KR101316806B1 - A Composition comprising glutathione and sylimarin having antioxidative effect and protective effect on the liver - Google Patents

A Composition comprising glutathione and sylimarin having antioxidative effect and protective effect on the liver Download PDF

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KR101316806B1
KR101316806B1 KR1020110076975A KR20110076975A KR101316806B1 KR 101316806 B1 KR101316806 B1 KR 101316806B1 KR 1020110076975 A KR1020110076975 A KR 1020110076975A KR 20110076975 A KR20110076975 A KR 20110076975A KR 101316806 B1 KR101316806 B1 KR 101316806B1
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정완식
박희규
김영준
김상범
권응기
판정훈
신동훈
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Abstract

본 발명은 글루타치온(glutathione)과 실리마린(sylimarin)을 유효성분으로 함유하는 항산화 및 간보호용 조성물에 관한 것이다.
본 발명에 의하면 글루타치온과 실리마린을 유효성분으로 함유하는 조성물은 글루타치온과 실리마린의 결합으로 인하여 항산화 및 간보호 효능에 있어서 현저한 상승효과가 있다. The present invention relates to an antioxidant and hepatoprotective composition containing glutathione and silymarin as active ingredients.
According to the present invention, a composition containing glutathione and silymarin as an active ingredient has a significant synergistic effect on antioxidant and hepatoprotective effects due to the combination of glutathione and silymarin.

Description

글루타치온과 실리마린을 유효성분으로 함유하는 항산화 및 간보호용 조성물{A Composition comprising glutathione and sylimarin having antioxidative effect and protective effect on the liver}A composition comprising glutathione and sylimarin having antioxidative effect and protective effect on the liver}

본 발명은 글루타치온(glutathione)과 실리마린(sylimarin)을 유효성분으로 함유하는 항산화 및 간보호용 조성물에 관한 것이다.The present invention relates to an antioxidant and hepatoprotective composition containing glutathione and silymarin as active ingredients.

글루타치온은 간에서 만들어지는 아미노산의 일종으로 강력한 항산화제로써 시스테인(cysteine)과 함께 글루타치온 과산화 효소를 증가시켜준다. 이 효소는 활성산소로부터 간, 폐, 심장과 혈액 등의 손상을 막아주며, 또한 DNA의 복구, 임파구의 활성, 기타 각종 항암활성을 지니고 있으며, 주로 간에서 70%가 만들어지고 폐에서 15%, 신장에서 15% 가량이 만들어진다고 한다.
Glutathione is a type of amino acid produced in the liver that is a powerful antioxidant that increases glutathione peroxidase along with cysteine. This enzyme prevents damage to the liver, lungs, heart and blood from free radicals, and also has DNA repair, lymphocyte activity, and other anticancer activities. It is produced 70% in the liver and 15% in the lungs. About 15% is made in the kidneys.

글루타치온은 인체의 항상성(homeostasis)을 유지시켜주는 중요한 역할을 한다고 알려져 있으며, 세포의 활성화는 물론 노화된 세포를 교체해 주고 또한 세포 내에 축적되어 있는 노폐물이나 독소를 제거하는 기능이 탁월하며 특히, 이상분열하는 세포를 억제함으로써 함암 효능도 탁월하다고 보고되어 있다.
Glutathione is known to play an important role in maintaining homeostasis of the human body, and it is excellent for cell activation, replacing old cells, and removing waste products and toxins accumulated in cells. It has been reported that the cancer-causing efficacy is also excellent by inhibiting the cells.

체내에서 글루타치온을 가장 많이 저장하고 있는 곳은 간이며 혈관으로 유입되어 적혈구를 맑게 해주고 백혈구를 보호해 준다. 또한 폐와 장에도 존재하여 탄수화물의 신진대사를 돕고 동맥경화를 유발하는 산화지방을 분해시켜주는 역할을 하며, 체내 세포들의 산화를 방지해 준다. 글루타치온이 결핍되면 신경계의 정신질환, 떨림증, 균형유지 결핍 등이 나타난다. 글루타치온은 사람이 나이가 들어감에 따라 감소하므로 글루타치온 결핍은 노화와 간접적으로 관련되어 있다. 암환자들에게 있어 글루타치온의 섭취는 필수적이며 혈액에 녹아있는 중성지방이나 LDL(Low Density Lipoprotein) 콜레스테롤을 낮추어 주고 HDL(High Density Lipoprotein) 콜레스테롤을 증가시켜준다.
The most stored glutathione in the body is the liver, which enters the blood vessels to clear red blood cells and protects white blood cells. It also exists in the lungs and intestines, which helps metabolize carbohydrates and breaks down oxidized fats that cause atherosclerosis and prevents the oxidation of cells in the body. Lack of glutathione can lead to mental illness, tremors, and lack of balance in the nervous system. Glutathione deficiency is indirectly related to aging because glutathione decreases with age. Glutathione intake is essential for cancer patients and lowers triglyceride or LDL (Low Density Lipoprotein) cholesterol in the blood and increases HDL (High Density Lipoprotein) cholesterol.

실리마린은 실리빈(silybin), 실리디아닌(silidianin), 그리고 실리크리스틴(silicristin)으로 구성되어 있으며, 실리빈이 실리마린의 약리적 효능에 있어서 대부분을 담당하고 있다. 다른 플라보노이드(flavonoid)와 마찬가지로 실리마린은 강력한 항산화제이다. 실리마린은 간세포의 단백질 합성을 활성화하여 간세포의 재생을 촉진시키며, 활성 산소 제거능이 있어 간 손상을 예방하고 간 기능을 강화하는 효능이 있다. 최근에는 간 조직에 존재하는 대식세포의 일종인 쿠퍼 세포(kupper cell)의 활성 억제에 의한 염증관련 인자들의 발현 억제가 실리마린의 간 보호 작용과 연관이 있다는 보고가 있다.
Silymarin consists of silybin, sililidianin, and sililicristin, and silybin is responsible for most of the pharmacological effects of silymarin. Like other flavonoids, silymarin is a powerful antioxidant. Silymarin promotes the regeneration of hepatocytes by activating protein synthesis of hepatocytes, and has the ability to remove free radicals, thus preventing liver damage and enhancing liver function. Recently, it has been reported that inhibition of the expression of inflammation-related factors by inhibition of the activity of kupper cells, a type of macrophages present in liver tissues, is associated with the liver protective action of silymarin.

상기한 바와 같이 글루타치온은 다양한 생리활성을 지니지만 체내에서의 합성량에는 한계가 있기 때문에 체외에서 공급할 필요가 있다. 그러나, 글루타치온의 단독섭취 시에는 소화기관을 거치면서 변성되어 효과가 없다고 보고되어 있고, 정맥주사를 통한 투여는 강력한 통증으로 인하여 적절치가 못하며, 또한 고가의 물질인 단점이 있다.
As mentioned above, glutathione has various physiological activities, but since it is limited in the amount of synthesis in the body, it is necessary to supply it externally. However, glutathione alone has been reported to be ineffective as it is denatured through the digestive tract, and administration via intravenous injection is not appropriate due to strong pain, and is an expensive substance.

실리마린을 유효성분으로 함유하는 제제의 경우 이미 임상적으로 간질환 치료에 널리 응용되고 있지만, 그 유효성분인 실리마린은 물에 거의 녹지 않는 난용성 물질로서 경구투여 시 체내 흡수율이 낮아 생체이용률이 약 20~40%에 불과하다. 따라서 약리적인 활성을 유지하면서 생체이용률이 향상된 실리마린 및 글루타치온이 함유된 제제의 개발은 중요한 과제이다.   The preparations containing silymarin as an active ingredient have already been widely applied in the treatment of liver disease, but its active ingredient, silymarin, is an insoluble substance that is almost insoluble in water. Only ~ 40%. Therefore, development of formulations containing silymarin and glutathione with improved bioavailability while maintaining pharmacological activity is an important task.

[선행기술][Prior technology]

[특허문헌][Patent Literature]

[문헌 1] 대한민국 등록특허 제1027555호 "혼합 생약 추출물을 유효성분으로 함유하는 간보호 및 간염의 예방 및 치료용 조성물"(2011. 3. 30. 등록)   [Document 1] Republic of Korea Patent No. 1027555 "Hepatoprotective and hepatitis preventive and therapeutic composition containing a mixed herbal extract as an active ingredient" (2011. 3. 30. registered)

[문헌 2] 대한민국 등록특허 제852737호 "털부처꽃 추출물을 포함하는 항산화 및 간보호 활성을 갖는 조성물"(2008. 8. 11. 등록)   [Document 2] Republic of Korea Patent No. 852737 "Composition having antioxidant and hepatoprotective activity, including hairy bud extract" (registered August 11, 2008)

[문헌 2] 대한민국 등록특허 제538983호 "생약추출물이 함유된 간기능 개선용 약학 조성물"(2005. 12. 20. 등록)   [Document 2] Republic of Korea Patent No. 538983 "Pharmaceutical composition for improving liver function containing herbal extracts" (December 20, 2005 registration)

본 발명의 목적은 in vitro 실험[DPPH(1,1-diphenyl-2-picrylhydrazyl)], cellular antioxidant activity(CAA), ABTS[2,2'-azinobis(3-ethylbenzothia zoline-6-sulfonate)], Ferric Reducing Antioxidant Power(FRAP), Oxygen Radical Absorbance Capacity(ORAC), Alcohol Dehydrogenase(ADH) 분석, Aldehyde Dehydrogenase(ALDH) 분석} 및 동물실험을 바탕으로 혈청분석 {아스파르트산 아미노기 전달효소(aspartate aminotransferase, AST), 알라닌 아미노기 전달효소(alanine aminotransferase, ALT) 수치 및 혈중 활성산소} 및 간 조직을 분석하여, 글루타치온과 실리마린을 유효성분으로 함유하는 조성물의 항산화 및 간보호 효능의 상승효과를 입증하기 위한 것이다.
The object of the present invention is in vitro experiment [DPPH (1,1-diphenyl-2-picrylhydrazyl)], cellular antioxidant activity (CAA), ABTS [2,2'-azinobis (3-ethylbenzothia zoline-6-sulfonate)], Serum analysis based on Ferric Reducing Antioxidant Power (FRAP), Oxygen Radical Absorbance Capacity (ORAC), Alcohol Dehydrogenase (ADH) analysis, Aldehyde Dehydrogenase (ALDH) analysis and animal experiments {aspartate aminotransferase (AST) , Alanine aminotransferase (ALT) levels and free radicals in blood} and liver tissues are analyzed to demonstrate the synergistic effect of antioxidant and hepatoprotective efficacy of compositions containing glutathione and silymarin as active ingredients.

본 발명의 발명자는 글루타치온과 실리마린을 유효성분으로 함유하는 조성물이 항산화능 및 간보호 효능에 있어서 글루타치온과 실리마린의 결합에 의하여 현저한 상승효과가 있음을 확인함으로써 본 발명을 완성하였다.   The inventor of the present invention completed the present invention by confirming that the composition containing glutathione and silymarin as an active ingredient has a significant synergistic effect by the combination of glutathione and silymarin in antioxidant activity and hepatoprotective effect.

본 발명에 의한 항산화 및 간보호용 조성물은, 글루타치온과 실리마린을 유효성분으로 함유하는 것을 특징으로 할 수 있다.
Antioxidant and hepatoprotective compositions according to the present invention may be characterized by containing glutathione and silymarin as active ingredients.

또한 상기 글루타치온은 효모추출물에 포함되어 있는 것임을 특징으로 할 수 있다.   In addition, the glutathione may be characterized in that it is contained in the yeast extract.

또한 상기 실리마린은 엉겅퀴 추출물에 포함되어 있는 것임을 특징으로 할 수 있다.
In addition, the silymarin may be characterized in that it is contained in thistle extract.

또한 상기 항산화 및 간보호용 조성물은 조성물 100 중량%에 대하여 효모추출물 60 내지 70 중량%, 엉겅퀴 추출물 30 내지 40 중량%를 혼합하여 조성하는 것을 특징으로 할 수 있다.
In addition, the antioxidant and hepatoprotective composition may be characterized in that the composition by mixing the yeast extract 60 to 70% by weight, thistle extract 30 to 40% by weight based on 100% by weight of the composition.

또한 상기 조성물은 식품조성물 또는 약학 조성물인 것을 특징으로 할 수 있다.   In addition, the composition may be characterized in that the food composition or pharmaceutical composition.

본 발명에 의하면 글루타치온과 실리마린을 유효성분으로 함유하는 조성물은 글루타치온과 실리마린의 결합으로 인하여 항산화 및 간보호 효능에 있어서 현저한 상승효과를 나타내었다. According to the present invention, a composition containing glutathione and silymarin as an active ingredient showed a significant synergistic effect on antioxidant and hepatoprotective effects due to the combination of glutathione and silymarin.

도 1a은 본 발명의 실험예 1에 의한 각 시료의 농도별 항산화 활성(DPPH 분석)을 비교한 그래프이다.
도 1b는 본 발명의 실험예 1에 의한 항산화 활성(CAA 분석)을 비교한 그래프이다.
도 1c는 본 발명의 실험예 1에 의한 항산화 활성(ABTS 분석)을 비교한 그래프이다.
도 1d는 본 발명의 실험예 1에 의한 항산화 활성(FRAP 분석)을 비교한 그래프이다.
도 1e는 본 발명의 실험예 1에 의한 항산화 활성(ORAC 분석)을 비교한 그래프이다.
도 2a는 본 발명의 실험예 2에 의한 간 손상에 대한 지표로써 혈중 내 ADH 효소 활성을 비교한 그래프이다.
도 2b는 본 발명의 실험예 2에 의한 간 손상에 대한 지표로써 혈중 내 ALDH 효소 활성을 비교한 그래프이다.
도 3a는 본 발명의 실험예 3에 의한 간 손상 정도를 확인하기 위하여 혈중 내 AST, ALT 값을 비교한 그래프이다.
도 3b는 본 발명의 실험예 3에 의한 혈중 내 활성산소 함량을 비교한 그래프이다.
도 3c는 본 발명의 실험예 3에 의한 간 손상 정도를 확인하기 위하여 간조직의 손상정도를 비교한 그래프이다. Figure 1a is a graph comparing the antioxidant activity (DPPH analysis) for each concentration of each sample by Experimental Example 1 of the present invention.
Figure 1b is a graph comparing the antioxidant activity (CAA assay) by Experimental Example 1 of the present invention.
Figure 1c is a graph comparing the antioxidant activity (ABTS assay) by Experimental Example 1 of the present invention.
Figure 1d is a graph comparing the antioxidant activity (FRAP assay) by Experimental Example 1 of the present invention.
Figure 1e is a graph comparing the antioxidant activity (ORAC analysis) by Experimental Example 1 of the present invention.
Figure 2a is a graph comparing the ADH enzyme activity in the blood as an indicator for liver damage by Experimental Example 2 of the present invention.
Figure 2b is a graph comparing the ALDH enzyme activity in the blood as an indicator for liver damage by Experimental Example 2 of the present invention.
Figure 3a is a graph comparing the blood AST, ALT values in order to confirm the degree of liver damage by Experimental Example 3 of the present invention.
Figure 3b is a graph comparing the active oxygen content in the blood by Experimental Example 3 of the present invention.
Figure 3c is a graph comparing the degree of damage to liver tissue to confirm the degree of liver damage by Experimental Example 3 of the present invention.

이하, 본 발명을 실시하기 위한 내용을 구체적으로 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the content for implementing this invention is demonstrated concretely.

본 발명자들은 체내 활성산소 및 자유라디칼(free radical) 제거에서 중요한 작용을 하는 글루타치온을 함유하는 효모추출물과 간독성 저해 효과가 우수한 실리마린을 함유하는 엉겅퀴 추출물을 함께 투여함으로써, 항산화능을 통해 간독성을 저해하고, 결과적으로 그 물질들이 가진 생리활성에 대한 상승효과를 나타낼 수 있을 것이라는 가설을 세우고, 이를 검증하기 위해 실험하였다.
The present inventors administered a yeast extract containing glutathione, which plays an important role in removing free radicals and free radicals in the body, and a thistle extract containing silymarin with excellent hepatotoxic inhibitory effect, thereby inhibiting hepatotoxicity through antioxidant activity. As a result, we hypothesized that the result could have synergistic effects on the physiological activity of the substances, and experimented to verify this.

효모(yeast)는 낭자균 혹은 구형 또는 난형의 단세포 미생물로서 건조효모 또는 효모추출물로 사용한다. 효모에는 단백질, 식이섬유, 비타민 B군, 무기질, 핵산, 글루타치온, 에르고스테롤(ergosterol) 등이 함유되어 있으므로 양질의 단백질 공급과 비타민 B군의 공급에 유용하며, 흡수성이 뛰어난 미네날의 공급과 장내 이용도가 높은 식이섬유의 함량이 높다. 효모 추출물에는 8% 이상의 글루타치온이 함유되어 있다.
Yeast is a cystic bacterium or a spherical or ovoid unicellular microorganism used as dry yeast or yeast extract. Yeast contains protein, dietary fiber, vitamin B group, minerals, nucleic acids, glutathione, ergosterol, etc., so it is useful for supplying high quality protein and vitamin B group. Higher dietary fiber content. Yeast extract contains more than 8% glutathione.

엉겅퀴(milk thistle)는 우유같이 하얀 잎맥과 함께 깊고 반짝이는 녹색의 식물이며 가장자리에는 물결무늬가 있고 가시도 있는 뿌리-잎사귀로 분할되는 가늘고 긴 식물이다. 이것은 제방 울타리와 버려진 땅에서 주로 발견된다. 식물의 유용한 부분은 허브 전체, 뿌리, 잎사귀, 씨, 그리고 겉껍질이며, 엉겅퀴 씨는 실리마린으로 알려진 플라보노이드(flavonoid) 화합물을 함유하고 있다. 엉겅퀴 씨 추출물에는 70~80%의 실리마린이 함유되어 있는데, 이 성분은 간세포의 신진대사를 촉진하고 독성으로부터 간세포의 손상을 막는 역할을 한다.
Milk thistle is a deep, shiny green plant with milky white leaf veins. It is a long, thin plant that is divided into a wavy, spiny root-leaves at the edges. It is mainly found in embankment fences and abandoned lands. Useful parts of the plant are whole herbs, roots, leaves, seeds, and husks, and thistle seeds contain flavonoid compounds known as silymarin. Thistle seed extract contains 70-80% of silymarin, which promotes metabolism of hepatocytes and prevents damage to hepatocytes from toxicity.

본 발명에서 효모 추출물과 엉겅퀴 추출물의 추출방법은 본 발명이 속하는 기술분야에서 통상적으로 알려져 있는 방법을 적의 선정하여 이용할 수 있으며, 제품화되어 시중에서 상품으로 판매되고 있는 것을 사용할 수도 있다.
Extraction method of the yeast extract and thistle extract in the present invention can be used by appropriately selecting a method commonly known in the art to which the present invention belongs, it may be used that is commercialized and sold as a commercial product.

본 발명은 본 발명의 조성물 100 중량%에 대하여 효모 추출물 60 내지 70 중량%, 엉겅퀴 추출물 30 내지 40 중량%를 혼합하여 조성하는 것을 특징으로 한다.
The present invention is characterized by mixing 60 to 70% by weight yeast extract, 30 to 40% by weight thistle extract with respect to 100% by weight of the composition of the present invention.

효모 추출물의 글루타치온 함량이 8% 수준이므로 효모추출물의 조성비가 60 중량% 미만이면 본 발명의 조성물에서 글루타치온의 함량이 너무 낮아지게 되어 본 발명의 효과를 기대하기 어려우며, 효모추출물의 조성비가 70 중량%를 초과하게 되면 실리마린의 조성비가 30 중량%에도 못 미치게 되어 본 발명의 조성물의 글루타치온과 실리마린의 결합에 의한 상승효과를 기대하기 어려운 점이 있다.   Since the glutathione content of the yeast extract is 8% level, if the composition ratio of the yeast extract is less than 60% by weight, the content of glutathione in the composition of the present invention is too low, so it is difficult to expect the effect of the present invention, and the composition ratio of the yeast extract is 70% by weight. If it exceeds the composition ratio of silymarin is less than 30% by weight, there is a point that it is difficult to expect a synergistic effect by the combination of glutathione and silymarin of the composition of the present invention.

본 발명의 조성물은 구체적인 양태에 있어서, 식품조성물로 제조될 수 있다. 본 발명의 식품 조성물에는 그 유효성분 이외에 감미제, 풍미제, 생리활성 성분, 미네랄 등을 포함하여 차, 주스 및 드링크의 형태로 제조하여 음용하거나, 과립화, 캡슐화 또는 분말화하여 섭취할 수 있다.
The composition of the present invention may be prepared in a food composition in a specific embodiment. The food composition of the present invention may be ingested in the form of tea, juice and drink, including sweeteners, flavors, physiologically active ingredients, minerals, etc., in addition to the active ingredient, and then consumed, ingested, granulated, encapsulated or powdered.

본 발명의 조성물은 구체적인 양태에 있어서, 약학 조성물로 제조될 수 있다. 본 발명의 약학 조성물은 그 유효성분 이외에 약학적으로 허용되는 담체, 부형제 등을 포함하여, 경구용 제형(정제, 현탁액, 과립, 에멀젼, 캡슐, 시럽 등), 비경구형 제형(멸균 주사용 수성 또는 유성 현탁액) 등으로 제조될 수 있다.
The composition of the present invention may be prepared as a pharmaceutical composition in a specific embodiment. Pharmaceutical compositions of the present invention, in addition to its active ingredients, include pharmaceutically acceptable carriers, excipients, and the like, oral dosage forms (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral dosage forms (sterile injectable aqueous or Oily suspensions), and the like.

<< 실험예Experimental Example 1> 본 발명의 조성물의  1 > The composition of the present invention 항산화능Antioxidant ability 조사 Research

효모 추출물(글루타치온 함량 8%)은 일본 KOHJIN 제품을 사용하였으며, 엉겅퀴 추출물(실리마린 함량 80%)은 중국 CHENGDU WACOTT에서 생산되는 제품을 사용하였다.
Yeast extract (glutathione content 8%) was used in Japan KOHJIN product, thistle extract (siliconin content 80%) used a product produced in China CHENGDU WACOTT.

본 발명의 조성물 100 중량%에 대하여 효모추출물 60 중량%, 엉겅퀴 추출물 40 중량%의 비율로 혼합하여 실험하였다. 실험결과는 SAS 프로그램을 이용하여 평균±표준편차 나타내었으며, 그룹간의 유의적인 통계차를 분석하기 위하여 p<0.05의 유의수준에서 던칸 다중범위 검정(Duncan's multiple range test)을 이용하여 사후검증을 하였다.
The mixture was tested by mixing 60% by weight of yeast extract and 40% by weight of thistle extract with respect to 100% by weight of the composition of the present invention. The experimental results showed the mean ± standard deviation using SAS program. To analyze the statistical difference between the groups, post-test was performed using Duncan's multiple range test at the significance level of p <0.05.

1) DPPH(1,1-diphenyl-2-picrylhydrazyl) 분석
1) DPPH (1,1-diphenyl-2-picrylhydrazyl) analysis

각 시료의 농도를 설정하기 위하여 시료 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1,000mg을 각각 100ml의 에탄올에 녹인 후, 시료와 표준용액 0.1ml을 50㎛ 농도의 DPPH 용액 2.9ml과 교반 시켜 어두운 곳에서 30분간 반응시킨 후, 517nm에서 흡광도를 측정하고 그 결과를 도 1a에 나타내었다. 도 1에서 보는 바와 같이 효모 추출물의 항산화 활성은 600 mg/mL의 농도에서, 엉겅퀴 추출물의 항산화 활성은 400 mg/mL에서 최대치를 나타내었다. 따라서 이후의 모든 실험에서는 상기 최대치의 농도에서 실험을 실시하였다.
To set the concentration of each sample, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, and 1,000 mg of sample were dissolved in 100 ml of ethanol, and 0.1 ml of the sample and the standard solution were dissolved in 50 μm DPPH. After stirring with 2.9 ml of the solution and reacting for 30 minutes in a dark place, the absorbance was measured at 517 nm and the results are shown in FIG. 1A. As shown in Figure 1, the antioxidant activity of the yeast extract at 600 mg / mL concentration, the antioxidant activity of thistle extract showed a maximum at 400 mg / mL. Therefore, in all subsequent experiments, the experiment was conducted at the concentration of the maximum value.

2) 세포의 항산화 활성 분석(Cellular antioxidant activity assay; CAA)
2) Cellular antioxidant activity assay (CAA)

DCFH-DA(2',7'-dichlorofluorescin diacetate)는 세포 내로 들어가면서 에스테르 가수분해효소(cellular esterase)에 의하여 아세트초산염(diacetate)이 잘려나가 더욱 극성을 띄는 DCFH(2',7'-dichlorofluorescin)로 된다. ABAP[2, 2'-azobis-(2-Amidino propane) di-Hydro Chloride]를 처리하게 되면 세포 내로 들어가서 자발적으로 페록시 라디칼(peroxyl radical)을 형성하는데, 이들은 더 많은 라디칼 생성을 위해 세포막을 공격하고, 세포 내의 DCFH를 형광을 띄는 DCF(2',7'-dichlorofluorescein)로 산화시킨다. 세포막을 투과해 세포 내로 들어간 항산화물질은 DCFH와 막지질의 산화를 막고 DCF의 형성을 줄여서 항산화 효능을 발휘한다. 이러한 일련의 기작을 이용, 형광도 측정을 측정하여 시료의 세포 내 항산화능을 측정하였다.
DCFH-DA (2 ', 7'-dichlorofluorescin diacetate) is a more polar DCFH (2', 7'-dichlorofluorescin) that is cut into the acetate by cellular esterase as it enters the cell. do. Treatment with ABAP [2, 2'-azobis- (2-Amidino propane) di-Hydro Chloride] enters the cell and spontaneously forms peroxyl radicals, which attack the cell membrane for more radicals. Then, DCFH in the cells is oxidized with fluorescent DCF (2 ', 7'-dichlorofluorescein). Antioxidants that penetrate the cell membrane into the cell prevent oxidation of DCFH and membrane lipids and reduce the formation of DCF, thus exerting antioxidant activity. Using this series of mechanisms, fluorescence measurements were measured to determine the intracellular antioxidant capacity of the samples.

간암세포주(HepG2)를 24시간동안 배양 후, 인산완충액(phosphate buffered saline; PBS)로 세척한 다음 25uM의 DCFH-DA와 시료를 처리하여 1시간 동안 배양 후 600uM ABAP를 처리하였다. 이를 485nm/538nm에서 60분 간 형광도의 변화 측정하여 곡선의 면적 값(Area Under Curve; AUC)을 구하여 항산화능을 검증하였다. Control은 ABAP만 처리한 그룹이다.
Hepatocellular carcinoma cell line (HepG2) was incubated for 24 hours, washed with phosphate buffered (phosphate buffered saline (PBS), and then treated with 25 uM of DCFH-DA and samples for 1 hour and then treated with 600 uM ABAP. The change in fluorescence for 60 minutes at 485 nm / 538 nm was measured to obtain an area under curve (AUC), and the antioxidant activity was verified. Control is a group that processes only ABAP.

도 1b에서 보는 바와 같이 엉겅퀴 추출물의 세포내 항산화 활성이 효모 추출물보다 높았으며, 본 발명의 조성물이 가장 높은 세포내 항산화 활성을 나타내었다.
As shown in Figure 1b the intracellular antioxidant activity of thistle extract was higher than the yeast extract, the composition of the present invention showed the highest intracellular antioxidant activity.

3) ABTS 분석
3) ABTS analysis

산화를 띈 물질인 ABTS2 -(매우 안정된 산화 자유라디칼)에 대한 시료의 자유라디칼 소거능을 보고, 항산화능을 측정 및 계산하였다. 각 시료의 농도별 항산화 능력을 테스트하고 항산화제로 효과를 인정받은 Vitamin C와 비교, 분석하였다.Of ABTS 2 material ttuin oxidation - see the free radical scavenging activity of the sample for the (very stable oxide free radicals), which was measured and calculated for antioxidant activity. Antioxidant ability of each sample was tested and compared and analyzed with Vitamin C, which was recognized as an antioxidant.

1.0mM의 AAPH [2,2’―azobis-(2-amidinopropane) HCl]와 2.5mM의 ABTS2 - [2,2’―azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt]를 100mL의 PBS에 녹여서 ABTS가 라디칼 활성을 띄도록 하였다. 이를 70 수조에서 30분 간 가열해주며 매 5분마다 교반하였다. 가열한 라디칼을 실온에서 식힌 후, Acrodisc LC13 PVDF(Polyvinylidene difluoride) 0.45um의 주사기 여과기를 이용하여 여과하면서 녹지 않은 부유물을 제거하였다. 여과된 라디칼을 734nm에서 PBS를 이용하여 0.63~0.67 사이로 흡광도 값을 보정하였다. 20ul의 시료와 980ul의 라디칼을 혼합하여 37에서 16분간 반응시킨 후 734nm에서 흡광도를 측정하였다. 흡광도를 측정하는 동안 라디칼의 온도를 항상 37로 유지하였다. 결과값을 비타민 C를 이용한 표준곡선에 대입하여 Vitamin C Equivalents Antioxidant Capacity (VCEAC)를 계산하였다.1.0mM of AAPH [2,2'-azobis- (2- amidinopropane) HCl] 2.5mM and ABTS of 2 - a [2,2'-azino-bis (3 -ethylbenzthiazoline-6-sulfonic acid) diammonium salt] 100mL It was dissolved in PBS so that ABTS has a radical activity. It was heated in a 70 bath for 30 minutes and stirred every 5 minutes. After cooling the heated radicals at room temperature, the undissolved suspension was removed by filtration using a syringe filter of Acrodisc LC13 Polyvinylidene difluoride (PVDF) 0.45 um. The filtered radicals were corrected for absorbance values between 0.63 and 0.67 using PBS at 734 nm. After mixing 20ul of sample and 980ul of radical and reacting at 37 for 16 minutes, the absorbance was measured at 734nm. The temperature of the radicals was always kept at 37 while absorbance was measured. The vitamin C Equivalents Antioxidant Capacity (VCEAC) was calculated by substituting the results into a standard curve using vitamin C.

도 1c에서 보는 바와 같이 효모 추출물의 항산화 활성이 엉겅퀴 추출물보다 높았으며, 본 발명의 조성물이 가장 높은 항산화 활성을 나타내었다.
As shown in Figure 1c the antioxidant activity of the yeast extract was higher than thistle extract, the composition of the present invention showed the highest antioxidant activity.

4) 환원력 측정 (Ferric reducing antioxidant power; FRAP)
4) Ferric reducing antioxidant power (FRAP)

시료를 1mL의 증류수에 희석하여 0.2M 인산나트륨 완충액(sodium phosphate buffer, pH 6.6) 1mL와 1% 페리시안화 칼륨(potassium ferricyanide) 1mL을 첨가하여 50에서 20~30분간 반응시킨 후, 10% 트라이클로로아세트산(trichloroacetic acid) 1mL을 첨가한 다음, 3000 rpm에서 10~20분간 원심분리하였다. 상층액 1mL에 증류수 1mL, 0.1% 염화제이철(ferric chloride) 0.1mL을 첨가하여 700nm에서 흡광도를 측정하였다. 결과값을 비타민 C를 이용한 표준곡선에 대입하여 Vitamin C Equivalents Antioxidant Capacity(VCEAC)를 계산하였다.
The sample was diluted in 1 mL of distilled water, and 1 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 1 mL of 1% potassium ferricyanide were added and reacted at 50 to 20-30 minutes, followed by 10% trichloro Acetic acid (trichloroacetic acid) 1mL was added and then centrifuged for 10-20 minutes at 3000 rpm. 1 mL of the supernatant was added 1 mL of distilled water and 0.1 mL of 0.1% ferric chloride, and the absorbance was measured at 700 nm. Vitamin C Equivalents Antioxidant Capacity (VCEAC) was calculated by substituting the results into a standard curve using vitamin C.

도 1d에 나타난 바와 같이 엉겅퀴 추출물의 환원력이 효모 추출물보다 높았으며, 본 발명의 조성물이 가장 높은 환원력을 나타내었다.
As shown in FIG. 1D, the reducing power of the thistle extract was higher than that of the yeast extract, and the composition of the present invention showed the highest reducing power.

5) Oxygen Radical Absorbance Capacity(ORAC) 분석
5) Oxygen Radical Absorbance Capacity (ORAC) Analysis

74uM의 PBS(pH 7.4)에 희석한 시료 20uL를 플루오레세인 (fluorescein) 160uL와 혼합한 후 37에서 15분간 반응시킨 다음, AAPH 100uL을 첨가 후 60분 동안 형광도를 측정하고(485/528nm), 각각의 면적(Area Under Curve; AUC)을 구하여 항산화능을 비교하였다.
20 uL of the sample diluted in 74 uM of PBS (pH 7.4) was mixed with 160 uL of fluorescein and reacted for 15 minutes at 37. Then, the fluorescence was measured for 60 minutes after the addition of 100 uL of AAPH (485/528 nm). The area under curve (AUC) was calculated to compare the antioxidant activity.

도 1e에 나타난 바와 같이 엉겅퀴 추출물의 항산화능이 효모 추출물보다 높았으며, 본 발명의 조성물이 가장 높은 항산화 활성을 나타내었다.
As shown in Figure 1e, the antioxidant capacity of the thistle extract was higher than the yeast extract, the composition of the present invention showed the highest antioxidant activity.

상기한 바와 같이 본 발명의 조성물은 효모 추출물, 엉겅퀴 추출물 각각에 비하여 항산화능이 유의적으로 높았으며, 또한 두 추출물의 혼합으로 상승효과가 있음을 확인할 수 있었다.
As described above, the composition of the present invention was significantly higher in antioxidant capacity than the yeast extract and thistle extract, respectively, and also confirmed that there is a synergistic effect by mixing the two extracts.

<< 실험예Experimental Example 2> 본 발명의 조성물을 이용한  2> using the composition of the present invention 간보호Liver protection 효과 조사 Investigation of effects

1) Alcohol Dehydrogenase(ADH) 분석
1) Alcohol Dehydrogenase (ADH) Analysis

15ml 튜브에 50mM Sodium Pyrophosphate Buffer 1.2ml, 95% 에탄올 0.1ml,β-NAD(β-Nicotinamide-adenine dinucleotide) 1.5ml, 시료 0.1ml, ADH 효소용액 0.1ml을 넣어 30초 간 교반한 후, 6분 간 340nm에서 흡광도를 측정하였다.
1.2 ml of 50 mM Sodium Pyrophosphate Buffer, 0.1 ml of 95% ethanol, 1.5 ml of β-NAD (β-Nicotinamide-adenine dinucleotide), 0.1 ml of sample, and 0.1 ml of ADH enzyme solution were stirred for 30 seconds in a 15 ml tube. Absorbance was measured at 340 nm of liver.

도 2a에서 보는 바와 같이 엉겅퀴 추출물의 알콜 분해능이 효모 추출물보다 높았으며, 본 발명의 조성물이 가장 높은 알콜 분해능을 나타냈다.
As shown in Figure 2a, the alcohol resolution of thistle extract was higher than the yeast extract, the composition of the present invention showed the highest alcohol resolution.

2) Aldehyde Dehydrogenase(ALDH) 분석
2) Aldehyde Dehydrogenase (ALDH) Analysis

15ml 튜브에 탈이온수(Deionized Water) 2.22ml, 1M Tris-Hydrochloride Buffer 0.3ml, β-NAD 0.1ml, 3M Potassium Chloride Solution 0.1ml, 100mM Acetaldehyde Solution 0.05ml, 1M 2-Mercaptoethanol Solution, 0.03ml, 시료 0.1ml, ALDH 효소용액 0.1ml을 넣어 30초 간 교반한 후, 5분 간 340nm에서 흡광도를 측정하였다.
Deionized Water 2.22ml, 1M Tris-Hydrochloride Buffer 0.3ml, β-NAD 0.1ml, 3M Potassium Chloride Solution 0.1ml, 100mM Acetaldehyde Solution 0.05ml, 1M 2-Mercaptoethanol Solution, 0.03ml, Sample 0.1 After putting ml and 0.1 ml of ALDH enzyme solution and stirring for 30 seconds, absorbance was measured at 340 nm for 5 minutes.

도 2b에서 보는 바와 같이 ADH 실험결과와 유사하게 엉겅퀴 추출물의 알콜 분해능이 효모 추출물보다 높았으며, 본 발명의 조성물이 가장 높은 알콜 분해능을 나타내었다.   As shown in FIG. 2B, the alcohol resolution of thistle extract was higher than that of the yeast extract, and the composition of the present invention showed the highest alcohol resolution similar to the results of ADH experiment.

상기한 바와 같이 본 발명의 조성물은 효모 추출물, 엉겅퀴 추출물 각각에 비하여 알콜 분해능이 유의적으로 높았으며, 또한 두 추출물의 혼합으로 상승효과가 있음을 확인할 수 있었다.
As described above, the composition of the present invention was significantly higher alcohol degradability than the yeast extract, thistle extract, respectively, and also confirmed that there is a synergistic effect by mixing the two extracts.

<< 실험예Experimental Example 3> 본 발명의 조성물을 이용하는 동물실험을 통한  3> through the animal experiment using the composition of the present invention 간보호Liver protection 효과 조사 Investigation of effects

총 40마리의 6주령 수컷 SD 랫츠(rats)를 사용하였으며, 1주간의 적응기간 동안 일반사료를 급여하면서 랫츠를 대조군(control group)과 4개의 실험군(test group) 등 모두 5개 그룹으로 8마리씩 그룹화한 후 4주간 실험을 진행하였다.
A total of 40 6-week-old male SD rats were used, and 8 rats were divided into 5 groups, including a control group and 4 test groups, while feeding normal feed during the 1-week adjustment period. After the grouping, the experiment was conducted for 4 weeks.

알콜 유도를 위해 공업용 에탄올이 아닌 발효 에탄올(주정; ethyl alcohol) 을 이용하여 대조군을 제외하고 모든 실험군에 4주간 매일 100% 주정을 물에 25%로 희석하여 1.75 mL씩 경구 투여하였다.
For alcohol induction, fermented ethanol (alcohol; ethyl alcohol), not industrial ethanol, was administered orally by diluting 100% alcohol to 25% in water daily for 4 weeks in all experimental groups except the control group.

실험군(test group)의 4개 그룹 중 3개 그룹에는 상기한 바와 같이 주정을 투여한 후 30분간 방치한 다음 시료를 물에 희석하여 1mL씩 경구 투여하였다. 이들 3개 그룹에는 각각 효모추출물 100중량%(Alc+YE), 엉겅퀴 추출물 100중량%(Alc+MT), 효모추출물 60 중량%+엉겅퀴 추출물 40중량% (Alc+YE+MT)을 투여하였다.
Three of the four groups in the test group were allowed to stand for 30 minutes after the administration of alcohol as described above, and then the samples were diluted in water and administered orally by 1 mL. Each of these three groups was administered 100% by weight of yeast extract (Alc + YE), 100% by weight of thistle extract (Alc + MT), 60% by weight of yeast extract + 40% by weight of thistle extract (Alc + YE + MT).

4주 후 혈액을 체취하여 ALT(Alanine aminotransferase), AST(Asparatate aminotransferase), 혈중 활성산소를 측정함으로써 간 손상 정도를 측정하였으며, 간 조직을 적출하여 간을 염색한 후, 촬영하여 손상정도를 시각적으로 나타내었다.
Four weeks later, the blood was collected and measured for ALT (Alanine aminotransferase), AST (Asparatate aminotransferase), and free radicals in the blood to determine the degree of liver damage. Indicated.

실험동물의 해부 후에 수집된 혈액은 3,000 rpm에서 10분 동안 원심분리하여 혈청을 취득한 후 분석 전까지 -70에서 보관하였고, AST, ALT 수치는 Cobas C111 analyzer를 이용하여 분석하였으며, 혈중 활성산소는 FORM analyzer를 이용하여 분석하였다.
Blood collected after dissection of the experimental animals was collected by centrifugation at 3,000 rpm for 10 minutes and serum was collected and stored at -70 before analysis. AST and ALT levels were analyzed using Cobas C111 analyzer. It was analyzed using.

쥐의 간 조직을 채취한 후, 면도날을 사용하여 wax panel에 놓고 모양을 다듬은 다음, 각각 1cm 정도의 정육면체 형태로 잘라준다. 다듬은 조직을 cassette에 넣고 10% 포르말린에서 약 6~12시간 넣어 고정한다. 수돗물로 약 2~4시간 세척해준 후, 70% 에탄올로 45분 동안 침지, 95% 에탄올로 45분 동안 침지 후, 다시 새로운 95% 에탄올로 45분 동안 침지, 100% 에탄올로 45분 동안 침지, 다시 새로운 100% 에탄올로 45분 동안 침지시켜준다. 자일렌(xylene)으로 30분 동안 반응 후, 새로운 자일렌으로 30분 동안 반응시켜준다. 60 오븐에서 액체 파라핀 용기에 조직을 넣고 잘 흔들어 기포를 제거한 후, 약 1시간 정도 넣어 둔다. 틀에 원하는 조직 부위가 바닥을 향하게 심는다. 파라핀이 중간에 굳지 않도록 빠르게 진행하고, 4 냉각판에 놓고 잘 식힌다.
After collecting the liver tissues of rats, use a razor blade to place them on the wax panel, shape them, and cut them into cubes of 1 cm each. Put the trimmed tissue in the cassette and fix it for about 6 to 12 hours in 10% formalin. After about 2-4 hours of washing with tap water, soak for 45 minutes in 70% ethanol, 45 minutes in 95% ethanol, then 45 minutes in fresh 95% ethanol, 45 minutes in 100% ethanol, Again immerse in fresh 100% ethanol for 45 minutes. After reacting with xylene for 30 minutes, it is reacted with fresh xylene for 30 minutes. 60 Place the tissue in a liquid paraffin container in the oven, shake well to remove air bubbles, and leave for about 1 hour. Plant the desired tissue area in the frame facing the floor. Proceed quickly so that paraffin does not harden in the middle, place on 4 cold plates and cool.

조직은 회전식 박절기 (rotary microtome HM 340E; Microm Co., Walldorf, Germany)를 사용하여 절개면 중앙부위에서 수직방향으로 4의 두께로 박절하여 50% 알코올을 묻힌 slide glass에 올려 부유온수조에 옮긴다. 부유온수조(43)에 띄워 파라핀 절편의 주름을 펴고, 이를 새 slide glass에 붙인 후, 실온에서 말린다. 말린 slide glass를 60 오븐에 넣어 약 하루 동안 탈파라핀을 행한 후, 실온으로 식힌 다음, 자일렌에 약 5분 간 넣어둔다(2회). 다시 100% 에탄올에서 2분 동안 침지(2회), 95% 에탄올에서 2분 동안 침지(2회), 70% 에탄올에서 2분 동안 침지, 마지막으로 수돗물에서 10분 동안 세척해준다. Harris hematoxylin으로 30초 동안 반응시켜준 후, 10분간 세척한 다음, 에오신(eosin)으로 1분 간 반응시켜준다. 다시 95% 에탄올로 10초 간 침지(2회), 100% 에탄올로 10초 간 침지(2회), 마지막으로 자일렌으로 2분 간 반응시켜준다. Canada balsam 용액을 자일렌에 섞어 slide glass위에 한 방울 떨어뜨리고 덮개유리(cover glass)를 덮어 실온에서 2일 간 말린 후 현미경으로 관찰하며, 간조직의 핵은 파란색으로, 세포질은 핑크색으로 나타난다.
The tissue is cut using a rotary microtome HM 340E (Microm Co., Walldorf, Germany) with a thickness of 4 in the vertical direction at the center of the incision and placed on a slide glass filled with 50% alcohol. Float the floating hot water tank (43) to straighten the wrinkles of the paraffin section, attach it to a new slide glass, and dry at room temperature. Place the dried slide glass in 60 oven to deparaffinize for about a day, cool to room temperature and place in xylene for about 5 minutes (twice). Again immersed in 100% ethanol for 2 minutes (2 times), 95% ethanol for 2 minutes (2 times), 70% ethanol for 2 minutes, then washed in tap water for 10 minutes. After reacting with Harris hematoxylin for 30 seconds, it is washed for 10 minutes and then reacted for 1 minute with eosin (eosin). Again immersed in 95% ethanol for 10 seconds (2 times), 100% ethanol for 10 seconds (2 times), and finally reacted with xylene for 2 minutes. Mix Canada balsam solution with xylene, drop one drop onto slide glass, cover glass cover, dry at room temperature for 2 days and observe under a microscope. The nucleus of liver tissue is blue and the cytoplasm is pink.

일반적으로 AST, ALT와 같은 효소들은 간세포에서 발현되는 효소로써 간이 어떠한 자극에 의해 손상을 입었을 때 혈액 내로 방출되어 간손상을 알리는 역할을 하게 된다. 간 손상에 대한 영향을 보기 위해 AST와 ALT 수치를 비교 분석한 결과, 도 3a에 도시된 바와 같이, AST와 ALT 수치에 있어서 주정에 의해 증가된 AST와 ALT 수치는 시료 처리에 의해 간손상을 상쇄하는 결과를 보였으며, 특히 엉겅퀴 추출물이 효모 추출물보다 더 높은 상쇄효과를 보였고, 본 발명의 조성물이 가장 높은 상쇄효과를 나타내었다.
Generally, enzymes such as AST and ALT are enzymes expressed in hepatocytes, which are released into the blood when the liver is damaged by any stimulus, thereby informing liver damage. As a result of comparing and analyzing the AST and ALT levels to see the effect on liver damage, as shown in FIG. 3A, AST and ALT levels increased by alcohol in the AST and ALT levels were offset by the sample treatment. In particular, thistle extract showed a higher offset effect than the yeast extract, the composition of the present invention showed the highest offset effect.

도 3b에 도시된 바와 같이, 혈중 활성산소 수치에 있어서 주정에 의해 증가된 활성산소는 시료처리에 의해 감소되었으며, 특히 본 발명의 조성물에 의해 가장 높은 감소결과를 보였다.
As shown in FIG. 3B, the active oxygen increased by alcohol in the free radicals in the blood was reduced by sample treatment, and the highest reduction was achieved by the composition of the present invention.

도 3c에 도시된 바와 같이, 간조직에 있어서 대조구가 가장 정상적인 간의 세포 조성을 나타내었고, 알콜 유도에 의해 간 조직의 괴사흔적을 볼 수 있었으며, 시료처리에 의해 간 조직의 손상을 보호해주는 결과를 나타내었다.
As shown in FIG. 3C, the control group showed the most normal liver cell composition in liver tissue, necrotic traces of liver tissue could be seen by alcohol induction, and resulted in protecting liver tissue damage by sample treatment. It was.

상기한 바와 같이 간 보호 효과에 있어서 본 발명의 조성물은 높은 생체이용률로 인해 혈액 내에 간 손상 유발 인자들을 감소시키는 결과를 나타내었으며, 또한 간 손상을 상쇄하는 결과를 나타내었다.   As described above, the composition of the present invention has a result of reducing liver damage causing factors in the blood due to high bioavailability, and also counteracts liver damage.

Claims (6)

삭제delete 삭제delete 삭제delete 조성물 100중량%에 대하여 글루타치온 함량 8%인 효모추출물 60중량%, 실리마린 함량 80%인 엉겅퀴 추출물 40중량%를 혼합하여 조성하는 것을 특징으로 하는 항산화 및 간보호용 조성물An antioxidant and hepatoprotective composition, comprising 60% by weight of glutathione content of yeast extract and 40% by weight of thistle extract with 80% of silymarin content, based on 100% by weight of the composition. 삭제delete 삭제delete
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KR20230126027A (en) 2022-02-22 2023-08-29 대전대학교 산학협력단 Method for preparing a food composition for improving liver function comprising Liriope platyphylla extract as an active ingredient and food composition prepared therefrom
KR20230135218A (en) 2022-03-15 2023-09-25 (주)이뮤노텍 Composition for Anti-diabetes

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