여 하였고 3일간은 뇨와 대변을 채취하였다.Because cadmium is more likely to be contaminated through drinking water in everyday life, it contains a low concentration of 50 ppm cadmium for chronic poisoning. The basic composition of cadmium drinking water and diet is shown in Tables 2 and 3. Drinking water and diet were freely ingested and bred for 10 and 20 weeks, respectively. In order to observe the body cadmium absorption and retention rate, 700 mL of cadmium was orally administered once every 5 weeks (corresponding to the daily intake of 50 ppm cadmium-containing drinking water). At 10 weeks and 20 weeks, respectively, they were placed in a metabolic cage 5 days before the end of the experiment, and 1 mL of 700 ppm of cadmium was administered daily. An equal amount of distilled water was orally administered to the cadmium-non-supplied group. Urine and feces were collected.

카드뮴 식수 구성Cadmium Drinking Water Composition 카테킨(식이중 %)Catechin (% of diet) 카드뮴(식수 중 50ppm의 카드뮴)Cadmium (50 ppm cadmium in drinking water) NormalNormal 00 -- Cd-0CCd-0C 00 ++ Cd-0.5CCd-0.5C 0.50.5 ++ Cd-1.0CCd-1.0C 1.01.0 ++ [주] 실험군과 대조군은 각각 식수에 50ppm의 카드뮴을 포함하거나포함하지 않고 공급하였다(CdCl2·½H2O).Normal : 카드뮴 무처리군Cd-0C : 카드뮴 처리, 카테킨 무공급군Cd-0.5C : 카드뮴 처리, 식이중 0.5% 카테킨 공급Cd-1.0C : 카드뮴 처리, 식이중 1.0% 카테킨 공급NOTES test and control groups were fed with or without a cadmium 50ppm of each water (CdCl 2 · ½H 2 O) .Normal: cadmium untreated group-0C Cd: cadmium treatment, catechin-free feed-group Cd 0.5 C: Cadmium treatment, 0.5% catechin supply in diet Cd-1.0C: Cadmium treatment, 1.0% catechin supply in diet

식이 구성Dietary composition 성분ingredient 양(g/㎏ diet)Amount (g / kg diet) 콘스타치1) Cornstarch 1) 668668 카제인2) Casein 2) 180180 DL-메티오닌3) DL-methionine 3) 22 콘오일4) Conoil 4) 5050 염혼합물5) Salt mixture 5) 4040 비타민혼합물6) Vitamin Mix 6) 1010 셀룰로오즈7) Cellulose 7) 5050 Kcal/㎏Kcal / kg 38503850 [주] 1)Pung Jin Chem.Co.,Seoul,Korea2)Lactic Casein, 30 mesh, New Zealand Dairy Board, Wington, N.Z.3)Sigma Chem.Co.,St.Louis,Missouri,U.S.A4)Dong Bang oil Co.,Seoul,Korea5)AIN-76 무기질 혼합물(g/㎏ mixture) : 인산칼슘, CaHPO4·2H2O 500, NaCl 74,K3C6H5O7·H2O 220, K2SO452, MgO 24, Manganious carbonate(45∼48% Mn)3.5,구연산철(16∼17% Fe) 6, Zinc carbonate(70% ZnO)1.6, Cuppric carbonate(53∼55% Cu)0.3, KIO30.01, Na2SeO3·5H2O 0.01, CrK(SO4)2·12H2O 0.55,Sucrose finely powedered, to make 1,0006)AIN-76 비타민 혼합물(㎎/㎏ mixture) : 티아민·염산 600, 리보플라빈 600,피리독신·염산 700, 니코틴산 3,000, D-칼슘 판토테네이트 1,600, 엽산 200,D-비오틴(VA) as stabilized powder to provide 400,000 IU VAactibity or120,000 retinol equivaleut, DL-α-토코페릴 아세테이트 5,000 IU,콜레칼시페롤(100,000IU, may be in powder form)2.5, Menaquinone(VK, Menadione)5, Sucrose fimely powdered, to make 1,0007)Sigma Chem.co.CMC(Sodium carboxyl methyl cellulose, mon-nutritive fiber),St.Louis, Missouri, U.S.A2) Pung Jin Chem.Co., Seoul, Korea 2) Lactic Casein, 30 mesh, New Zealand Dairy Board, Wington, NZ3) Sigma Chem.Co., St.Louis, Missouri, USA4) Dong Bang oil Co. , Seoul, Korea5) AIN-76 mineral mixture (g / ㎏ mixture): calcium phosphate, CaHPO 4 · 2H 2 O 500 , NaCl 74, K 3 C 6 H 5 O 7 · H 2 O 220, K 2 SO 4 52 , MgO 24, Manganious carbonate (45-48% Mn) 3.5, Iron citrate (16-17% Fe) 6, Zinc carbonate (70% ZnO) 1.6, Cuppric carbonate (53-55% Cu) 0.3, KIO 3 0.01, Na 2 SeO 3 · 5H 2 O 0.01, CrK (SO 4 ) 2 · 12H 2 O 0.55, Sucrose finely powedered, to make 1,0006) AIN-76 Vitamin mixture (mg / kg mixture): Thiamine hydrochloride 600, Riboflavin 600, pyridoxine / hydrochloric acid 700, nicotinic acid 3,000, D-calcium pantothenate 1,600, folic acid 200, D-biotin (V A ) as stabilized powder to provide 400,000 IU V A actibity or120,000 retinol equivaleut, DL-α-tocopheryl Acetate 5,000 IU, cholecalciferol (100,000 IU, may be in powder form) 2.5, Menaquinone (V K , Menadione) 5, Sucrose fimely powdered, to make 1,0007 Sigma Chem.co.CMC (Sodium carboxyl methyl cellulose, mon-nutritive fiber), St. Louis, Missouri, USA

제 2 단계 :사육동물의 사료섭취량, 체중증가량 및 식이효율Stage 2: Feed Intake, Weight Gain and Dietary Efficiency of Breeding Animals

사료와 식수 섭취량 및 체중은 전 실험기간을 통하여 매일 일정한 시간에 측정하였다. 식이효율은 전체중증가량을 같은 기간동안의 사료섭취량으로 나누어줌으로써 하기 계산식에 의해 계산하였다.Feed, water intake and body weight were measured at regular times every day throughout the entire experimental period. The dietary efficiency was calculated by the following formula by dividing the total increase in weight by the feed intake during the same period.

흰쥐를 20주간 만성적으로 카드뮴을 중독시키면서 실험기간동안의 체중증가량을 관찰하였다. 실험결과 도 1에 나타낸 바와 같이, 정상군은 실험기간동안 계속적인 증가를 보인 반면 카테킨을 공급하지 않은 카드뮴 중독군은 카드뮴 식수공급 후부터 실험기간동안 정상군에 비해 계속적으로 낮은 체중증가량을 나타내었으며, 카테킨공급군은 Cd-0C군에 비해 높은 체중증가량을 나타내었다. 실험결과는 표 4와 같이, 식이 섭취량은 정상군에 비해 카테킨을 공급하지 않으면서 카드뮴을 만성적으로 중독시킨 Cd-0C군만이 유의적으로 감소하였고 카테킨 공급군은 정상수준이었다. 체중증가는 카드뮴을 투여한 군에서 유의적으로 감소하였으나, 카테킨을 공급한 Cd-0.5C군과 Cd-1.0C군에서는 Cd-0C군에 비하여 증가율이 높았다(p<0.05).The rats were chronically intoxicated with cadmium for 20 weeks and the weight gain was observed during the experiment. As shown in FIG. 1, the normal group showed a continuous increase during the experimental period, while the cadmium poisoning group without the catechin supply showed a lower weight gain than the normal group after the cadmium drinking water supply. The catechin supply group showed higher weight gain than the Cd-0C group. As shown in Table 4, the dietary intake was significantly decreased in the Cd-0C group that chronically intoxicated cadmium without supplying catechin, and the catechin supply group was normal compared to the normal group. Body weight gain was significantly decreased in the cadmium-treated group, but was higher in the catechin-supplied Cd-0.5C and Cd-1.0C groups than in the Cd-0C group (p <0.05).

식이효율은 정상군에 비해 카드뮴 투여군 모두가 감소되었으며, 카테킨 투여군(Cd-0.5C군, Cd-1.0C군)에서 Cd-0C군에 비해 유의적으로 증가하였다(p<0.05). 또한 식수섭취량에 따른 카드뮴 섭취량은 카드뮴군간의 유의적인 차이는 없었다.Dietary efficiency was decreased in both cadmium-treated groups compared to the normal group, and significantly increased in the catechin-treated group (Cd-0.5C group, Cd-1.0C group) compared to the Cd-0C group (p <0.05). There was no significant difference in cadmium intake by cadmium group.

녹차 카테킨이 체중, 식이섭취, 식이효율 및 카드뮴섭취에 미치는 영향Effect of Green Tea Catechin on Weight, Dietary Intake, Dietary Efficiency and Cadmium Intake GroupGroup 식이섭취(g/day)Dietary Intake (g / day) 체중(g)Weight (g) 식이효율Dietary efficiency 수분섭취(mL/day)Intake of water (mL / day) Cd 섭취(ppm/day)Cd intake (ppm / day) NormalNormal 14.27 ±0.12a 14.27 ± 0.12 a 309.00 ±5.51a 309.00 ± 5.51 a 0.15 ±0.01a 0.15 ± 0.01 a 32.20 ±0.58a 32.20 ± 0.58 a -- Cd-0CCd-0C 13.07 ±0.06b 13.07 ± 0.06 b 252.25 ±7.32b 252.25 ± 7.32 b 0.13 ±0.01b 0.13 ± 0.01 b 24.82 ±2.44b 24.82 ± 2.44 b 1.38 ±0.14NS 1.38 ± 0.14 NS Cd-0.5CCd-0.5C 14.00 ±0.47a 14.00 ± 0.47 a 284.40 ±5.08c 284.40 ± 5.08 c 0.14 ±0.01ac 0.14 ± 0.01 ac 27.58 ±2.54ab 27.58 ± 2.54 ab 1.46 ±0.161.46 ± 0.16 Cd-1.0CCd-1.0C 14.42 ±0.27a 14.42 ± 0.27 a 286.25 ±8.70c 286.25 ± 8.70 c 0.14 ±0.01ac 0.14 ± 0.01 ac 28.13 ±3.57ab 28.13 ± 3.57 ab 1.41 ±0.141.41 ± 0.14 [주] 1.모든 값은 ±SE(n=10)를 의미한다.2.상기 표의 a,b,c,ab,ac,NS는 Tukey's test의 p<0.05에 의해 유의적인 차이가있다는 의미이다.3.실험조건은 도2와 같다.NOTE 1. All values mean ± SE (n = 10). 2, a, b, c, ab, ac, and NS in the above table means that there is a significant difference by p <0.05 of Tukey's test. .3. Experimental conditions are the same as in FIG.

제 3 단계 :사육 동물로부터 시료채취Step 3: Sampling from Breeding Animals

실험 종료 후 실험 동물을 가벼운 에테르 마취하에서 1 mL의 3.8% 구연산나트륨을 미리 넣어둔 22 게이지의 주사기로 복부 대동맥으로부터 혈액을 채혈하여 실온에서 30분간 방치한 후 1500 ×g에서 각각 15분간 원심분리하여 혈청과 혈소판을 얻었다. 간장과 신장 등을 적출하여 생리식염수로 씻어내고 무게를 측정한 후 -80 ℃에 보관하였다. 무기질 분석을 위한 시료로 이용될 간장, 신장, 경골 및 대퇴골은 무게를 칭량한 후 110℃의 드라이오븐에서 말린 후 데시케이터에 보관하였다. 또한 뇨와 변은 12시간을 절식시킨 날을 제외한 실험종료 전 3일간 대사케이지에서 채취하였으며, 뇨는 총량을 측정한 다음 1500 ×g에서 10분간 원심분리한 후 상등액을 냉동보관하였고, 변은 무게를 측정한 다음 110 ℃의 드라이오븐에서 말린 후 데시케이터에 보관하였다.After completion of the experiment, the animals were collected from the abdominal aorta with a 22 gauge syringe preloaded with 1 mL of 3.8% sodium citrate under light ether anesthesia, left at room temperature for 30 minutes, and then centrifuged at 1500 × g for 15 minutes each. Serum and platelets were obtained. Soy sauce and kidneys were extracted, washed with physiological saline, weighed, and stored at -80 ° C. Soy sauce, kidney, tibia and femur to be used as samples for mineral analysis were weighed and dried in a dry oven at 110 ℃ and stored in a desiccator. In addition, urine and stool were collected in the metabolism cage for 3 days before the end of the experiment except the day of fasting 12 hours, urine was measured for total amount, centrifuged at 1500 × g for 10 minutes, and the supernatant was stored frozen. Was measured and dried in a dry oven at 110 ℃ and stored in a desiccator.

실험결과 표 5에 나타낸 바와 같이, 간장의 무게는 10주 20주 모두 정상군에 비하여 카드뮴 중독군에서 유의적으로 감소하였으며, 카테킨 공급군에서는 카테킨 비공급군인 Cd-0C군에 비해 증가하였으나 유의적인 차이는 없었다. 신장무게는 실험 10주에는 Cd-0C군과 Cd-0.5C군은 정상군에 비해 유의적으로 감소하였으며 Cd-1.0C군만이 정상군 수준이었으나 20주에서는 카테킨 공급군 모두 Cd-0C군에 비해 유의적으로 높았다(p<0.05).As shown in Table 5, the weight of the liver was significantly decreased in the cadmium poisoning group compared to the normal group in all 10 weeks and 20 weeks, and increased in the catechin-supply group compared to the Cd-0C group, which was not the catechin-supply group. There was no difference. Kidney weight was significantly decreased in the Cd-0C and Cd-0.5C groups compared to the normal group at 10 weeks, and only the Cd-1.0C group was normal, but at 20 weeks, the catechin supply group was significantly lower than the Cd-0C group. Significantly higher (p <0.05).

간장무게를 체중으로 나눈 단위체중당 간장 중량은(표 5) 10주에는 실험군간의 유의적인 차이가 없었으나 20주에는 정상군에 비하여 Cd-0C군은 8.6% 증가되었고, 카테킨 공급군에서(Cd-0.5C군, Cd-1.0C군)에서는 정상군 수준이었다. 단위체중당 신장 중량도 10주에는 실험군간의 유의적인 차이가 없었으나 20주에는 정상군에 비해 Cd-0C군만이 유의적으로 높았다(p<0.05).The liver weight per unit weight divided by the weight of the liver (Table 5) was not significantly different among the experimental groups at 10 weeks, but at 20 weeks the Cd-0C group increased 8.6% compared to the normal group, and the catechin supply group (Cd -0.5C group, Cd-1.0C group) was the normal group level. The kidney weight per unit weight was not significantly different among the experimental groups at 10 weeks, but at 20 weeks, only the Cd-0C group was significantly higher than the normal group (p <0.05).

만성 카드뮴중독 쥐의 간장, 신장 중량 및 단위체중당 간장, 신장 중량에 녹차 카테킨이 미치는 영향Effect of Green Tea Catechin on the Liver, Kidney Weight, and Hepatic and Kidney Weight of Chronic Cadmium Poisoned Rats 간장 중량(g)Soy weight (g) 신장 중량(g)Kidney weight (g) 10주10 Weeks 20주20 Weeks 10주10 Weeks 20주20 Weeks NormalNormal 8.63 ±0.37a 8.63 ± 0.37 a 8.97 ±0.75a 8.97 ± 0.75 a 2.81 ±0.06a 2.81 ± 0.06 a 2.94 ±0.05a 2.94 ± 0.05 a Cd-0CCd-0C 6.98 ±0.86b 6.98 ± 0.86 b 7.17 ±0.65b 7.17 ± 0.65 b 2.58 ±0.02b 2.58 ± 0.02 b 2.43 ±0.13b 2.43 ± 0.13 b Cd-0.5CCd-0.5C 7.19 ±0.53b 7.19 ± 0.53 b 8.42 ±0.38b 8.42 ± 0.38 b 2.62 ±0.02bc 2.62 ± 0.02 bc 2.66 ±0.14c 2.66 ± 0.14 c Cd-1.0CCd-1.0C 7.21 ±0.30b 7.21 ± 0.30 b 8.29 ±0.59b 8.29 ± 0.59 b 2.66 ±0.06ac 2.66 ± 0.06 ac 2.72 ±0.04c 2.72 ± 0.04 c 단위체중당 간장 중량(g/100g bw)Soy weight per unit weight (g / 100g bw) 단위체중당 신장 중량(g/100g bw)Elongation weight per unit weight (g / 100g bw) NormalNormal 2.44 ±0.12NS 2.44 ± 0.12 NS 2.55 ±0.10a 2.55 ± 0.10 a 0.64 ±0.03NS 0.64 ± 0.03 NS 0.62 ±0.01a 0.62 ± 0.01 a Cd-0CCd-0C 2.70 ±0.152.70 ± 0.15 2.77 ±0.03b 2.77 ± 0.03 b 0.67 ±0.020.67 ± 0.02 0.68 ±0.01b 0.68 ± 0.01 b Cd-0.5CCd-0.5C 2.58 ±0.072.58 ± 0.07 2.52 ±0.07a 2.52 ± 0.07 a 0.65 ±0.020.65 ± 0.02 0.63 ±0.01a 0.63 ± 0.01 a Cd-1.0CCd-1.0C 2.56 ±0.072.56 ± 0.07 2.57 ±0.05a 2.57 ± 0.05 a 0.63 ±0.020.63 ± 0.02 0.65 ±0.01a 0.65 ± 0.01 a

제 4 단계 :각 조직의 무기질 함량 측정Fourth Step: Determination of Mineral Content in Each Tissue

간장, 신장, 대퇴골과 경골 및 변의 카드뮴 함량은 상기 3단계에서 채취한 시료의 일정량을 취하여 550 ℃의 muffle furnace에서 24시간 동안 건식분해시켜 농질산으로 녹인후 1 N 염산으로, 혈액 및 뇨의 카드뮴함량은 시료의 일정량을 킬달플라스크(kjedahl flask)에 넣고 습식 분해시킨 후 증류수로 희석하여 파장 228.8nm에서 원자 흡수 분광광도기(AAs;atomic absorption spectrophotomer, Cambridge, UK)로 측정하였다. 또, 대퇴골, 경골과 변의 칼슘 함량은 건식회화하고 혈액과 뇨는 각각 습식회화하여 대퇴골, 경골, 혈액, 뇨와 변에서 칼슘은 AOAC법에의하여 422.7 nm에서 AAs로 측정하였다.The cadmium content of the liver, kidney, femur, tibia and feces is taken in a predetermined amount of the sample taken in step 3, and then dried in a muffle furnace at 550 ° C. for 24 hours, dissolved in concentrated nitric acid, and then dissolved in 1 N hydrochloric acid. A predetermined amount of the silver sample was placed in a Kjedahl flask, wet-digested, diluted with distilled water, and measured with an atomic absorption spectrophotometer (AAs) at a wavelength of 228.8 nm. The calcium content of the femur, tibia and feces was dry calcified, and the blood and urine were wet calcified, respectively. The calcium in femur, tibia, blood, urine and feces was measured by AAs at 422.7 nm by AOAC method.

실험결과 표 6에 실험 10주와 20주의 간장 및 신장조직에서의 카드뮴 축적변화를 나타냈다. 간장에서의 카드뮴 함량은 정상군에서는 거의 존재하지 않으나 카드뮴을 만성적으로 투여한 10주에서는 정상군보다 Cd-0C군에서는 85배, Cd-0.5C군은 60배, Cd-1.0C군에서는 45.5배 증가하였으며, 카테킨을 공급한 Cd-0.5C군과 Cd-1.0C군은 카테킨 비공급군인 Cd-0C군에 비해 29%, 46%씩 감소하였다. 또한 20주에서 간조직의 카드뮴 함량은 10주보다 높았으나 실험군간의 차이는 유사하였다. 신장조직에서의 카드뮴 함량변화는 10주와 20주 모두 카테킨 비공급군인 Cd-0C군에 비하여 카테킨 공급군에서는 유의적으로 감소하였다. 또한 10주에서 신장조직의 카드뮴 함량은 간조직의 ½정도로 함량이 적었으나, 20주간 중독시켰을 때에는 신장조직의 카드뮴 함량이 간조직에서 보다 약 2배이상 많았다. 또한 10주에 비하여 20주의 신장에서의 카드뮴 함량은 4배정도 증가하였다.Table 6 shows the changes in cadmium accumulation in the liver and kidney tissues of the 10 and 20 weeks of the experiment. Cadmium content in the liver is rarely present in the normal group, but at 10 weeks of chronic administration of cadmium, 85 times in the Cd-0C group, 60 times in the Cd-0.5C group, and 45.5 times in the Cd-1.0C group. The Cd-0.5C and Cd-1.0C groups fed the catechins decreased by 29% and 46%, respectively, compared to the Cd-0C group, which was not supplied with the catechins. Also, at 20 weeks, the cadmium content of liver tissue was higher than that of 10 weeks. Changes in cadmium content in renal tissues were significantly decreased in the catechin-supply group compared to the Cd-0C group, which was no catechin group. At 10 weeks, the cadmium content in kidney tissue was less than ½ of liver tissue. However, when intoxicated for 20 weeks, the cadmium content in kidney tissue was more than twice that of liver tissue. In addition, the cadmium content in kidneys at 20 weeks increased by 4 times compared to 10 weeks.

만성카드뮴중독 쥐의 간장, 신장 카드뮴 함량에 녹차 카테킨이 미치는 영향Effect of Green Tea Catechin on Hepatic and Kidney Cadmium Content in Chronic Cadmium Poisoning Rats 간장(㎍/g wet wt)Soy (㎍ / g wet wt) 신장(㎍/g wet wt)Kidney (μg / g wet wt) 10주10 Weeks 20주20 Weeks 10주10 Weeks 20주20 Weeks NormalNormal 0.23 ±0.01a 0.23 ± 0.01 a 0.53 ±0.03a 0.53 ± 0.03 a 1.50 ±0.01a 1.50 ± 0.01 a 2.50 ±0.01a 2.50 ± 0.01 a Cd-0CCd-0C 19.52 ±1.49b 19.52 ± 1.49 b 27.54 ±0.94b 27.54 ± 0.94 b 8.63 ±0.22b 8.63 ± 0.22 b 35.79 ±2.03b 35.79 ± 2.03 b Cd-0.5CCd-0.5C 13.80 ±0.92c 13.80 ± 0.92 c 13.14 ±0.43c 13.14 ± 0.43 c 7.58 ±0.21c 7.58 ± 0.21 c 19.55 ±1.10c 19.55 ± 1.10 c Cd-1.0CCd-1.0C 10.47 ±0.76d 10.47 ± 0.76 d 11.59 ±1.49c 11.59 ± 1.49 c 7.38 ±0.36c 7.38 ± 0.36 c 20.25 ±0.51c 20.25 ± 0.51 c

혈액 중의 카드뮴 함랑을 측정한 결과는 도 2에 나타낸 바와 같이, 10주에서 혈액중의 카드뮴 함량은 카테킨 비공급군인 Cd-0C군(21.9±0.90)에 비하여 카테킨공급군인 Cd-0.5C군(10.0±0.9)과 Cd-1.0C군(12.6 ±0.4)에서 각각 54%, 32%씩 감소하였다. 20주에는 Cd-0C군(29.1±0.9)에 비하여 Cd-0.5C군(13.6±1.5)과 Cd-1.0C군(15.0±0.4)은 유의적(p<0.05)으로 감소하였으며 이는 10주와 유사한 경향이었다.As a result of measuring the cadmium dilution in the blood, as shown in Figure 2, the cadmium content in the blood at 10 weeks compared to the Cd-0C group (21.9 ± 0.90), catechin supply group Cd-0.5C group (10.0) ± 0.9) and Cd-1.0C group (12.6 ± 0.4) decreased by 54% and 32%, respectively. At 20 weeks, the Cd-0.5C group (13.6 ± 1.5) and the Cd-1.0C group (15.0 ± 0.4) decreased significantly (p <0.05) compared to the Cd-0C group (29.1 ± 0.9). There was a similar trend.

경골 및 대퇴골에서의 카드뮴 함량 변화는 표 7과 같았다. 경골에서의 카드뮴 함량은 10주에서는 카테킨 비공급군인 Cd-0C군에 비하여 카테킨 공급군에서 감소하였으나 카테킨 공급수준에 따른 차이는 없었다. 20주에는 Cd-0C군에 비하여 Cd-0.5C군과 Cd-1.0C군이 34%, 31%씩 각각 유의적으로 감소하였다(p<0.05). 대퇴골에서의 카드뮴 함량은 10주에서는 카테킨 비공급군인 Cd-0C군에 비하여 카테킨 공급군이 약 44%씩 감소하였으며 카테킨 공급수준에 따른 군간의 차이는 없었다. 20주에는 Cd-0C군(3.94±0.11)에 비하여 Cd-0.5C군(2.64±0.08)과 Cd-1.0C군 (2.39±0.04)이 33%, 39%씩 각각 유의적으로 감소하였다(p<0.05).Changes in the cadmium content in the tibia and femur were shown in Table 7. The cadmium content in the tibia was decreased in the catechin-supply group at 10 weeks compared to the Cd-0C group, which was not the catechin group, but there was no difference in catechin supply level. At 20 weeks, the Cd-0.5C and Cd-1.0C groups were significantly decreased by 34% and 31%, respectively, compared to the Cd-0C group (p <0.05). The cadmium content in the femur was decreased by about 44% in the catechin supply group compared to the Cd-0C group, which was not supplied to the catechin group at 10 weeks. At 20 weeks, Cd-0.5C group (2.64 ± 0.08) and Cd-1.0C group (2.39 ± 0.04) were significantly decreased by 33% and 39%, respectively, compared to Cd-0C group (3.94 ± 0.11) (p <0.05).

뇨와 대변 중의 카드뮴 배설량을 측정한 결과 표 8과 같이, 10주에는 정상군에서 뇨중 카드뮴 배설량은 극히 미량이나 카드뮴 투여군에서는 11.64㎍/day 이었으며, Cd-0.5C군과 Cd-1.0C군에서는 각각 16.08㎍/day 및 19.78㎍/day로 카테킨에 의한 뇨중으로 배설량이 다소 촉진됨을 알 수 있었다. 20주에서 10주에서와 뇨중 배설량도 유사한 경향이었다.As a result of measuring the amount of cadmium excretion in urine and feces, the amount of urine cadmium excretion was very low in the normal group at 10 weeks, but was 11.64 µg / day in the cadmium-treated group, and in the Cd-0.5C and Cd-1.0C groups, respectively. Excretion was slightly promoted by catechin at 16.08 µg / day and 19.78 µg / day. Urine excretion was similar between 20 and 10 weeks.

중금속의 주된 배설경로인 대변중으로 카드뮴의 배설을 보면 10주에서는 카테킨 투여군인 Cd-0.5C군과 Cd-1.0C군에서 비투여군인 Cd-0C군에 비해 2.6배, 4.3배로 현저하게 증가된 것을 알 수 있었다. 20주에서도 10주에서와 마찬가지로 카테킨 비공급군에 비하여 카테킨 공급군에서 현저히 증가하였으며 또한 카테킨의 함량에 따라서도 대변 중 배설량이 차이가 나타나는 것을 볼 수 있었다.The excretion of cadmium in feces, the main excretion pathway of heavy metals, showed a significant increase of 10 and 4.3 times in the catechin-administered group, Cd-0.5C and Cd-1.0C, compared with the non-administered Cd-0C group. Could know. In 20 weeks, as in 10 weeks, catechin was significantly increased in the catechin-supply group, and fecal excretion was also different according to the content of catechin.

만성 카드뮴중독 쥐의 뇨와 대변 중의 카드뮴 배설량에 녹차 카테킨이 미치는 영향Effect of Green Tea Catechin on Cadmium Excretion in Urine and Stool in Chronic Cadmium Poisoning Rats 뇨(㎍/day)Urine (µg / day) 대변(㎍/day)Stool (㎍ / day) 10주10 Weeks 20주20 Weeks 10주10 Weeks 20주20 Weeks NormalNormal 0.3 ±0.08a 0.3 ± 0.08 a 0.5 ±0.07a 0.5 ± 0.07 a 2.4 ±0.21a 2.4 ± 0.21 a 2.0 ±0.11a 2.0 ± 0.11 a Cd-0CCd-0C 11.6 ±3.05b 11.6 ± 3.05 b 12.8 ±2.44b 12.8 ± 2.44 b 101.2 ±10.67b 101.2 ± 10.67 b 128.48 ±12.68b 128.48 ± 12.68 b Cd-0.5CCd-0.5C 16.1 ±4.76bc 16.1 ± 4.76 bc 19.9 ±2.35c 19.9 ± 2.35 c 262.4 ±4.88c 262.4 ± 4.88 c 253.03 ±19.80c 253.03 ± 19.80 c Cd-1.0CCd-1.0C 19.8 ±2.66c 19.8 ± 2.66 c 23.4 ±3.14c 23.4 ± 3.14 c 431.2 ±47.76d 431.2 ± 47.76 d 455.45 ±39.30d 455.45 ± 39.30 d

제 5 단계 :간장 및 신장의 메탈로사이오닌(MT) 함량 측정5th step: Determination of metallocyionine (MT) content in liver and kidney

간장 및 신장조직의 일정량을 취하여 potter-elvejhem homogenizer를 사용하여 10mM tris/HCl(pH 8.2), 0.25M sucrose, 2mM 2-mercaptoethanol, 10mM sodium azide, 0.1mM PMSF(phenylmethyl sulfonyl fluoride)의 50%용액으로써 마쇄하여 105,000 ×g에서 60분간 초원심분리하여 얻은 상층액(cytosol)을 total MT(metallothionein) 분석용으로 사용하였다.Take a certain amount of liver and kidney tissue and use a potter-elvejhem homogenizer as a 50% solution of 10 mM tris / HCl (pH 8.2), 0.25 M sucrose, 2 mM 2-mercaptoethanol, 10 mM sodium azide, 0.1 mM PMphenyl (phenylmethyl sulfonyl fluoride) The supernatant (cytosol) obtained by ultracentrifugation at 105,000 × g for 60 minutes was used for total MT (metallothionein) analysis.

상기의 방법으로 얻은 시토솔(cytosol)에서 Hidalgo 등의 방법에 의하여 MT를 측정하였다. 즉, 일정량의 시토솔에 카드뮴용액(25㎍ Cd/㎖ 시토솔)을 넣어 30분 동안 실온에 방치하면서 시토솔 내에 존재하는 여러 종류의 금속과 결합되어 있는 MT를 Cd-MT로 치환시킨 후 60∼80% 아세톤 침전 단백질 분획을 얻어서 다시 아세톤으로 씻어낸 후 증류수에 재구성시켜 AAs(Solar 929, UK)로 228.8nm에서 측정하였다.MT was measured in the cytosol obtained by the above method by the method of Hidalgo et al. That is, a cadmium solution (25 ㎍ Cd / ㎖ cytosol) in a predetermined amount of the cytosol and allowed to stand at room temperature for 30 minutes while replacing the MT combined with various metals in the cytosol to Cd-MT 60 The 80% acetone precipitated protein fraction was obtained, washed again with acetone and reconstituted in distilled water and measured at 228.8 nm with AAs (Solar 929, UK).

실험결과 표 9에 나타난 바와 같이, 10주에서의 간조직 중의 Cd-MT 함량은 정상군에 비해 카테킨 비공급 카드뮴군(Cd-0C), 0.5% 카테킨 공급군(Cd-0.5C) 및 1% 카테킨 공급군(Cd-1.0C)에서 각각 19.8배, 23배 및 21.2배씩 증가되었다. 20주에서의 간조직 중의 Cd-MT 함량은 10주와 유사한 경향이었으나, 카테킨 공급군에서의 Cd-MT 합성량은 다소 증가되었다.As shown in Table 9, the Cd-MT content in liver tissue at 10 weeks was compared with the catechin-free cadmium group (Cd-0C), 0.5% catechin supply group (Cd-0.5C) and 1% compared to the normal group. In the catechin feed group (Cd-1.0C), they were increased by 19.8, 23 and 21.2 times, respectively. Cd-MT content in liver tissue at 20 weeks tended to be similar to 10 weeks, but the amount of Cd-MT synthesis in the catechin fed group was slightly increased.

10주와 20주 모두 신장조직 중의 Cd-MT 함량은 정상군에 비해 카드뮴 공급군에서 유의적(p<0.05)으로 증가하였으며, 10주에는 카테킨 비공급군(Cd-0C)에 비하여 Cd-0.5C군 및 Cd-1.0C군에서 각각 1.3배 및 1.2배씩 증가되었다. 20주에는 10주에 비하여 신장중의 Cd-MT 합성량이 다소 증가된 경향이었으나 10주와 유사한 경향이었다.In both 10 and 20 weeks, Cd-MT content in renal tissue was significantly increased (p <0.05) in the cadmium-supplied group compared to the normal group, and at 10 weeks, Cd-0.5 was increased compared to the catechin-free group (Cd-0C). 1.3- and 1.2-fold increase in group C and Cd-1.0C, respectively. At 20 weeks, the amount of Cd-MT synthesis in kidneys tended to increase slightly compared to 10 weeks, but it was similar to that of 10 weeks.

만성 카드뮴중독 쥐의 간장, 신장 메탈로티오닌 합성량에 녹차 카테킨이 미치는 영향Effect of Green Tea Catechin on Hepatic and Renal Metallothionine Synthesis in Chronic Cadmium Poisoning Rats 간장(㎍ MT/g 간장중량)Soy Sauce (µg MT / g Soy Weight) 신장(㎍ MT/g 신장중량)Height (μg MT / g Height) 10주10 Weeks 20주20 Weeks 10주10 Weeks 20주20 Weeks NormalNormal 7.06 ±0.69a 7.06 ± 0.69 a 12.35 ±3.17a 12.35 ± 3.17 a 8.66 ±0.87a 8.66 ± 0.87 a 18.66 ±10.87a 18.66 ± 10.87 a Cd-0CCd-0C 139.82 ±13.91b 139.82 ± 13.91 b 120.47 ±12.89b 120.47 ± 12.89 b 51.88 ±3.78b 51.88 ± 3.78 b 61.88 ±13.78b 61.88 ± 13.78 b Cd-0.5CCd-0.5C 162.59 ±7.08b 162.59 ± 7.08 b 182.12 ±20.89c 182.12 ± 20.89 c 66.48 ±3.88c 66.48 ± 3.88 c 76.48 ±13.88c 76.48 ± 13.88 c Cd-1.0CCd-1.0C 149.84 ±6.66b 149.84 ± 6.66 b 169.68 ±11.34c 169.68 ± 11.34 c 61.46 ±2.34c 61.46 ± 2.34 c 71.46 ±12.34c 71.46 ± 12.34 c

제 6 단계 :체내 카드뮴 흡수율 및 보유율Step 6: Absorption and retention rate of cadmium in the body

카드뮴의 체내 흡수율 및 보유율은 상기 제 4단계에서 얻은 카드뮴Absorption rate and retention rate of cadmium in the body is obtained by the cadmium

경구투여량과 변배설량 및 뇨배설량을 하기 계산식에 대입하여 계산하였다.Oral administration, fecal excretion, and urine excretion were calculated by substituting the following formula.

(카드뮴경구투여량, 변배설량 및 뇨배설량의 단위는 ㎍임)(The unit of cadmium oral administration, fecal excretion and urine excretion is μg)

카드뮴의 체내 흡수율과 보유율을 구한 결과 표 10과 같이, 흡수율 과 보유율 모두 카테킨 비공급군에 비하여 카테킨 공급군에서 현저하게 감소되었다. 흡수율의 경우, 10주에는 Cd-0C군에 비하여 Cd-0.5C군과 Cd-1.0C군에서 27%, 55%씩 각각 감소하였고, 20주에는 22%, 57%씩 감소하였다. 또한, 보유율의 경우에는 10주에 Cd-0C군에 비하여 Cd-0.5C군과 Cd-1.0C군이 28%, 58%씩 유의적으로 감소하였으며, 20주에는 23% 60%씩 감소하였다.As a result of the body absorption and retention rate of cadmium, as shown in Table 10, both absorption rate and retention rate were significantly decreased in the catechin supply group compared to the catechin non-feeding group. Absorption rate was decreased by 27% and 55% in the Cd-0.5C and Cd-1.0C groups at 10 weeks and by 22% and 57% at 20 weeks, respectively. In addition, retention rates of Cd-0.5C and Cd-1.0C groups were significantly decreased by 28% and 58% at 10 weeks, and 23% and 60% at 20 weeks, respectively.

만성 카드뮴중독 쥐의 카드뮴 흡수율, 보유율에 녹차 카테킨이 미치는 영향Effect of Green Tea Catechin on Cadmium Absorption and Retention Rates in Chronic Cadmium Poisoning Rats 흡수율(%)Absorption rate (%) 보유율(%)Retention Rate (%) 10주10 Weeks 20주20 Weeks 10주10 Weeks 20주20 Weeks NormalNormal -- -- -- -- Cd-0CCd-0C 85.54 ±1.00a 85.54 ± 1.00 a 81.65 ±0.98a 81.65 ± 0.98 a 83.88 ±1.00a 83.88 ± 1.00 a 78.87 ±0.98a 78.87 ± 0.98 a Cd-0.5CCd-0.5C 62.52 ±0.99b 62.52 ± 0.99 b 63.90 ±0.97b 63.90 ± 0.97 b 60.22 ±0.99b 60.22 ± 0.99 b 61.01 ±0.97b 61.01 ± 0.97 b Cd-1.0CCd-1.0C 38.43 ±0.93c 38.43 ± 0.93 c 34.92 ±0.94c 34.92 ± 0.94 c 35.57 ±0.93c 35.57 ± 0.93 c 31.60 ±0.94c 31.60 ± 0.94 c

실시예 2 :녹차 카테킨의 골 생리적 기능 향상 효과Example 2 Bone Physiological Function Enhancement Effect of Green Tea Catechin

상기 실시예 1에서 사육한 만성 카드뮴 중독 쥐로부터 채취한 시료 혈액 중백혈구와 적혈구 측정은 각각 Cell dyn 1300(Abdott Co., USA)을 이용하여 함량을 측정하였고, 혈액 중 헤모글로빈과 헤마토크리트치는 각각 Cell dyn 1300(Abdott Co., USA)을 이용하여 함량을 측정하였다.Measurement of blood white blood cells and erythrocytes sampled from the chronic cadmium poisoning rats bred in Example 1 was measured using the Cell dyn 1300 (Abdott Co., USA), respectively, hemoglobin and hematocrit in the blood cell dyn The content was measured using 1300 (Abdott Co., USA).

실험결과 도 3에 나타난 바와 같이, 적혈구 함량은 정상군과 카드뮴 투여군간의 유의적인 차이가 없었고, 백혈구 수치는 정상군(11.6±0.59)에 비하여 카드뮴 투여군이 모두 감소되었다. 그러나 카테킨 공급군인 Cd-0.5C군(8.90±0.73)과 Cd-1.0C군(9.90±1.12)은 Cd-0C군(8.20±0.88)에 비해 증가되었다.As shown in FIG. 3, the erythrocyte content was not significantly different between the normal group and the cadmium-administered group, and the white blood cell count was decreased in the cadmium-administered group compared to the normal group (11.6 ± 0.59). However, Cd-0.5C group (8.90 ± 0.73) and Cd-1.0C group (9.90 ± 1.12), which were fed catechins, were increased compared to Cd-0C group (8.20 ± 0.88).

또한, 빈혈의 척도인 헤모글로빈과 헤마토크리트치를 관찰한 결과는 도 4에 나타낸 바와 같이, 헤모글로빈 함량은 정상군(16.8±0.4)에 비해 카드뮴 투여군(Cd-0C군: 14.7±0.2)에서는 유의적으로 감소되었으나, 카테킨 공급군(Cd-0.5C군: 15.01±0.3, Cd-1.0C군: 15.2±0.2)에서는 정상군 수준으로 증가하였다. 헤마토크리트치도 Cd-0C군(36.3±1.41)에서는 25% 감소되었으며 카테킨 공급군(Cd-0.5C: 45.7±0.7, Cd-1.0C군: 45.8±1.0)에서는 Cd-0C군에 비해 유의적으로 증가하였다.In addition, as a result of observing hemoglobin and hematocrit, which are the measures of anemia, the hemoglobin content was significantly decreased in the cadmium-administered group (Cd-0C group: 14.7 ± 0.2) compared to the normal group (16.8 ± 0.4). However, the catechin supply group (Cd-0.5C group: 15.01 ± 0.3, Cd-1.0C group: 15.2 ± 0.2) increased to the normal group level. Hematocrit levels were decreased by 25% in the Cd-0C group (36.3 ± 1.41) and significantly increased in the catechin supply group (Cd-0.5C: 45.7 ± 0.7 and Cd-1.0C group: 45.8 ± 1.0). It was.

실시예 3 :녹차 카테킨의 골대사 지표 효과Example 3 Bone Metabolism Index Effect of Green Tea Catechin

실험예 1 :뇨중 데옥시피리디놀린, 크레아틴 및 크로스링크치 측정Experimental Example 1 Determination of Deoxypyridinolin, Creatine and Crosslink in Urine

실시예 1에서 사육한 만성 카드뮴 중독 쥐로부터 얻은 뇨시료를 원심분리하여 뇨량을 잰 다음 pyrilinks-D kit(Metra biosystem, U.S.A.)를 사용하여 ELISA(enzyme linked immunosorbent assay)법으로 골흡수지표인데옥시피리디놀린(deoxypridinoline)을 측정하였다.Urine samples obtained from chronic cadmium poisoning rats bred in Example 1 were centrifuged to measure urine volume, and then bone absorption index indeoxypyri (ELISA) using pyrilinks-D kit (Metra biosystem, USA). Dinoline (deoxypridinoline) was measured.

또한, 뇨중 크로스링크치(crosslink value)는 sicdia creatinine reagent kit를 이용하여 크레아티닌을 측정하여 뇨중 크레아티닌에 대한 피리디놀린의 비로 나타내었다.In addition, the urine crosslink value (crosslink value) was expressed as the ratio of pyridinolin to creatinine in urine by measuring creatinine using a sicdia creatinine reagent kit.

실험결과 표 11에 나타낸 바와 같이, 뇨중 데옥시피리디놀린을 측정한 결과 정상군(3176±18.5)에 비해 Cd-0C군(3512±65.8)은 11% 정도 높았으나 1% 카테킨 공급군인 Cd-1.0C군(3215±31.9)은 정상군 수준이었다. 뇨중 크레아틴은 정상군에 비하여 Cd-0C군에서 다소 감소하였으나 실험군 모두 유의적인 차이는 없었다. 뇨중 크로스링크치는 뇨중 크레아틴에 대한 데옥시피리디놀린의 비로 나타낸다. 크로스링크치는 정상군(370±4.88)비해 카드뮴 공급군(Cd-0C: 420±12.1, Cd-0.5C: 397±12.2)에서 모두 증가하였으나 Cd-1.0C군(383±6.36)은 정상군 수준으로 회복되었다.As shown in Table 11, the results of measuring urinary deoxypyridinolin were 11% higher in the Cd-0C group (3512 ± 65.8) than in the normal group (3176 ± 18.5), but were 1% catechin-supplied Cd- The 1.0C group (3215 ± 31.9) was normal. Urinary creatine was slightly decreased in the Cd-0C group compared to the normal group, but there was no significant difference in the experimental group. Urine crosslinks are expressed as the ratio of deoxypyridinolin to creatine in urine. Crosslink values were increased in the cadmium supply group (Cd-0C: 420 ± 12.1, Cd-0.5C: 397 ± 12.2) compared to the normal group (370 ± 4.88), but the Cd-1.0C group (383 ± 6.36) was normal. Recovered.

20주동안 카드뮴을 투여한 쥐의 뇨중 데옥시피리디놀린, 크레아틴, 크로스링크치에 녹차 카테킨이 미치는 영향Effects of Green Tea Catechin on Deoxypyridinolin, Creatine, and Crosslink Levels in Urine of Cadmium-treated Rats for 20 Weeks 데옥시피리디놀린(nM)Deoxypyridinolin (nM) 크레아틴(mM)Creatine (mM) 크로스링크값(nM D-pyr/mM 크레아틴)Crosslink value (nM D-pyr / mM creatine) NormalNormal 3176 ±18.5a 3176 ± 18.5 a 8.63 ±0.53NS 8.63 ± 0.53 NS 370 ±4.88a 370 ± 4.88 a Cd-0CCd-0C 3512 ±65.8b 3512 ± 65.8 b 7.89 ±0.977.89 ± 0.97 420 ±12.06b 420 ± 12.06 b Cd-0.5CCd-0.5C 3380 ±24.3c 3380 ± 24.3 c 8.46 ±0.738.46 ± 0.73 397 ±12.19b 397 ± 12.19 b Cd-1.0CCd-1.0C 3215 ±31.9a 3215 ± 31.9 a 8.90 ±0.118.90 ± 0.11 383 ±6.36ab 383 ± 6.36 ab

실험예 2 :혈액중 오스테오칼신 함량 측정Experimental Example 2 Measurement of Osteocalcin Content in Blood

실시예 1에서 얻은 혈액 시료의 혈중 오스테오칼신(osteocalcin)은The blood osteocalcin of the blood sample obtained in Example 1 is

osteocalcin myria kit(Techno genetis Co. Italy)을 이용하여 γ-counter로 레디오엑티비티(radioactivity)를 측정하였다.Radioactivity was measured by γ-counter using osteocalcin myria kit (Techno genetis Co. Italy).

실험결과 도 5에 나타난 바와 같이, 혈청 오스테오칼신은 논콜라겐프로틴(noncollagen protein)이며 골세포간질(bone matrix)에 특이적으로 발견되는 성분으로 골아세포에 의해 합성되며 골형성 지표로 이용된다.As shown in Figure 5, serum osteocalcin is a non-collagen protein (noncollagen protein) is a component found specifically in the bone marrow interstitial (bone matrix) is synthesized by osteoblasts and used as an indicator of bone formation.

정상군(0.19±0.004)에 비해 Cd-0C군(0.23±0.009)에서는 약 21% 높았으나 Cd-0.5C군(0.21±0.014)과 Cd-1.0C군(0.19±0.003)은 정상군과 유의적인 차이가 없었다.The Cd-0C group (0.23 ± 0.009) was about 21% higher than the normal group (0.19 ± 0.004), but the Cd-0.5C group (0.21 ± 0.014) and Cd-1.0C group (0.19 ± 0.003) were significantly different from the normal group. There was no difference.

실험예 3 :대퇴골 및 신장조직의 형태학적 변화 관찰Experimental Example 3 Observation of Morphological Changes of Femur and Kidney Tissue

실시예 1에서 제조한 시료 대퇴골 조직의 광학 현미경적 관찰을 위해 각 군의 실험동물을 희생시킨 후 오른쪽 대퇴부의 골편을 절취하여 5% 질산용액에서 2∼3일간 탈회시킨후 에탄올 계열로 탈수하고 통상방법에 따라 파라핀에 포매하였다. 포매된 조직은 마이크로톱으로 4∼5㎛로 절편을 만들고 헤마토실린-에오신 (Hematoxylin-Eosin)염색을 한 후 광학현미경(Nikon, Japan)으로 관찰하였다. 또한 신장의 광학 현미경적 관찰은 실험동물의 우측 신장을 적출하여 10% neutrol formalin으로 수세하고 탈수하여 파라핀으로 포매한 후 4∼5 μm 두께로 절단하여 헤마토실린-에오신 시약으로 발색시킨 후 관찰하였다. 이어서 동일한 조직을 전자현미경으로 다음과 같이 관찰하였다. 절취한 신장 조직 절편을 1 ㎣의 크기로 세절하여 2.5% 글루타르알데하이드 용액(0.1M 인산완충제, pH 7.4)으로 0∼4 ℃에서 2시간 전고정, 0.1 M PBS로 세척한 후 1% OsO4용액에 2시간 후 고정하고 같은 완충용액으로 세척하여 에탄올로 탈수하였다. 절취한 대퇴골은 1 ㎝ 정도로 잘라서 2.5% 글루타르알데하이드 용액에 2시간 고정한 후 다시 단면을 만들어서 2시간 고정하였다. 탈회는 EDTA(ethylene diamine tetra acetic acid) 탈회법을 이용하여 조직을 교반기로 교반하면서 실온에서 탈회하였다. 탈회액은 격일로 교환하고 침이 통과할 정도의 경도까지 탈회하였다. 종료 후에는 0.25M 스크로오즈를 포함한 완충액에 넣어 하룻밤을 냉장실에서 세척하였다. 세척 후 1% OsO4용액에 후고정하였으며 다른 조직과 같은 방법으로 탈수하였다. 프로필렌 옥사이드(propylene oxide)로 치환한 후 Luft 방법에 의한 epon혼합물로 포매하여 37 ℃에 12시간, 60℃에 48시간 동안 방치하여 열중합시켰다. 포매된 조직을 1μm두께로 박절하여 toluidine blue염색을 하였으며 관찰 부위를 결정한 다음 초박절은 sorval MT 6000형 초박절기에 Dupont 다이아몬드 칼로 회백색(40∼60nm)의 간섭색을 나타내는 초박절편을 얻었다. Grid에 부착하여 Watson 및 Reynolds 방법에 의한 우라닐 아세테이트(uranyl acetate)와 lead citrate로 이중전자염색을 실시하여 Hitachi H-600 투과전자현미경(Japan)으로 관찰하였다. 대퇴골과 신장 조직을 광학현미경으로 관찰한 실험결과, 대퇴골의 정상군은 조직학적 단면에서 밖은 치밀뼈로 되어있는 피질과 뼈잔기둥(trabeculae)이 서로 얽히어 그물모양을 이루고 있는 수질부위로 구분되어져 있었다. 피질은 비교적 얇았으며 층판구조로 되어있고 층판 안에는 뼈세포가 들어있는 작은 공간인 뼈세포방이 층판에 따라 배열되어 있었다. 수질부의 뼈잔기둥 및 뼈침(bony spicule)들의 가장자리에는 뼈모세포(osteoblast)들이 한 층으로 나열되어 있으며 가끔씩 골파괴세포(osteoblast)들도 한 두개씩 보였다. 나머지 공간들은 골수세포와 지방세포들이 거의 같은 양으로 메워져있었다. 골수에는 혈구성 및 골수성의 모세포들이 약 1:3 내지는 1:4정도로 분포되어 있었으며 가끔 혈소판의 모세포인 거대핵세포(megakaryocyte)도 관찰되었다. 이들 모세포들 이외에도 적혈구 및 다양한 숫자의 과립구 및 림프구도 관찰되었다(도 6A). 카드뮴 투여군에서는 피질의 치밀뼈가 얇아지거나 두꺼워지지 않았고 수질부의 뼈잔기둥도 숫자나 배열에 이상이 없었으며, 골세포 및 골모세포 등도 형태학적 이상이 없었다. 골수세포의 분포상의 변화도 없었으며 세포형태의 이상소견도 없었다(도 6B,C,D).For the optical microscopic observation of the sample femoral tissue prepared in Example 1, after sacrifice of the experimental animals of each group, the bone fragments of the right thigh were cut and demineralized in 5% nitric acid solution for 2-3 days, followed by dehydration with ethanol series. It was embedded in paraffin according to the method. The embedded tissue was sectioned with a microtop with 4-5 μm, stained with hematoxylin-Eosin, and observed with an optical microscope (Nikon, Japan). In addition, optical microscopic observation of the kidney was performed after extraction of the right kidney of the animal, washed with 10% neutrol formalin, dehydrated, embedded with paraffin, cut to 4-5 μm thickness, and developed with hematoxylin-eosin reagent. . The same tissue was then observed by electron microscopy as follows. Cut the resected kidney tissue into 1 ㎣ size and fix it with 2.5% glutaraldehyde solution (0.1M phosphate buffer, pH 7.4) for 2 hours at 0-4 ° C, wash with 0.1 M PBS, and then wash with 1% OsO 4 After fixing for 2 hours, the solution was washed with the same buffer and dehydrated with ethanol. The cut femur was cut to about 1 cm and fixed in 2.5% glutaraldehyde solution for 2 hours, and then sectioned again to fix for 2 hours. Deliming was delimed at room temperature while stirring the tissue with a stirrer using ethylene diamine tetra acetic acid (EDTA) deliming method. The deliming liquor was exchanged every other day and demineralized to the extent that the saliva passed. After termination, the resultant was placed in a buffer containing 0.25M rhodes and washed overnight in the refrigerator. After washing, it was post-fixed in 1% OsO 4 solution and dehydrated in the same manner as other tissues. Substituted with propylene oxide (propylene oxide) and embedded in the epon mixture by the Luft method, and thermally polymerized by standing for 12 hours at 37 ℃, 48 hours at 60 ℃. Toluidine blue was stained by cutting the embedded tissue to a thickness of 1 μm. After determining the observation site, the ultrathin section was obtained by using an ultra-thin section of gray white (40 ~ 60nm) with a Dupont diamond knife in a sorval MT 6000 ultrathin section. Attached to the grid, double electron staining with uranyl acetate and lead citrate by Watson and Reynolds method was observed with Hitachi H-600 transmission electron microscope (Japan). Experimental results of femoral and renal tissue under an optical microscope show that the normal group of femurs is divided into cortical and trabeculae of the dense bones in the histological section and the medulla, which is reticulated. there was. The cortex was relatively thin and had a lamellar structure. In the lamina, the bone cell room, a small space containing bone cells, was arranged along the lamina. Osteoblasts are arranged in layers on the edges of the medulla and bony spicules, and sometimes osteoblasts are seen one or two. The remaining spaces filled almost the same amount of bone marrow cells and fat cells. In the bone marrow, hematopoietic and myeloid blasts were distributed at about 1: 3 or 1: 4, and sometimes megakaryocytes, which are platelet blasts, were also observed. In addition to these blasts, red blood cells and various numbers of granulocytes and lymphocytes were also observed (FIG. 6A). In the cadmium-administered group, the dense bones of the cortex were not thinned or thickened, and there were no abnormalities in the number or arrangement of bone residues in the medulla. There was no change in the distribution of myeloid cells and no abnormalities in cell morphology (FIGS. 6B, C, D).

전자현미경으로 관찰한 결과는, 피질은 골기질이 규칙적인 층계를 이루고 있으며 중간 중간에 뼈세포방이 있고 안에는 뼈세포가 들어있다. 골수세포들은 세포질에 다양한 형태의 과립이 관찰되었다. 핵은 성숙정도에 따라 단핵인 경우와 분엽의 경우 등으로 나타났다(도 7A). 카드뮴 투여군에서는 피질의 골기질의 분포와 양에서도 이상 소견이 없었고 골세포 및 골모세포 등도 형태학적 이상이 없었으며, 골수 세포의 핵과 과립에도 이상소견이 없었다(도7B,C,D).The results of electron microscopy show that the cortex has a regular stratification of bone matrix, with bone cells in the middle and bone cells inside. In the bone marrow cells, various types of granules were observed in the cytoplasm. The nucleus was found to be mononuclear and soybean, depending on the degree of maturity (Fig. 7A). In the cadmium-administered group, there were no abnormalities in the distribution and quantity of bone matrix in the cortex, no morphological abnormalities in bone cells and osteoblasts, and no abnormalities in the nuclei and granules of bone marrow cells (Fig. 7B, C, and D).

실시예 4 :녹차 카테킨이 골밀도 감소와 골무기질 함량에 미치는 효과Example 4 Effect of Green Tea Catechin on Bone Mineral Density and Bone Mineral Content

small animal total body option을 가지고 있는 dual energy x-rayabsorptiometry(이하 DEXA : Lunar DPX-L, USA)를 이용하여 실험, 5, 10, 15, 20주째 되는 날 카드뮴 중독 흰쥐의 척추, 골반, 오른쪽 대퇴골 및 경골의 골밀도(bone mineral density, 이하 BMD), 골무기질 함량(bone mineral content, BMC)과 총골칼슘함량(total bone calcium contents, TBCa)을 측정하였다.Experiments using dual energy x-rayabsorptiometry (DEXA: Lunar DPX-L, USA) with small animal total body options, spine, pelvis, right femur and The bone mineral density (BMD), bone mineral content (BMC) and total bone calcium contents (TBCa) of the tibia were measured.

요추 골밀도는 전후면 투영(anteroposterior projection, AP)으로 측정하고, 요추 골밀도로 표현되는 수치는 제 2요추에서 제 4요추까지의 골밀도의 평균 수치를 사용하였다.The lumbar spine BMD was measured by anteroposterior projection (AP), and the lumbar spine BMD was used as an average value of bone density from the second lumbar spine to the fourth lumbar spine.

실험결과, 전체 골밀도는 도 8과 같이, 실험초기 0주부터 20주까지 기간에 따라 점차적으로 증가하였으나 Cd-0C군은 10주에서부터는 증가가 둔화되었다. 척추 골밀도를 측정한 결과 도 9a와 같이, 실험초기부터 20주까지 실험기간이 지남에 따라 전반적으로 증가는 하였으나 Cd-0C군만은 10주에서부터 실험기간별로 유의성이 없었다. 골반 골밀도를 측정한 결과 도 9b와 같이, Cd-0.5C군을 제외한 실험군 모두가 실험 기간별로는 10주에서부터는 기간에 따른 유의적인 차이는 없었으며 Cd-0.5C군은 10주때에 비해 20주에 유의적으로 골밀도가 증가하였다. 경골에서의 골밀도를 관찰한 결과 도 10a와 같이, 실험초기에서 20주간의 골밀도 증가율은 정상군은 169%인 반면 Cd-0C군은 148%로 정상군에 비해 골밀도 증가율이 약 20% 낮았다. 대퇴골의 골밀도를 측정한 결과 도 10b와 같이, 실험군별로는 Cd-0C군을 제외한 모든 실험군에서 15주까지 실험기간별로 유의적으로 증가하였으나 Cd-0C군에서는 10주(0.294±0.007)에서부터 20주(0.308±0.007)까지 기간에 따른 유의성은 없었다.As a result, as shown in Figure 8, the total bone density gradually increased with the period from 0 weeks to 20 weeks at the beginning of the experiment, but the increase was slowed from 10 weeks in the Cd-0C group. As a result of measuring the spinal bone density, as shown in FIG. 9A, the overall increase from the beginning of the experiment to the 20-week experimental period increased, but the Cd-0C group was not significantly different from the 10-week experimental period. As a result of measuring the pelvic bone density, as shown in FIG. 9B, all the experimental groups except for the Cd-0.5C group had no significant difference according to the period from 10 weeks to the experimental period, and the Cd-0.5C group at 20 weeks compared with the 10 weeks. Bone density increased significantly. As a result of observing the bone density in the tibia, as shown in FIG. 10A, the bone density increase rate of 20 weeks at the beginning of the experiment was 169% in the normal group, and 148% in the Cd-0C group, which was about 20% lower than in the normal group. As a result of measuring the BMD of the femur, as shown in FIG. 10B, the experimental groups significantly increased by 15 weeks in all experimental groups except the Cd-0C group, but in the Cd-0C group, 10 weeks (0.294 ± 0.007) to 20 weeks ( Up to 0.308 ± 0.007).

몸 전체 골무기질 함량을 카드뮴 섭취 기간별로 관찰한 결과 도 11과 같이, 실험시작 0주부터 20주까지 지속적으로 증가하여 전체 골밀도와 유사한 경향을 보였다.As a result of observing the total bone mineral content by the period of intake of cadmium, as shown in Figure 11, it was continuously increased from 0 weeks to 20 weeks from the beginning of the experiment showed a tendency similar to the total bone density.

척추 골무기질 함량을 측정한 결과 도 12a와 같이, 정상군은 실험시작 0주부터 20주까지 기간이 지남에 따라 전반적으로 증가를 하였으며 Cd-0C군은 10주에서부터 유의적인 증가는 되지 않았고 카테킨군은 15주까지 유의적으로 증가되었다(p<0.05). 골반에서 골무기질 함량을 측정한 결과 도 12b와 같이, 실험기간별로 10주까지는 모든 군에서 유의적으로 증가하였으며, 특히 Cd-1.0C군은 20주까지 전반적으로 지속적으로 증가되었다. 경골중의 골무기질 함량을 측정한 결과 도 13a와 같이, 실험기간 별로 15주까지는 유의적으로 증가하였다(p<0.05). 그러나 골무기질 함량 증가율을 비교하면 정상군은 실험 초기에 비해 20주에는 498%의 증가율을 보이는 반면 Cd-0C군은 445%의 증가율을 보임으로써 정상군에 비해 약 40%의 낮은 증가율을 보였다. 대퇴골의 골무기질 함량 역시 도 13b와 같이, 타조직의 골무기질 함량과 비슷하게 실험기간 별로 실험군 모두 10주까지 유의적으로(p<0.05) 증가하였으며 정상군은 20주의 실험기간동안 약 760%, Cd-0C군은 56%, 카테킨 공급군인 Cd-0.5C군과 Cd-1.0C군은 각각 738%, 712%씩 증가하였다.As a result of measuring the bone mineral content of the spine, as shown in FIG. 12A, the normal group increased as the period from 0 week to 20 weeks, and the Cd-0C group was not significantly increased from 10 weeks. Increased significantly by week 15 (p <0.05). As a result of measuring the bone mineral content in the pelvis, as shown in Figure 12b, the experimental period was significantly increased in all groups up to 10 weeks, especially in the Cd-1.0C group was continuously increased up to 20 weeks. As a result of measuring the bone mineral content of the tibia, as shown in FIG. 13a, the experimental period increased significantly up to 15 weeks (p <0.05). However, compared with the increase rate of bone mineral content, the normal group showed an increase rate of 498% at 20 weeks compared with the initial experiment, while the Cd-0C group showed an increase rate of 445%, which was about 40% lower than the normal group. The bone mineral content of the femur also increased significantly (p <0.05) in all experimental groups up to 10 weeks for each experimental period, similar to the bone mineral content of other tissues. In the -0C group, the catechin supply group increased by 738% and 712% in the Cd-0.5C and Cd-1.0C groups, respectively.

전체 골조직중의 칼슘 함량은 도 14와 같이, 실험기간별로 전 실험군에서 15주까지 유의적으로 증가하였다(p<0.05). 실험기간 20주동안 정상군은 514% 증가하였으며 Cd-0C군은 442% 증가하는 반면 0.5% 카테킨 공급군은 489%, 1% 카테킨 공급군은 498% 증가함으로서 카드뮴 중독시 카테킨을 공급함으로서 골무기질 함량이 약40∼50% 증가되었다.The calcium content in the total bone tissue was significantly increased up to 15 weeks in all experimental groups by the experimental period, as shown in Figure 14 (p <0.05). During the 20-week period, the normal group increased 514%, the Cd-0C group increased 442%, while the 0.5% catechin supply group increased 489% and the 1% catechin supply group increased 498%. The content increased about 40-50%.

실시예 5 :녹차 카테킨의 항혈전 및 항염증에 미치는 효과Example 5 Effect of Green Tea Catechin on Antithrombosis and Anti-inflammatory

PRP(Platelete rich plasma)를 조제하기 위하여 카드뮴 중독 흰쥐를 디에틸 에테르로 마취시킨 후, 복부 대동맥으로부터 채혈하였다. 이때 항응고제인 트리소디움 시트레이트(trisodium citrate) 3.8% 용액이 전체의 10%가 되도록 하였다. PRP(platelet rich plasma)와 PPP(platelet poor plasma)는 분별원심분리법에 의해 얻는데 200 ×g에서 15분간 원심분리를 행하여 침전된 적혈구와 백혈구 등을 제거한 상등액을 PRP라 하고, PRP를 150 ×g에서 10분간 더 원심분리하여 침전을 제거한 상등액을 PPP라 하였다. 혈소판 응집능 실험에 사용한 PRP는 cell count(Toa Co.)를 이용하여 PRP중의 혈소판 수를 계측하고 PPP로 희석하여 약 3 ×108cells/mL이 되도록 만들며, 제조한 PRP는 응집측정시까지 실온중에 보관하였고 1시간 30분 이내에 사용하였다.Cadmium poisoned rats were anesthetized with diethyl ether to prepare platelet rich plasma (PRP), and blood was collected from the abdominal aorta. The anticoagulant trisodium citrate 3.8% solution was 10% of the total. PRP (platelet rich plasma) and PPP (platelet poor plasma) are obtained by fractional centrifugation. The supernatant from which precipitated erythrocytes and leukocytes are removed by centrifugation at 200 × g for 15 minutes is called PRP. The supernatant from which precipitate was removed by centrifugation for 10 minutes was referred to as PPP. The PRP used for platelet aggregation ability was measured using a cell count (Toa Co.) to measure the platelet count in PRP and diluted with PPP to make it about 3 x 10 8 cells / mL. Was stored and used within 1 hour 30 minutes.

WPS(Washed platelet suspension)은 상기 방법으로 얻은 PRP를 1,500 ×g에서 15분간 원심분리하여 얻은 침전을 2 mM Tris-HCl(PH 7.4, 130 mM NaCl, 2 mM EDTA 함유)로 3번 세척한 후, 0.03% BSA가 함유된 modified tyrode's buffer(150 mM NaCl, 0.55 mM NaH2PO4, 7 mM NaHCO3, 2.7 mM KCl, 0.5 mM MgCl2, 5.6 mM glucose, 5 mM HEPES)에 재현탁시켰다.Washed platelet suspension (WPS) was washed three times with 2 mM Tris-HCl (containing PH 7.4, 130 mM NaCl, and 2 mM EDTA) after centrifuging the PRP obtained by the above method at 1,500 × g for 15 minutes. Resuspended in modified tyrode's buffer (150 mM NaCl, 0.55 mM NaH 2 PO 4 , 7 mM NaHCO 3 , 2.7 mM KCl, 0.5 mM MgCl 2 , 5.6 mM glucose, 5 mM HEPES) containing 0.03% BSA.

실험예 1 :포스포리파아제 AExperimental Example 1: Phospholipase A 22 (PLA(PLA 22 ) 활성 측정Activity measurement

3H-아라키도닉산(3H-arachidonic acid) 표지 혈소판의 조제는 혈소판 현탄액 2.5 mL를 취하여 25??Ci의〔5,6,8,9,11,12,14,15-3H〕아라키도닉산과 400 mM의 propyl gallate(시클로옥시게나아제와 리폭시게나아제의 억제제; 유리된 아라키도닉산의 대사를 막음)를 넣은 후 37 ℃에서 2시간 동안 항온한 후, 여기에 PPP 5 mL를 넣고 1,500 ×g에서 15분간 원심분리하여 pellet을 얻은 후 20 mM-EDTA를 함유한 HEPES-tyrode buffer 5 mL를 넣고 1,500g에서 15분간 원심분리함으로써 유입되지 않은3H-아라키도닉산을 제거하였다.3H-아라키도닉산 표지 혈소판은 이 pellet을 EDTA를 함유하지 않은 HEPES tyrode buffer를 가해서 1 ×109platelets/mL가 되도록 현탁하였다. 3 H- Araki donik acid (3 H-arachidonic acid) Preparation of labeled platelets is 5,6,8,9,11,12,14,15-3H [] of 25 Ci ?? ml of platelet suspension 2.5 mL Araki Add donic acid and 400 mM propyl gallate (inhibitor of cyclooxygenase and lipoxygenase; prevent free metabolism of arachidonic acid) and incubate at 37 ° C for 2 hours, add 5 mL of PPP to 1,500 The pellet was obtained by centrifugation at xg for 15 minutes, and 5 mL of HEPES-tyrode buffer containing 20 mM-EDTA was added thereto, followed by centrifugation at 1,500 g for 15 minutes to remove 3 H-arachidonic acid. 3 H-arachidonic acid labeled platelets were suspended in pellets of 1 × 10 9 platelets / mL in HEPES tyrode buffer containing no EDTA.

포스포리파아제 A2(Phospholipase A2) 활성은 다음과 같이 측정하였다.Phospholipase A 2 (Phospholipase A 2) activity was measured as follows.

유리된 아라키도닉산의 대사를 방지하기 위하여 얻어진 혈소판 현탄액 500 ㎕에 400 mM propyl gallate 50 ㎕를 가하였다. 트롬빈 1U를 가하고 다시 5분 후 엽산과 EDTA가 포함된 CHCl3/MeOH(1/2)용액으로 반응을 중지시켰다. 이 반응 중지액은 ice bath상에서 4 ℃ 상태를 유지하였으며 반응이 중지된 시료에 CHCl3와 2 M-KCl을 넣고 교반하였다. 3,000 ×g에서 10분간 원심분리하여 상분리를 한 후 하층은 취하고 상층에는 다시 CHCl3를 넣고 교반하여 층을 취하여 먼저 취해놓은 하층과합하였다. 얻어진 하층은 상온에서 질소가스로 evaporation한 후 CHCl31mL에 용해시켜서 에틸아세테이트/이소옥탄/아세트산/물(90:50:20:100)의 상층을 전개용매로 사용하여 TLC한 뒤 iodine chamber에서 발색시킨 후 아라키도닉산 밴드를 긁어 liquid scintillation count(LSC, 6000IC, Beckman, USA) 하였다.To prevent metabolism of free arachidonic acid, 50 µl of 400 mM propyl gallate was added to 500 µl of the platelet suspension obtained. Thrombin 1U was added and after 5 minutes the reaction was stopped with CHCl 3 / MeOH (1/2) solution containing folic acid and EDTA. The reaction stopper was maintained at 4 ℃ in the ice bath, CHCl 3 and 2 M-KCl was added to the sample was stopped and stirred. After centrifugation at 3,000 × g for 10 minutes to separate the phases, the lower layer was taken, CHCl 3 was added to the upper layer, stirred, and the layers were combined with the lower layer taken first. The obtained lower layer was evaporated with nitrogen gas at room temperature, dissolved in 1 mL of CHCl 3 , TLC using an upper layer of ethyl acetate / isooctane / acetic acid / water (90: 50: 20: 100) as a developing solvent, followed by color development in an iodine chamber. The arachidonic acid band was then scratched and subjected to a liquid scintillation count (LSC, 6000IC, Beckman, USA).

실험결과, PLA2활성은 도 15와 같이, 정상군 (919±30.9)에 비해 Cd-0C군(2012±35.9)에서는 117%의 가장 많이 증가되었고, Cd-0.5C군(1475±26.2)에서는 60% 증가되었으나 카테킨 다량 공급군인 Cd-1.0C군(956±18.9)은 정상군 수준이었다. 따라서 PLA2는 만성 카드뮴 중독시에 그 활성이 증가되고 카테킨을 공급했을때는 그 활성이 저하되어 녹차 카테킨 공급수준과 혈소판 PLA2활성은 역비례하였다.As a result, PLA 2 activity was increased by 117% in the Cd-0C group (2012 ± 35.9) compared to the normal group (919 ± 30.9), as shown in Figure 15, in the Cd-0.5C group (1475 ± 26.2) The Cd-1.0C group (956 ± 18.9), a large catechin-supply group, increased by 60%. Therefore, PLA 2 activity was increased during chronic cadmium poisoning, and the activity was decreased when catechin was supplied, and green tea catechin supply level and platelet PLA 2 activity were inversely proportional.

실험예 2 :사이클로옥시게나아제 활성 측정Experimental Example 2 Measurement of Cyclooxygenase Activity

실시예 5에서 분리한 혈소판은 WPS를 3 ×108cell/mL 농도로 준비하여 450 ㎕을 취하고 여기에 최종농도가 10 ??M되게 아라키도닉산을 넣고 6분간 항온시킨 후 인도메타신을 넣어 반응을 정지시키고 12,000 ×g에서 2분간 원심하여 얻은 상층액을 -70℃에서 냉동보관하였다. 이때 과량의 아라키도닉산을 넣어 COX(cyclooxygenase)를 활성화시켜 생성된 TXB2를 COX활성의 지표로 하였다. 그러므로 RIA Kit(Amersham RPA516)로서 TXB2를 측정하였다.The platelets isolated in Example 5 were prepared with WPS at 3 × 10 8 cell / mL concentration, 450 μl, and arachidonic acid was added thereto to a final concentration of 10 ?? M, followed by incubation for 6 minutes, followed by indomethacin reaction. The supernatant obtained by centrifugation at 12,000 xg for 2 minutes was frozen and stored at -70 ° C. At this time, an excess of arachidonic acid was added to activate COX (cyclooxygenase), resulting in TXB 2 as an index of COX activity. Therefore TXB 2 was measured as RIA Kit (Amersham RPA516).

실험결과 도 16과 같이, 정상군(93±5.8)에 비해 Cd-0C군(356±13.3)에서 284%의 현저한 증가가 되었고 Cd-0.5C군(271±7.4), Cd-1.0C군(228±8.2)에서도 각각 193%, 147%씩 증가되었다. 그러나 Cd-0.5C군과 Cd-1.0C군은 Cd-0C군에 비해 24%, 36%씩 감소되었다(p<0.01). 이와 같이 COX 활성은 혈소판 PLA2활성과 같이 녹차 카테킨 공급수준과 역비례하여 활성화되었다.As shown in FIG. 16, there was a significant increase of 284% in the Cd-0C group (356 ± 13.3) and the Cd-0.5C group (271 ± 7.4) and Cd-1.0C group (93 ± 5.8). 228 ± 8.2) increased by 193% and 147%, respectively. However, the Cd-0.5C and Cd-1.0C groups decreased by 24% and 36%, respectively, compared to the Cd-0C group (p <0.01). As such, COX activity was activated in inverse proportion to the level of green tea catechin supply, such as platelet PLA 2 activity.

실험예 3 :혈소판 트롬복산 AExperimental Example 3 Platelet Thromboxane A 22 (TXA(TXA 22 ) 및 프로스타그란딘 D) And prostaglandin D 22 (PGD(PGD 22 ) 생성능 측정) Generation capacity measurement

트롬복산 B2(TXB2;Thromboxane B2)의 생성능을 측정하기 위하여 TXB2를 생성 지표로 하여 TXB2RIA kit(Amersham TRK 890)을 사용하여 측정하였고, 상기에서 얻은 반응 상등액 100 ㎕를 PBS-gel(0.1% gelatin/PBS buffer, pH 7.4)에 희석하여 300 ㎕로 만든 후, 여기에〔125I〕-TXB2용액과 antiserum을 각각 100 ㎕을 가하고 TXB2-antibody 500㎕를 가하여 잘 혼합하고, 4℃ cold chamber내에서 15시간 방치하여 반응액 중의 TXB2와 〔125I〕-TXB2가 antiserum과 경쟁적으로 결합하도록 하였다. 반응하여 생성된 TXB2-TXB2antibody complex를 제거하기 위하여 500 ㎕의 anti-TXB2-antibody(secondary antibody)를 가해 섞은 후, 실온에서 10분간 방치하였다. 생성된 TXB2-TXB2antibody-secondary TXB2antibody complex는 2,000 ×g로10분간 원심분리를 행하여 침전시켜 상층액을 버리고 침전물인〔125I〕-TXB2양을 γ-counter를 이용하여 측정하였다. 검체중의 TXB2의 양은 작성된 검량선으로부터 산출하였다.In order to measure the ability of thromboxane B 2 (TXB 2 ; Thromboxane B 2 ), TXB 2 was used as a production index and measured using a TXB 2 RIA kit (Amersham TRK 890). After diluting in gel (0.1% gelatin / PBS buffer, pH 7.4) to make 300 μl, add 100 μl of [ 125 I] -TXB 2 solution and antiserum, and add 500 μl TXB 2 -antibody to each well. Incubated for 15 hours in a cold chamber at 4 ° C, TXB 2 and [ 125 I] -TXB 2 in the reaction solution were competitively bound to the antiserum. To remove the resulting TXB 2 -TXB 2 antibody complex, 500 μl of anti-TXB 2 -antibody (secondary antibody) was added to the mixture and allowed to stand at room temperature for 10 minutes. The resulting TXB 2 -TXB 2 antibody-secondary TXB 2 antibody complex was precipitated by centrifugation at 2,000 × g for 10 minutes to discard the supernatant, and the amount of precipitate [ 125 I] -TXB 2 was measured using γ-counter. . The amount of TXB 2 in the sample was calculated from the prepared calibration curve.

또한, 프로스타그란딘류의 생성능 측정은 WPS를 제조하는 방법에 의해 혈소판을 재현탁시킨 반응액을 37 ℃에서 5분간 예비항온시키고, 여기에 콜라겐(최종농도 50 ㎍/mL)이나 트롬빈(최종농도 0.5U/mL)용액 50 ㎕을 가하여 5분간 반응시켰다. 이 반응은 indomethacin 용액 100 ㎕를 가하여 중지시킨다. 이때 indometacin 최종농도는 20 μM이 되게 하였는데, 이 농도에서는 시클로옥시게나제가 완전히 억제되어 프로스타그란딘류가 더 이상 생성되지 못하였다. 이어 4 ℃에서 11,000 ×g으로 2분간 원심분리를 행하여 얻은 상등액을 -70 ℃에서 TXB2를 측정할 때까지 보관하였다.In addition, the measurement of the production ability of prostaglandins was pre-incubated for 5 minutes at 37 ℃ the reaction solution resuspended platelets by the method for producing WPS, collagen (final concentration 50 ㎍ / mL) and thrombin (final concentration 0.5U) 50 mL of the solution was added and allowed to react for 5 minutes. The reaction is stopped by adding 100 μl of indomethacin solution. The final concentration of indometacin was 20 μM. At this concentration, cyclooxygenase was completely inhibited and prostaglandins could no longer be produced. Subsequently, the supernatant obtained by centrifugation at 4 ° C. at 11,000 × g for 2 minutes was stored at -70 ° C. until TXB 2 was measured.

프로스타그란딘 D2(PGD2:prostaglandin D2) 생성능은 WPS를 제조하는 방법으로 얻은 아라키도닉산 대사산물이 함유된 반응액 100 ㎕를 가하여 잘 섞은 후, PGD2-antiserum 100 ㎕를 가했다. 반응액 중에 존재하는 PGD2와 [3H]-PGD2가 antierum에 경쟁적으로 결합하도록 4℃에서 16시간 인큐베이션하였다. 반응액이 담긴 시험관을 꺼내어 냉각수조상에 방치하면서 잘 흔들어 균질화시킨 dextran-coated charcoal 용액 500 ㎕를 가해 교반하여 antiserum과 결합하지 않은 PGD2및[3H]-PGD2가 흡착되도록 하였다. 냉각수조상에서 10분간 방치한 후 2,000 ×g에서 10분간 원심분리함으로써 charcoal을 침전시켜 제거하였다. Antiserum과 결합한 [3H]-PGD2를 함유하고 있는 상등액을 이용하여 LSC(Beckman Co., USA)로서 동위원소 활성을 측정하였다(Amersham 890).Prostaglandin D 2 (PGD 2 : prostaglandin D 2 ) production ability was added to 100 μl of the reaction solution containing the arachidonic acid metabolite obtained by the method of preparing WPS, followed by mixing well, and then 100 μl of PGD 2 -antiserum. PGD 2 and [ 3 H] -PGD 2 present in the reaction solution were incubated at 4 ° C. for 16 hours to competitively bind to the antierum. The test tube containing the reaction solution was taken out, and the mixture was stirred in a cooling water bath, and 500 µl of a homogenized dextran-coated charcoal solution was added thereto, followed by stirring to adsorb PGD 2 and [ 3 H] -PGD 2 which did not bind to antiserum. After leaving for 10 minutes on a cooling water tank, the charcoal was precipitated and removed by centrifugation at 2,000 × g for 10 minutes. Isotope activity was measured as LSC (Beckman Co., USA) using a supernatant containing [ 3 H] -PGD 2 bound to Antiserum (Amersham 890).

프로스타사이클린은 반감기가 짧으므로 생리적으로 안정된 대사 물질인 6-케토 프로스타그란딘 F1??(6-ketoprostaglandin F1α)를 측정하여 PGI2생성량을 대신하였다.Because prostacycline has a short half-life, 6-keto prostaglandin F 1 ?? (6-ketoprostaglandin F 1α ) was measured to replace PGI 2 production.

시료제조를 위하여 대동맥은 떼내어 지방조직을 제거하고 생리식염수로 세척한 후, 약 1cm로 잘라 폴리에틸렌 튜브에 넣고 0.05M tris buffer solution 2ml, pH 7.4를 첨가하여 37℃의 shaking water bath에서 30분간 인큐베이션시켜 6-케토 프로스타그란딘 F1??의 생산을 자극하였다. 배양이 끝난 후 대동맥은 건져내고 배양액에 4.8M 엽산을 첨가하여 반응을 종료시키고 분석 전까지 -70℃에 냉동보관 하였다. 건져낸 대동맥 조각은 100℃에서 하룻밤동안 건조시킨 후 n-핵산으로 지방을 제거하고 다시 하룻밤동안 100℃에서 건조시켜 무게를 측정하였다. 6-케토 프로스타그란딘-F1??의 생산은 건조된 대동맥의 단위무게당으로 표시하였다.To prepare the sample, remove the aorta, remove the adipose tissue, wash it with physiological saline solution, cut it into about 1cm, put it in a polyethylene tube, and incubate for 30 minutes in a shaking water bath at 37 ℃ by adding 0.05M tris buffer solution 2ml and pH 7.4. 6-keto prostaglandin F 1 ?? Stimulated the production of. After the incubation, the aorta was removed and the reaction was terminated by adding 4.8M folic acid to the culture and frozen at -70 ° C until analysis. The harvested aortic slices were dried overnight at 100 ° C., then freed of fat with n-nucleic acid and dried at 100 ° C. overnight to weigh. 6-keto prostaglandin-F 1 ?? Production was expressed as unit weight of the dried aorta.

6-케토 프로스타그란딘 F1??함량은 냉동보관했던 배양액을 녹인 후[I125]가 표지된 RIA kit(Amersham TRK 790)로 측정하였다.6-keto prostaglandin F 1 ?? The content was measured using a RIA kit (Amersham TRK 790) labeled [I 125 ] after thawing the culture solution stored in the frozen.

실험결과 표 12에 나타낸 바와 같이, TXB2는 정상군(216±22.5)에 비해 Cd-0C군(555±32.2)과 Cd-0.5C군(442±30.9)에서는 157% 및 105%씩 현저한 증가를 보였으나, 카테킨 다량 첨가군인 Cd-1.0C군(283±18.0)에서는 31%만 증가되었다. 이와같이 혈소판 TXA2의 간접측정 지표인 TXB2농도는 카테킨 공급시는 그 생성량이 현저하게 감소되었다.As shown in Table 12, TXB 2 was significantly increased by 157% and 105% in Cd-0C group (555 ± 32.2) and Cd-0.5C group (442 ± 30.9) compared to normal group (216 ± 22.5). However, only 31% was increased in the Cd-1.0C group (283 ± 18.0). As such, the TXB 2 concentration, which is an indirect measure of platelet TXA 2 , was significantly reduced in catechin supply.

혈소판 프로스타그라딘 D2(PGD2)는 표 12에서와 같이 정상군(5.57±0.26)에 비해 Cd-0C군(9.84±0.67), Cd-0.5C군(8.85±0.53)에서 각각 77%, 59%씩 증가되었으나 Cd-1.0C군(6.10±0.31)은 정상군 수준이었다. 한편 PGD2/TXA2비는 정상군(0.027±0.003)에 비해 Cd-0C군(0.018±0.001), Cd-0.5C군(0.021±0.001)은 유의적으로 감소되었으나, 카테킨 다량투여군인 Cd-1.0C군(0.022±0.001)은 유의적인 차이가 없었다(p<0.05).Platelet prostaglandin D 2 (PGD 2 ) was 77% in Cd-0C group (9.84 ± 0.67) and Cd-0.5C group (8.85 ± 0.53), respectively, compared to normal group (5.57 ± 0.26), as shown in Table 12. In addition, the Cd-1.0C group (6.10 ± 0.31) was normal. The PGD 2 / TXA 2 ratio was significantly decreased in the Cd-0C group (0.018 ± 0.001) and the Cd-0.5C group (0.021 ± 0.001) compared to the normal group (0.027 ± 0.003), but the Cd- high dose group, Cd- There was no significant difference in the 1.0C group (0.022 ± 0.001) (p <0.05).

20주동안 카드뮴을 튜여한 쥐의 TXA2, PGD2, PGD2/TXA2에 녹차 카테킨이 미치는 영향Effects of Green Tea Catechin on TXA 2 , PGD 2 and PGD 2 / TXA 2 in Cadmium-Treated Rats for 20 Weeks TXA2(pg/1.5 ×109 platelet)TXA 2 (pg / 1.5 × 109 platelet) PGD2(pg/1.5 ×109 platelet)PGD 2 (pg / 1.5 × 109 platelet) PGD2/TXA2(ratio)PGD 2 / TXA 2 (ratio) NormalNormal 216 ±22.5a 216 ± 22.5 a 5.57 ±0.26a 5.57 ± 0.26 a 0.027 ±0.003a 0.027 ± 0.003 a Cd-0CCd-0C 555 ±32.2b 555 ± 32.2 b 9.84 ±0.67b 9.84 ± 0.67 b 0.018 ±0.001b 0.018 ± 0.001 b Cd-0.5CCd-0.5C 442 ±30.9c 442 ± 30.9 c 8.85 ±0.53b 8.85 ± 0.53 b 0.021 ±0.001c 0.021 ± 0.001 c Cd-1.0CCd-1.0C 283 ±18.0d 283 ± 18.0 d 6.10 ±0.31a 6.10 ± 0.31 a 0.022 ±0.001ac 0.022 ± 0.001 ac

대동맥에서 생성되는 혈관이완 및 항혈소판 응집물질인 PGI2는 매우 불안정한물질로서 생리적인 반감기가 불과 수초에 불과하여 생성즉시 안정된 대사산물인 6-케토 프로스타그란딘 F1α(6-ketoPGF1α)로 자동전환되므로 6-keto PGF1α의 함량을 측정하여 PGI2의 생성량으로 간접적으로 나타내었다. 대동맥의 PGI2함량(표 13)은 정상군에 비해 Cd-0C군과 Cd-0.5C군은 23%, 18%씩 감소되었으나 Cd-1.0C군은 정상군 수준이었다.(p<0.05).PGI 2 , a vasorelaxant and antiplatelet aggregate produced in the aorta, is very unstable and has a physiological half-life of only a few seconds, so it is automatically converted into a stable metabolite 6-keto prostaglandin F 1α (6-ketoPGF 1α ). The content of 6-keto PGF 1α was measured and expressed indirectly as the amount of PGI 2 produced. The PGI 2 content of the aorta (Table 13) was decreased by 23% and 18% in the Cd-0C and Cd-0.5C groups compared to the normal group, but the Cd-1.0C group was normal (p <0.05).

한편 혈전생성 지표인 PGI2/TXA2ratio(도 17)는 6-keto PGF1α/TXB2로 나타낼 수 있는데, Cd-0C군(1.74±0.14), Cd-0.5C군(2.41±0.26)은 정상군(5.85±0.59)에 비해 70%, 59％씩 감소되었으나 Cd-1.0C군(3.96±0.31)은 32％만 감소되었다. 이와같이 PGI2생성량과 PGI2/TXA2비는 카테킨의공급이 증가됨에 따라 증가되었다.On the other hand, PGI 2 / TXA 2 ratio (FIG. 17), which is an index of thrombogenesis, may be represented by 6-keto PGF 1α / TXB 2 , and Cd-0C group (1.74 ± 0.14) and Cd-0.5C group (2.41 ± 0.26) Compared to the normal group (5.85 ± 0.59), 70%, 59% was decreased, but only 32% of the Cd-1.0C group (3.96 ± 0.31). As such, PGI 2 production and PGI 2 / TXA 2 ratios increased with increased catechin supply.

20주동안 카드뮴을 투여한 쥐의 대동맥의 PGI2에 녹차 카테킨이 미치는 영향Effect of Green Tea Catechin on PGI 2 in Aorta of Cadmium-treated Rats for 20 Weeks PGI2(pg/㎎ fat free arota)PGI 2 (pg / mg fat free arota) NormalNormal 1231 ±90.3a 1231 ± 90.3 a Cd-0CCd-0C 942 ±52.2b 942 ± 52.2 b Cd-0.5CCd-0.5C 1013 ±49.5bc 1013 ± 49.5 bc Cd-1.0CCd-1.0C 1114 ±47.0ac 1114 ± 47.0 ac

실험예 4 :다형핵성 백혈구에서 5'-리폭시게나아제 활성 및 루코트리언 BExperimental Example 4 5'-lipoxygenase activity and leukotrien B in polymorphonuclear leukocytes 44 합성synthesis

Koshihara법을 이용하며 PMNL pellet를 인큐베이션 완충제(50 mM 인산염 완충제 + 1 mM EDTA + 0.1% 젤라틴)에 현탁(0.1 ×107cell/mL)시킨 후 hemogenizer를이용하여 homogenate를 만들고 원심분리(10,000 ×g, 10분, 4 ℃)하여 얻은 상층액은 5'-리폭시게나아제(lipoxygenae) 분획으로 하여 실험하였다.Using the Koshihara method, the PMNL pellet is suspended in incubation buffer (50 mM phosphate buffer + 1 mM EDTA + 0.1% gelatin) (0.1 × 10 7 cells / mL), and then homogenate is made using a hemogenizer and centrifuged (10,000 × g). , 10 minutes, 4 ℃) was obtained as a 5'-lipoxygenase (lipoxygenae) fraction was tested.

효소분획을 배양 시험관 당 1.8mL씩 취한 다음 CaCl2용액을 1×10-3M 되게 주가하고 37 ℃에서 5분간 예비배양한 후 기질인 아라키도닉산의 알코올용액을 50 μM이 되게 주가하여 총용량을 2.0 mL가 되도록 하였다. 각 2.0 mL의 배양시험관을 37 ℃에서 5분간 배양하고 빙냉하여 반응을 정지시키고 internal standard로 PGB1을가한 후 Sep-pak C18카트리지에 흡착시키고 무수 MeOH로 용해시킨 후 HPLC(Waters Co., USA)로 정량하였으며 280nm에서 흡광도를 측정하여 LTB4에 양을 정량하였다. 이 때 column은 Nucleosil C18(4.6×150mm)을 사용하고 유출 용매는 CH3CN: MeOH: H2O: AcOH(33.6: 5.4: 61.1: 1.0 v/v, pH 5.6)을 사용하였다.The enzyme fraction was taken 1.8mL per culture tube, and the CaCl 2 solution was added to 1 × 10 -3 M and pre-incubated at 37 ° C. for 5 minutes, and then the alcohol solution of arachidonic acid as the substrate was added to 50 μM. 2.0 mL was made. Incubate each 2.0 mL culture tube at 37 ° C for 5 minutes, stop the reaction by ice-cooling, add PGB 1 to internal standard, adsorb to Sep-pak C 18 cartridge, dissolve in anhydrous MeOH, and then use HPLC (Waters Co., USA). The amount was determined in LTB 4 by measuring absorbance at 280 nm. In this case, Nucleosil C 18 (4.6 × 150mm) was used as a column and CH 3 CN: MeOH: H 2 O: AcOH (33.6: 5.4: 61.1: 1.0 v / v, pH 5.6) was used.

민의 방법(민경락, 이승희, 천태희, 김학성, 김만욱 : 인삼 Saponin 분획의 루코트리언 B(leukotriene B) 합성억제효과에 관한 연구. 약학논문집 1 : 30∼36, 1986)에 의해 채취한 PMNL pellet을 30mM HEPES와 L(+)-글루타민이 함유된 Eagle's minimum essential medium(EMEM)으로 현탁(1×107cells/㎖)하였다. Ionophore A23187soln.을 최종농도가 5 μM이 되게 주가하고 생성된 아라키도닉산의 대사물과 다형핵성백혈구로 분리한 후 5'-LPx와 동일한 방법으로 HPLC(Waters Co., USA)에서 측정하였다.Min's Method (Min, Kyung-Rak, Seung-Hee Lee, Tae-Hee Chun, Hak-Sung Kim, Hak-Sung Kim, Man-Wook Kim: A Study on the Inhibitory Effect of Ginseng Saponin Fraction on the Synthesis of leukotriene B. 30mM PMNL pellets collected by Pharmacology Journal 1: 30-36, 1986 It was suspended (1 × 10 7 cells / ml) with Eagle's minimum essential medium (EMEM) containing HEPES and L (+)-glutamine. Ionophore A 23187 soln. Was added to a final concentration of 5 μM, isolated from the resulting arachidonic acid metabolite and polymorphonuclear leukocytes, and measured by HPLC (Waters Co., USA) in the same manner as 5'-LPx. .

실험결과 도 18에 나타낸 바와 같이, 아라키도닉산 대사의 두 경로 중 루코트리언을 합성하는 경로인 리폭시게나아제를 관찰하였다. 다형핵성 백혈구에서의 5'-리폭시게나아제(5'-LPx) 활성을 측정한 결과, 정상군(69.2±7.83)에 비해 Cd-0C군(97.1±9.82)에서만이 40%의 현저한 증가가 있었고(p<0.05) 카테킨 공급군(Cd-0.5C군: 70.5±4.63, Cd-1.0C군: 69.5±8.95)에서는 정상군 수준이었다. 이와 같이 5'-LPx 활성은 COX와 같이 카테킨 공급 수준과 역비례하여 활성화되었다.As shown in FIG. 18, lipoxygenase, a pathway for synthesizing leukotrien, was observed among two pathways of arachidonic acid metabolism. As a result of measuring 5'-lipoxygenase (5'-LPx) activity in polymorphonuclear leukocytes, there was a significant increase of 40% in the Cd-0C group (97.1 ± 9.82) compared to the normal group (69.2 ± 7.83). (p <0.05) The catechin supply group (Cd-0.5C group: 70.5 ± 4.63, Cd-1.0C group: 69.5 ± 8.95) was normal. Thus 5'-LPx activity was activated in inverse proportion to the catechin supply level, such as COX.

또한 루코트리언 B4(LTB4) 생성량을 측정한 결과(도 19), 정상군(9.65±1.69)에 비해 Cd-0C군(14.8±0.39)에서는 54%의 현저한 증가를 보였으나(p<0.01), 카테킨 첨가군인 Cd-0.5C군(11.1±1.31)과 Cd-1.0C군(11.6±0.46)에서는 정상군 수준으로 감소하였다. 이와같이 LTB4는 카테킨 공급시는 그 생성량이 5'-리폭시게나아제 활성감소와 같이 현저하게 감소되었다.In addition, as a result of measuring the production amount of rucotrian B 4 (LTB 4 ) (FIG. 19), the Cd-0C group (14.8 ± 0.39) showed a significant increase of 54% compared to the normal group (9.65 ± 1.69) (p < 0.01), and Cd-0.5C group (11.1 ± 1.31) and Cd-1.0C group (11.6 ± 0.46), which were added to the catechin group, decreased to normal levels. As such, LTB 4 was significantly reduced in catechin feeding, such as 5′-lipoxygenase activity.

실시예 6 :녹차 카테킨이 신장기능에 미치는 효과Example 6 Effect of Green Tea Catechin on Renal Function

뇨중 β2-마이크로글로불린(microglobulin) 측정을 위하여 동물 희생 전 6일간 매일 24시간동안의 뇨를 수집하여 DSL-6200 RIA kit(U.S.A)를 사용하여 1분간 γ-counter하여 뇨중 β2-마이크로글로불린을 측정하였다. 또한, 사구체 여과율(GFR) 측정은 혈청과 뇨의 크레아티닌 측정은 sicdia creatinine reagent kit를 이용하였다. 혈청과 뇨의 크레아틴 값으로 부터 하기식을 이용하여 GFR을 구하였다.In order to measure urine β 2 -microglobulin, urine was collected for 24 hours every day for 6 days prior to animal sacrifice and γ-counter for 1 minute using DSL-6200 RIA kit (USA) to measure urine β 2 -microglobulin. Measured. In addition, glomerular filtration rate (GFR) was measured using sicdia creatinine reagent kit for serum and urine creatinine measurement. From the serum and urine creatine values, GFR was calculated using the following equation.

혈청중의 ACE(angiotensin converting enzyme) 활성은 10㎕의 혈청과 substrate 용액을 넣고 37 ℃에서 30분간 항온시킨 후, 소디움 텅스테이트(sodium tungstate)와 황산을 넣어 반응을 중지시켜 교반한 후 증류수를 넣고 2,000 ×g에서 10분간 원심분리하여 상층액을 취하였다. 여기에 borate buffer, TNBS 용액을 넣고 37 ℃에서 15분간 항온시키고 30분간 실온에 방치한 후 420nm에서 측정하였다.The activity of ACE (angiotensin converting enzyme) in serum was added to 10 μl of serum and substrate solution and incubated at 37 ° C. for 30 minutes. Then, sodium tungstate and sulfuric acid were added to stop the reaction, followed by stirring. The supernatant was taken by centrifugation at 2,000 xg for 10 minutes. Borate buffer and TNBS solution was added thereto, incubated at 37 ° C. for 15 minutes, and allowed to stand at room temperature for 30 minutes, and then measured at 420 nm.

실험결과 만성 카드뮴 중독시 뇨중 β2-마이크로글로불린은 정상군 (0.38±0.014)에 비해 Cd-0C군(0.44±0.014)은 16% 증가하였고 카테킨 공급군(Cd-0.5C: 0.41±0.019, Cd-1.0C : 0.40±0.009)은 정상군 수준으로 감소되어 카테킨이 만성 카드뮴 중독 흰쥐 신장의 세뇨관 손상을 완화시킴을 알 수 있었다.In chronic cadmium poisoning, urinary β 2 -microglobulin was increased by 16% in the Cd-0C group (0.44 ± 0.014) compared to the normal group (0.38 ± 0.014) and the catechin supply group (Cd-0.5C: 0.41 ± 0.019, Cd). -1.0C: 0.40 ± 0.009) was reduced to normal level, indicating that catechins alleviate tubular damage in chronic cadmium-addicted rat kidneys.

사구체 여과율은 정상군(2.03±0.08)에 비해 Cd-0C군(1.75±0.07)만이 14% 감소하였고 카테킨 공급군(Cd-0.5C군: 2.02±0.08, Cd-1.0C군: 1.97±0.05)은 정상군 수준으로 감소되었다. 혈청중의 angiotensin converting enzyme(ACE) 활성은 정상군(56.8±6.1)에 비해 Cd-0C군(105.2±10.3)에서는 85%의 현저한 증가를 보였으나, 카테킨 첨가군인 Cd-0.5C군(62.7±1.0)과 Cd-1.0C군(67.0±2.3)에서는 정상군 수준으로 감소하였다.The glomerular filtration rate was 14% lower in the Cd-0C group (1.75 ± 0.07) than in the normal group (2.03 ± 0.08) and the catechin supply group (Cd-0.5C group: 2.02 ± 0.08, Cd-1.0C group: 1.97 ± 0.05) Decreased to normal. Serum angiotensin converting enzyme (ACE) activity increased by 85% in the Cd-0C group (105.2 ± 10.3) compared to the normal group (56.8 ± 6.1), but in the catechin-added group Cd-0.5C (62.7 ± 1.0) and the Cd-1.0C group (67.0 ± 2.3) decreased to normal.

실시예 7 :녹차 카테킨이 심장기능에 미치는 효과Example 7 Effect of Green Tea Catechin on Cardiac Function

쥐를 28 ℃ 항온통에서 15분정도 두어 안정한 상태에서 pentobabital로 약하게 마취시킨 다음 헤파린이 채워진 polyethylene catheter(PE20)를 좌측대퇴동맥과 정맥에 삽입한 후 다시 대퇴동맥 catheter 끝을 statham P50 압력 transducer에 연결한 후 physiograph(Narco Biosystem Mark- IV-P) 상에서 혈압과 심박수를 측정하였다.The rats were placed in a constant temperature thermostat for 15 minutes at low temperature and weakly anesthetized with pentobabital.Then, heparin-filled polyethylene catheter (PE20) was inserted into the left femoral artery and vein. Blood pressure and heart rate were measured on a physiograph (Narco Biosystem Mark- IV-P).

만성카드뮴 중독에 따른 혈압 변화는 실험결과 표 14에 나타난 바와 같이 최고, 최저 혈압 모두 정상군에 비해 Cd-0C군에서 증가되었으나 Cd-0C군에 비하여 Cd-1.0C군에서 유의적으로 감소되었다(p<0.05). 따라서 카테킨이 혈압을 안정시켜 주는 역할을 하는 것이 관찰되었다. 또 심박수를 관찰한 결과(표 14) 정상군에 비해 카드뮴 투여군에서 다소 증가하였나 실험군간의 유의성은 없었다. 따라서 카테킨은 혈압과 더불어 심박수를 안정화시키는 역할을 하였다.As shown in Table 14, the changes in blood pressure due to chronic cadmium intoxication were increased in the Cd-0C group compared to the normal group, but significantly decreased in the Cd-1.0C group compared to the Cd-0C group. p <0.05). Therefore, catechins were observed to play a role in stabilizing blood pressure. The heart rate was also increased in the cadmium-treated group compared to the normal group, but there was no significant difference between the experimental groups. Therefore, catechins played a role in stabilizing heart rate as well as blood pressure.

20주동안 카드뮴을 투여한 쥐의 혈압, 심박수에 녹차 카테킨이 미치는 영향Effects of Green Tea Catechin on Blood Pressure and Heart Rate in Cadmium-treated Rats for 20 Weeks 혈압(mmHg)Blood pressure (mmHg) 심박수(beats/min)Heart rate (beats / min) systolsystol diastolediastole NormalNormal 108.00±7.24a 108.00 ± 7.24 a 79.00±1.58a 79.00 ± 1.58 a 180.33±19.01NS 180.33 ± 19.01 NS Cd-0CCd-0C 125.20±4.54b 125.20 ± 4.54 b 88.40±1.78b 88.40 ± 1.78 b 193.44±5.91193.44 ± 5.91 Cd-0.5CCd-0.5C 114.50±7.44ab 114.50 ± 7.44 ab 80.33±4.39ab 80.33 ± 4.39 ab 191.67±18.09191.67 ± 18.09 Cd-1.0CCd-1.0C 103.6±10.67a 103.6 ± 10.67 a 75.60±6.44a 75.60 ± 6.44 a 188.40±7.70188.40 ± 7.70

상기 실시예를 통해 설명한 바와 같이, 녹차 카테킨을 유효성분으로 함유하는 본 발명 중금속 해독용 조성물은 카드뮴의 체내 축적을 억제시키고, 골조직 대사를 원활히 하며, 카드뮴 중독으로 인한 신장 기능 장애를 개선시키고 혈액순환 장애 개선 작용에 뛰어난 효과가 있으므로 본 발명은 식품의약상 매우 유용한 발명인 것이다.As described through the above embodiment, the heavy metal detoxifying composition of the present invention containing green tea catechin as an active ingredient inhibits the accumulation of cadmium in the body, facilitates bone tissue metabolism, improves renal dysfunction due to cadmium poisoning, and blood circulation The present invention is a very useful invention for food medicine because it has an excellent effect on the disorder improving effect.

Claims (1)

녹차 카테킨을 유효성분으로 포함하는 중금속 해독용 조성물.Heavy metal detox composition comprising green tea catechin as an active ingredient.
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CN113768995A (en) * 2020-06-09 2021-12-10 株式会社爱茉莉太平洋 Composition for improving respiratory health from sustained exposure to a mote atmosphere
KR102659091B1 (en) * 2023-12-04 2024-04-18 이권호 Composition for detoxificating lead and improving sperm motility

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113768995A (en) * 2020-06-09 2021-12-10 株式会社爱茉莉太平洋 Composition for improving respiratory health from sustained exposure to a mote atmosphere
US11938163B2 (en) 2020-06-09 2024-03-26 Amorepacific Corporation Composition for improving respiratory health continuously exposed to particulate matter atmosphere
CN113768995B (en) * 2020-06-09 2024-07-30 株式会社爱茉莉太平洋 Composition for improving respiratory organ health due to continuous exposure to a fine dust atmosphere
KR102659091B1 (en) * 2023-12-04 2024-04-18 이권호 Composition for detoxificating lead and improving sperm motility

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