KR102359165B1 - Dendropanax morbifera complex extracts for improving liver and kidney, Manufacturing method thereof and Health functional food containing the same - Google Patents
Dendropanax morbifera complex extracts for improving liver and kidney, Manufacturing method thereof and Health functional food containing the same Download PDFInfo
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
본 발명은 황칠 추출물을 포함하는 복합 추출물, 이의 제조방법에 관한 것으로서, 황칠 추출물 외에 다른 식물 추출물을 최적 조성 및 조성비로 포함하는 복합 추출물로서, 손상된 간 세포 및/또는 신장 세포의 기능을 개선 효능이 우수한 복합 추출물, 이를 이용한 건강기능식품 및 이를 제조하는 방법에 관한 것이다.The present invention relates to a complex extract comprising a hwangchil extract, and a method for producing the same, and as a complex extract comprising other plant extracts in addition to the hwangchil extract in an optimal composition and composition ratio, the effect of improving the function of damaged liver cells and/or kidney cells It relates to an excellent complex extract, a health functional food using the same, and a method for manufacturing the same.
Description
본 발명은 간 및 신장 기능 개선용 황칠계 복합 추출물과 그것의 제조방법 및 그것을 포함하는 건강기능식품에 관한 것이다.The present invention relates to a hwangchil-based complex extract for improving liver and kidney function, a manufacturing method thereof, and a health functional food containing the same.
간은 생체 내 다양한 대사가 활발하게 일어나는 장기로서, 지방 성분이 포함된 음식이나 알코올의 과다 섭취, 바이러스의 감염, 각종 약품과 같은 유해물질, 영양부족 등 다양한 원인에 의해 급성 또는 만성의 장애가 일어날 수 있다. 또한, 간은 우리 몸에 독성 물질에 노출될 때 제거하는 중추적인 역할을 하는 기관으로 독성물질에 의해 손상될 가능성이 높음. 간세포의 손상은 독성 물질을 제거하는 해독기능 장애를 나타내고, 면역체계에 이상을 가져와 지방간, 간염, 황달, 간경화, 간암 등을 야기시킬 수 있다.The liver is an organ in which various metabolisms actively occur in the body, and acute or chronic disorders may occur due to various causes such as excessive intake of food or alcohol containing fat, virus infection, harmful substances such as various drugs, and nutritional deficiency. have. In addition, the liver is an organ that plays a central role in removing toxins from our body when exposed to them, and it is highly likely to be damaged by toxic substances. Damage to liver cells indicates a detoxification function that removes toxic substances, and may cause abnormalities in the immune system, such as fatty liver, hepatitis, jaundice, cirrhosis, and liver cancer.
또한, CCl4는 간세포 내 산화적 스트레스를 유발하게 되는데, 이는 HO-1 등과 같은 항산화 효소의 발현을 억제하며, 미토콘드리아에서의 세포사멸 단백질인 Bax를 억제하여 자연사멸을 유도함. 또한, 항산화 유전자 관련 단백질인, HO-1, Nrf2, NFκB를 억제함으로써 염증과 자연 사멸을 유도하는 것으로 알려져 있다(도 1a 참조). In addition, CCl 4 induces oxidative stress in hepatocytes, which suppresses the expression of antioxidant enzymes such as HO-1, and induces natural death by inhibiting the apoptosis protein Bax in mitochondria. In addition, it is known to induce inflammation and natural death by inhibiting antioxidant gene-related proteins, HO-1, Nrf2, and NFκB (see FIG. 1a ).
CCl4는 산업에서 많이 발생하는 유해물질로 CCl4가 과산화 작용기전을 활성화시켜 간세포를 손상시킨다는 보고가 있는데, CCl4는 실험동물에서 간세포 사멸, 지방간, 섬유증 및 암을 유발시키는 물질로 간 손상을 유발시키는데 많이 사용해 왔다. CCl4가 간세포를 손상시키는 기전을 살펴보면 사염화탄소는 smooth endoplasmic reticulum에서 시토크롬 P450(cytochrome P450)에 의해 트리클로로메틸 자유라디칼(trichloromethyl free radical, CCl3)로 전환되고 이것은 산소와 반응하여 Cl3C-O-O"E를 형성함. CCl3와 Cl3C-O-O는 DNA와 지방산의 과산화를 촉진시켜 미토콘드리아막이나 세포막의 불포화지방산에 과산화 반응을 일으켜 막의 구조와 기능을 손상시켜 간세포 사멸을 촉진한다. 또한, CCl4로 간 손상을 유발시키면 단백질 합성, 글리코겐의 저장량을 감소시키고 간세포 파괴로 인한 혈청 ALT와 AST 농도, GGT 활성을 증가시키며, 또한, 조직학적으로는 간세포의 응고괴사, 지방변성, 염증 증가, 수종 변성 등을 나타낸다고 알려져 있다(도 1b 참조). CCl 4 is a toxic substance that occurs a lot in industry, and there are reports that CCl 4 activates the peroxidation mechanism and damages hepatocytes. It has been used a lot to provoke. Looking at the mechanism by which CCl 4 damages hepatocytes, carbon tetrachloride is converted into trichloromethyl free radical (CCl 3 ) by cytochrome P450 in smooth endoplasmic reticulum, which reacts with oxygen to Cl 3 COO"E CCl 3 and Cl 3 COO promote peroxidation of DNA and fatty acids, causing a peroxidation reaction on unsaturated fatty acids in the mitochondrial membrane or cell membrane, damaging the structure and function of the membrane and promoting hepatocyte death. Inducing damage reduces protein synthesis and glycogen storage and increases serum ALT and AST concentrations and GGT activity due to hepatocyte destruction is known to show (see Fig. 1b).
그리고, 도 2에 작용기작으로 나타낸 바와 같이, 활성산소종(O2 -, ·OH, H2O2)의 증가는 NFκB와 세포사멸에 영향을 주는 p53 인자의 발현을 증가시켜 세포사멸이 유도되며, 이는 신장 질환의 원인이 된다. 항산화능이 우수한 물질은 p58의 발현을 억제함으로써 세포사멸 유도를 막음. 즉, 신장 질환은 활성산소와 밀접한 관련이 있는 것으로 알려져 있다.And, as shown as a mechanism of action in FIG. 2 , an increase in reactive oxygen species (O 2 - , OH, H 2 O 2 ) increases the expression of NFκB and p53 factor affecting apoptosis, leading to apoptosis. and cause kidney disease. Substances with excellent antioxidant activity block the induction of apoptosis by inhibiting the expression of p58. That is, kidney disease is known to be closely related to free radicals.
황칠 나무(Dendropanax morbifera)는 최대 높이 15 m에 달하는 두릅나무과의 난대성 상록활엽수로서 한국남부 해안지역과 일본의 난대, 아열대 및 타이완에 분포하고 있다. 학명은 덴드로 파낙스(Dendropanax morbifera Nakai)이며, 황칠 나무의 수피에서 채취되는 수액인 황칠은 안식향을 함유하여 예로부터 약리효과가 탁월한 나무로 일찍이 주목 받았으며, 과거에는 천연도료로 쓰였다. 최근의 연구 결과에 따르면 항암, 당뇨 치료, 경조직 세포 재생 등에 효과적인 것으로 밝혀져 있다. 그러나, 황칠 나무 추출물의 약리 효과에 대해 밝혀진 것은 극히 일부에 불과하며, 이들의 주요 성분 내지 함량에 따른 약리활성 또는 이에 따른 최적의 약리 효과 등에 대해서는 알려진 바가 없다. 황칠 나무를 비롯한 다수의 천연물들은 이들에 함유된 구성 성분 내지 함량 차이에 따라 효과의 차이가 크게 발생할 수 있는 바, 원하는 효과를 도출하기 위하여 이들 천연물 자체뿐만 아니라 최적의 효과를 도출할 수 있는 이의 조성, 함량 등에 대한 연구가 더욱 필요한 실정이다.Hwangchil (Dendropanax morbifera) is a temperate evergreen broad-leaved tree of the Araliaceae family reaching a maximum height of 15 m, and is distributed in the southern coastal area of Korea, Japan's temperate, subtropical and Taiwanese regions. The scientific name is Dendropanax morbifera Nakai, and Hwangchil, the sap from the bark of the Hwangchil tree, contains benzoin and has been attracting attention as a tree with excellent pharmacological effects since ancient times, and was used as a natural paint in the past. According to recent research results, it has been found to be effective in anticancer, diabetes treatment, and hard tissue cell regeneration. However, only a few have been revealed about the pharmacological effects of hwangchil tree extract, and there is no known about the pharmacological activity according to their main components or content or the optimal pharmacological effect thereof. Numerous natural products, including hwangchil wood, can have a large difference in effect depending on the difference in composition or content contained therein. There is a need for further research on , content, etc.
CCl4 등에 의해 손상된 간 및/또는 신장 기능을 개선하기 위한 방법을 연구한 결과, 황칠 나무 등의 천연 식물 소재를 이용한 천연 복합 추출물의 최적 조성 및 조성비를 알게 되어 본 발명을 완성하였다. 즉, 본 발명은 간 및 신장 기능 개선용 황칠계 복합 추출물, 이를 제조하는 방법 및 이를 이용한 건강기능식품을 제공하고자 한다.As a result of studying a method for improving liver and/or kidney function damaged by CCl 4 , etc., the optimal composition and composition ratio of a natural complex extract using natural plant materials such as hwangchil tree was found, and the present invention was completed. That is, the present invention is to provide a Hwangchil-based complex extract for improving liver and kidney function, a method for preparing the same, and a health functional food using the same.
상기의 목적을 달성하기 위한, 본 발명의 간 및 신장 기능 개선용 황칠계 복합 추출물은 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 포함한다.Hwangchil-based complex extract for improving liver and kidney function of the present invention for achieving the above object includes fermented hwangchil, longan meat extract, aronia extract and complex hot water extract.
본 발명의 다른 목적은 상기 간 및 신장 기능 개선용 황칠계 복합 추출물을 제조하는 방법으로서, 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 각각 준비하는 1단계; 및 상기 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 혼합하는 2단계;를 포함하는 공정을 수행하여 제조한다.Another object of the present invention is to provide a method for producing a hwangchil-based complex extract for improving liver and kidney function, the first step of preparing each of a hwangchil fermented product, a longan meat extract, an aronia extract and a complex hot water extract; And the second step of mixing the fermented hwangchil, longan meat extract, aronia extract and complex hot water extract; is prepared by performing a process comprising a.
본 발명의 또 다른 목적은 상기 간 및 신장 기능 개선용 황칠계 복합 추출물을 포함하는 건강기능식품을 제공하는데 있다.Another object of the present invention is to provide a health functional food comprising the Hwangchil-based complex extract for improving liver and kidney function.
본 발명의 황칠계 복합 추출물은 손상된 간 세포 및/또는 신장세포 내 활성 산소를 효과적으로 억제하여 할 수 있으며, 특히 간 손상의 경우 활성 산소 억제에 의한 지질의 과산화 반응이 억제되는 효과가 있으며, 간 세포 및/또는 신장 세포에 대한 독성도 없는 바, 손상된 간 및/또는 신장의 산화적 스레스 완화 등을 통한 간, 신장의 기능 개선 효과가 있다.The Hwangchil-based complex extract of the present invention can effectively inhibit active oxygen in damaged liver cells and/or kidney cells, and in particular, in the case of liver damage, has the effect of inhibiting the peroxidation of lipids by inhibiting active oxygen, liver cells And/or there is no toxicity to kidney cells, so there is an effect of improving liver and kidney function through relief of oxidative stress in the damaged liver and/or kidney.
도 1a은 CCl4에 의해 유도된 산화적 스트레스에 의한 간세포 내 해독 작용기전 모식도이고, 도 2b는 CCl4에 의한 간 손상 작용 기전 모식도이다.
도 2는 활성산소에 의한 신장 질환 유도 작용 모식도이다.
도 3a는 실시예 및 비교예에서 제조한 추출물을 건조한 샘플 사진이며, 도 3b는 실시예 및 비교예에서 제조한 추출물의 용액 상태를 찍은 사진이다.
도 4a는 실험예 1에서 측정한 HepG2 세포에 대한 실시예 1 및 비교예 1 ~ 4의 세포 독성 실험 결과이며, 도 4b는 실험예 1에서 측정한 CCl4로 산화적으로 손상이 유도된 HepG2 세포에 실시예 1 및 비교예 1 ~ 4의 세포 보호 효과 측정 결과이다.
도 5a ~ 도 5e는 실험예 2에서 실시한 비교예 1 ~ 4 및 실시예 1의 HepG2 세포에 대한 산화적 손상 예방 효과를 관찰한 실험으로서, ROS 증가 억제 측정 결과이다.
도 6a는 비교예 1 ~ 4 및 실시예 1 농도에 따른 CCl4로 산화적 손상이 유도된 HepG2 세포의 ALT 변화 측정 결과이고, 도 6b는 AST 변화 측정 결과이다.
도 7은 비교예 1 ~ 4 및 실시예 1 농도에 따른 CCl4로 산화적 손상이 유도된 HepG2 세포의 GSH(glutathione level) 변화 측정 결과이다.
도 8은 비교예 1 ~ 4 및 실시예 1 농도에 따른 CCl4로 산화적 손상이 유도된 HepG2 세포의 GGT(gamma-glutamyl transpeptidasel) 활성 측정 결과이다.
도 9는 비교예 1 ~ 4 및 실시예 1 농도에 따른 CCl4로 산화적 손상이 유도된 HepG2 세포의 MDA(malondialdehyde) 함량 측정 결과이다.
도 10은 oil red O 염색된 HepG2 세포의 세폭 독성 측정 결과로서, 비교예 1 ~ 4 및 실시예 1 농도에 따른 측정 결과이다.
도 11a는 LLC-PK1 세포에 대한 실시예 1 및 비교예 1 ~ 4의 세포 독성 실험 결과이며, 도 11b는 AAPH로 산화적으로 손상이 유도된 LLC-PK1 세포에 실시예 1 및 비교예 1 ~ 4의 세포 보호 효과 측정 결과이다.
도 12a ~ 도 12e 각각은 비교예 1 ~ 4 및 실시예 1의 LLC-PK1 세포에 대한 산화적 손상 예방 효과를 관찰한 실험으로서, ROS 증가 억제 측정 결과이다.1a is a schematic diagram of a detoxification mechanism in hepatocytes by oxidative stress induced by CCl 4 , and FIG. 2b is a schematic diagram of a mechanism of action of liver damage by CCl 4 .
Figure 2 is a schematic diagram of the kidney disease inducing action by free radicals.
Figure 3a is a photograph of a sample dried extract prepared in Examples and Comparative Examples, Figure 3b is a photograph taken of the solution state of the extract prepared in Examples and Comparative Examples.
Figure 4a is the cytotoxicity test results of Examples 1 and 1 to 4 on HepG2 cells measured in Experimental Example 1, Figure 4b is HepG2 cells oxidatively induced by CCl 4 measured in Experimental Example 1 In Example 1 and Comparative Examples 1 to 4 are the measurement results of the cytoprotective effect.
5a to 5e are experiments for observing the effect of preventing oxidative damage on HepG2 cells of Comparative Examples 1 to 4 and Example 1 carried out in Experimental Example 2, and ROS increase inhibition measurement results.
6a is a measurement result of ALT change in HepG2 cells induced by oxidative damage with CCl 4 according to the concentration of Comparative Examples 1 to 4 and Example 1, and FIG. 6b is a measurement result of AST change.
7 is a measurement result of changes in glutathione level (GSH) of HepG2 cells induced by oxidative damage with CCl 4 according to the concentrations of Comparative Examples 1 to 4 and Example 1. FIG.
8 is a measurement result of gamma-glutamyl transpeptidasel (GGT) activity of HepG2 cells induced by oxidative damage with CCl 4 according to the concentrations of Comparative Examples 1 to 4 and Example 1. FIG.
9 is a result of measuring the MDA (malondialdehyde) content of HepG2 cells induced by oxidative damage with CCl 4 according to the concentrations of Comparative Examples 1 to 4 and Example 1. FIG.
10 is a measurement result of narrowing toxicity of oil red O-stained HepG2 cells, and is a measurement result according to the concentrations of Comparative Examples 1 to 4 and Example 1.
11A is a cytotoxicity test result of Examples 1 and Comparative Examples 1 to 4 on LLC-PK1 cells, and FIG. 11B is an Example 1 and Comparative Examples 1 to LLC-PK1 cells induced by AAPH oxidative damage. 4 is the result of measuring the cytoprotective effect.
12a to 12e are experiments for observing the effect of preventing oxidative damage on LLC-PK1 cells of Comparative Examples 1 to 4 and Example 1, respectively, and measurement results of ROS increase inhibition.
이하 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 황칠계 복합 추출물은 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 포함하며, 바람직하게는 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 1 : 0.5 ~ 1.2 : 0.5 ~ 1.5 : 3.5 ~ 6.5 중량비로 포함하고, 더욱 바람직하게는 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 1 : 0.8 ~ 1.2 : 0.8 ~ 1.2 : 4.5 ~ 5.5 중량비로 포함할 수 있다. Hwangchil-based complex extract of the present invention includes fermented hwangchil, longan meat extract, aronia extract and complex hot water extract, and preferably contains fermented hwangchil, longan meat extract, aronia extract and complex hot water extract 1: 0.5 to 1.2: 0.5 to 1.5: it may include in a weight ratio of 3.5 to 6.5, and more preferably, fermented hwangchil, longan meat extract, aronia extract and complex hot water extract in a weight ratio of 1: 0.8 to 1.2: 0.8 to 1.2: 4.5 to 5.5 by weight. .
본 발명의 상기 황칠계 복합 추출물은 황칠 발효물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 각각 준비하는 1단계; 및 상기 황칠 추출물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 혼합하는 2단계;를 포함하는 공정을 수행하여 제조할 수 있다.The hwangchil-based complex extract of the present invention is a
1단계에서 상기 황칠 발효물은 황칠나무 줄기 건조물 및 황칠나무 잎 건조물의 혼합물을 염수 처리하는 단계; 염수 처리된 혼합물을 수세한 후, 설탕 1.0 ~ 3.5 중량%, 소금 0.001 ~ 0.04 중량% 및 잔량의 수세한 혼합물을 혼합하는 단계; 및 혼합물에 균주를 접종시킨 후, 발효 및 정제하여 황칠 발효물을 제조하는 단계;를 포함하는 공정을 수행하여 제조할 수 있다.In
상기 염수 처리는 한의학적으로 뜨거운 성질을 가지는 황치의 열적 성질을 약화시키기 위해 수행하는 것으로서, 0.8 ~ 2 부피% 농도의 염수에 8 ~ 16시간 동안 침적시켜서, 바람직하게는 1.0 ~ 1.6 부피% 농도의 염수에 10 ~ 14시간 동안 침적시켜서 수행할 수 있다.The brine treatment is performed to weaken the thermal properties of Hwangchi, which has hot properties in Oriental medicine, and is immersed in saline at a concentration of 0.8 to 2% by volume for 8 to 16 hours, preferably in brine at a concentration of 1.0 to 1.6% by volume. It can be carried out by immersing it in the for 10 to 14 hours.
상기 균주로는 락토바실러스균을 포함할 수 있고, 바람직하게는 Lactobacillus plantarum ilchiwhangchil 1785(미생물 수탁번호: KACC 92131P) 및 Lactobacillus plantarum ilchiwhangchil 2020(미생물 수탁번호: KACC 92132P)을 1:1 ~ 3.5 중량비로, 더욱 바람직하게는 1:1.5 ~ 2.5 중량비로 포함할 수 있다. The strain may include Lactobacillus, preferably Lactobacillus plantarum ilchiwhangchil 1785 (microbial accession number: KACC 92131P) and Lactobacillus plantarum ilchiwhangchil 2020 (microorganism accession number: KACC 92132P) in a weight ratio of 1:1 to 3.5, More preferably, it may be included in a weight ratio of 1:1.5 to 2.5.
그리고, 상기 발효는 32 ~ 40℃ 하에서, 바람직하게는 35 ~ 37℃ 하에서 5 ~ 8시간 동안 수행할 수 있다.And, the fermentation can be performed under 32 ~ 40 ℃, preferably under 35 ~ 37 ℃ 5 ~ 8 hours.
1단계에서 상기 용안육 추출물은 열수 추출물로서, 용안육 100 중량부 및 물 500 ~ 700 중량부를 혼합한 후, 혼합용액을 제조하는 1단계; 상기 혼합용액을 20 ~ 35℃ 하에서 20분 ~ 1시간 동안 침적시키는 2단계; 및 침적된 용액을 감압농축 및 정제하여 추출액을 수득하는 3단계;를 포함하는 공정을 수행하여 제조할 수 있다.In
또한, 1단계에서 상기 아로니아 추출물은 열수 추출물로서, 아로니아 100 중량부 및 물 500 ~ 700 중량부를 혼합한 후, 혼합용액을 제조하는 1계; 상기 혼합용액을 20 ~ 35℃ 하에서 20분 ~ 1시간 동안 침적시키는 2단계; 및 침적된 용액을 감압농축 및 정제하여 추출액을 수득하는 3단계;를 포함하는 공정을 수행하여 제조할 수 있다.In addition, in
또한, 상기 복합 열수 추출물은 건무화과, 갈근, 대추, 비수리, 산수유, 복분자, 진피, 호로파 및 노니를 혼합한 혼합물을 제조하는 1단계; 상기 혼합물 100 중량부 및 물 650 ~ 900 중량부를 혼합한 후, 혼합용액을 제조하는 2단계; 상기 혼합용액을 20 ~ 35℃ 하에서 20분 ~ 1시간 동안 침적시키는 3단계; 및침적된 용액을 감압농축 및 정제하여 추출액을 수득하는 4단계;를 포함하는 공정을 수행하여 제조할 수 있다.In addition, the complex hot water extract is prepared in the first step of preparing a mixture of dried figs, ground root, jujube, bisuri, cornflower oil, bokbunja, dermis, fenugreek and noni; After mixing 100 parts by weight of the mixture and 650 to 900 parts by weight of water, a second step of preparing a mixed solution;
상기 용안육 추출물, 아로니아 추출물 및/또는 복합 열수 추출물의 감압농축은 감압농축기에서 80 ~ 95℃에서 4 ~ 8시간 정도 수행할 수 있다. The concentration of the longan meat extract, the aronia extract and/or the complex hot water extract under reduced pressure may be performed in a vacuum concentrator at 80 to 95° C. for 4 to 8 hours.
그리고, 복합 열수 추출물 제조시 상기 1단계의 혼합물은 건무화과, 갈근, 대추, 비수리, 산수유, 복분자, 진피, 호로파 및 노니를 포함할 수 있으며, 바람직하게는 건무화과 100 중량부에 대하여, 갈근 80 ~ 150 중량부, 대추 50 ~ 150 중량부, 비수리 80 ~ 200 중량부, 산수유 30 ~ 85 중량부, 복분자 50 ~ 90 중량부, 진피 50 ~ 90 중량부, 호로파 30 ~ 85 중량부 및 노니 20 ~ 80 중량부를 포함할 수 있고, 더욱 바람직하게는 건무화과 100 중량부에 대하여, 갈근 80 ~ 120 중량부, 대추 70 ~ 110 중량부, 비수리 80 ~ 140 중량부, 산수유 45 ~ 80 중량부, 복분자 60 ~ 85 중량부, 진피 50 ~ 80 중량부, 호로파 50 ~ 85 중량부 및 노니 40 ~ 80 중량부를 포함할 수 있다. In addition, when preparing the complex hot water extract, the mixture of
이렇게 제조된 본 발명의 황칠계 복합 추출물은 액상 타입일 수도 있고, 농축된 후 건조시킨 건조물 타입일 수 도 있다.The hwangchil-based complex extract of the present invention prepared in this way may be of a liquid type, or may be of a dried type after concentration and drying.
본 발명의 황칠계 복합 추출물은 간 세포 및/또는 신장 세포에 대한 독성이 없으면서, 산화적으로 손상된 간 세포 및/또는 신장 세포를 보호하고, 보호하여 간 세포 및/또는 신장 세포의 기능을 개선하는 효과가 우수하다. 좀 더 구체적으로는 손상된 간 세포 내 ALT(alanine transminase), AST(aspartate transaminase), GGT(gamma-glutamyl transpeptidase) 활성도 및 MDA(malondialdehyde) 함량을 감소시키고, ROS(활성산소)를 억제시키며, GSH(glutathione level)를 증가시킴으로써, 간 세포 기능을 개선시킬 수 있다. 또한, 손상된 신장 세포 내 ROS 억제를 통한 지질의 과산화 반응 억제 등을 통한 신장 세포 기능을 개선시킬 수 있다.Hwangchil-based complex extract of the present invention, without toxicity to liver cells and / or kidney cells, protects and protects oxidatively damaged liver cells and / or kidney cells to improve the function of liver cells and / or kidney cells The effect is excellent. More specifically, it reduces ALT (alanine transminase), AST (aspartate transaminase), GGT (gamma-glutamyl transpeptidase) activity and MDA (malondialdehyde) content in damaged liver cells, inhibits ROS (active oxygen), and GSH ( By increasing glutathione level), liver cell function can be improved. In addition, it is possible to improve renal cell function through inhibition of lipid peroxidation through inhibition of ROS in damaged renal cells.
이러한 본 발명의 황칠계 복합 추출물은 간 기능 및/또는 신장 기능 개선을 위한 다양한 건강기능식품으로 제공될 수 있다.The hwangchil-based complex extract of the present invention may be provided as various health functional foods for improving liver function and/or kidney function.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
[실시예][Example]
준비예 1 : 황칠 발효물의 제조Preparation Example 1: Preparation of Hwangchil Fermented Product
보길도 황칠나무 10년~15년근의 줄기와 잎의 건조물을 준비하였다.
다음으로, 상기 황칠나무 줄기, 잎 건조물의 혼합물을, 소금 1.2 부피% 농도의 염수에 투입한 후, 23 ~ 25℃ 하에서 12시간 동안 염수 처리하였다.Next, the mixture of the stems of the hwangchil tree and the dried leaves was added to brine having a salt concentration of 1.2% by volume, followed by brine treatment at 23 to 25° C. for 12 hours.
다음으로, 염수 처리한 상기 혼합물을 수세한 후, 설탕 2 중량%, 소금 0.02 중량% 및 잔량의 수세한 혼합물을 혼합한 다음 균주 (3 ~ 4)X105 CFU를 접종한 다음, 잔량의 물(균주 제외한 총 중량 100 중량% 중 나머지 함량)을 혼합한 후, 38℃ 하에서 15 시간 동안 발효시켜서 발효물을 수득하였다.Next, after the brine-treated mixture is washed with water, 2 wt% of sugar, 0.02 wt% of salt and the remaining amount of the washed mixture are mixed, and then strain (3 ~ 4)X10 5 CFU is inoculated, and then the remaining amount of water ( The remaining content of 100% by weight of the total weight excluding the strain) was mixed, and then fermented at 38° C. for 15 hours to obtain a fermented product.
상기 균주는 Lactobacillus plantarum ilchiwhangchil 1785(미생물 수탁번호: KACC 92131P) 및 Lactobacillus plantarum ilchiwhangchil 2020(미생물 수탁번호: KACC 92132P)을 1:2 중량비로 접종하였다.The strain was inoculated with Lactobacillus plantarum ilchiwhangchil 1785 (microorganism accession number: KACC 92131P) and Lactobacillus plantarum ilchiwhangchil 2020 (microorganism accession number: KACC 92132P) at a weight ratio of 1:2.
다음으로, 상기 발효물을 정제(38 μm, 400 mesh)한 후, 황칠 발효물을 수득하였다.Next, after the fermented product was purified (38 μm, 400 mesh), a fermented hwangchil was obtained.
준비예 2 : 용안육 열수 추출물의 제조Preparation Example 2: Preparation of Longan Meat Hot Water Extract
용안육 100 중량부를 물 600 중량부와 혼합한 후, 25 ~ 27℃ 하에서 30분 동안 침적하였다. After mixing 100 parts by weight of longan meat with 600 parts by weight of water, it was immersed for 30 minutes at 25 ~ 27 ℃.
다음으로, 용안육이 침전된 용액을 감압농축기에서 90℃에서 6시간 처리한 추출액을 정제하여 최종 용안육 열수 추출액을 수득하였다 Next, the solution in which the longan meat was precipitated was purified in a vacuum concentrator at 90° C. for 6 hours to obtain a final hot water extract of longan meat.
준비예 3 : 아로니아 열수 추출물의 제조Preparation Example 3: Preparation of Aronia hot water extract
아로니아 100 중량부를 물 600 중량부와 혼합한 후, 25 ~ 27℃ 하에서 30분 동안 침적하였다. After mixing 100 parts by weight of aronia with 600 parts by weight of water, it was immersed for 30 minutes at 25 ~ 27 ℃.
다음으로, 아로니아가 침적된 용액을 감압농축기에서 90℃에서 6시간 처리한 추출액을 정제하여 최종 아로니아 열수 추출액을 수득하였다 Next, the final aronia hot water extract was obtained by purifying the extract obtained by treating the aronia-infused solution at 90° C. for 6 hours in a vacuum concentrator.
준비예 4 : 복합 열수 추출물의 제조Preparation Example 4: Preparation of complex hot water extract
건무화과, 갈근, 대추, 비수리, 산수유, 복분자, 진피, 호로파 및 노니를 각각 준비하였다.Dried figs, garlic root, jujube, bisuri, cornflower oil, bokbunja, dermis, fenugreek and noni were prepared, respectively.
다음으로, 건무화과 100 중량부에 대하여, 갈근 100 중량부, 대추 100 중량부, 비수리 100 중량부, 산수유 77.4 중량부, 복분자 77.4 중량부, 진피 77.4 중량부, 호로파 77.4 중량부 및 노니 77.2중량부를 혼합한 혼합물을 제조하였다.Next, based on 100 parts by weight of dried figs, 100 parts by weight of ground root, 100 parts by weight of jujube, 100 parts by weight of nonsuri, 77.4 parts by weight of cornflower oil, 77.4 parts by weight of bokbunja, 77.4 parts by weight of dermis, 77.4 parts by weight of fenugreek and 77.2 parts by weight of noni A mixture of parts was prepared.
다음으로, 상기 혼합물 100 중량부에 대하여 물 800 중량부와 혼합한 후, 25 ~ 27℃ 하에서 30분 동안 침적하였다. Next, after mixing with 800 parts by weight of water based on 100 parts by weight of the mixture, the mixture was immersed under 25 to 27° C. for 30 minutes.
다음으로, 침적된 용액을 감압농축기에서 130℃에서 6시간 처리한 추출액을 정제하여 최종 복합 열수 추출액을 수득하였다 Next, the extracted solution was purified by treating the deposited solution at 130° C. for 6 hours in a vacuum concentrator to obtain a final composite hot water extract.
실시예 1 : 황칠계 복합 추출물의 제조Example 1: Preparation of Hwangchil-based complex extract
준비예 1의 황칠 발효물, 준비예 2의 용안육 추출물, 준비예 3의 아로니아 추출물 및 준비예 4의 복합 열수 추출물을 하기 표 1의 조성으로 혼합 및 교반하여 황칠계 복합 추출물을 제조하였다.The hwangchil fermented product of Preparation Example 1, the longan meat extract of Preparation Example 2, the aronia extract of Preparation Example 3 and the complex hot water extract of Preparation Example 4 were mixed and stirred with the composition shown in Table 1 below to prepare a Hwangchil-based complex extract.
제조한 황칠계 복합 추출물의 액상 사진을 3b의 (E)에 나타내었고, 이를 동결 건조시킨 시료 사진을 도 3a에 나타내었다.A liquid photograph of the prepared hwangchil-based complex extract is shown in (E) of 3b, and a photograph of a sample obtained by freeze-drying it is shown in FIG. 3a.
비교예 1Comparative Example 1
준비예 1의 황칠 발효물을 비교예로 준비하였다. 이의 액상 사진을 3b의 (A)에 나타내었고, 이를 동결 건조시킨 시료 사진을 도 3a에 나타내었다.The fermented hwangchil of Preparation Example 1 was prepared as a comparative example. A photograph of its liquid phase is shown in (A) of 3b, and a photograph of a sample obtained by freeze-drying it is shown in FIG. 3a.
비교예 2Comparative Example 2
준비예 2의 용안육 열수 추출물을 비교예로 준비하였다. 이의 액상 사진을 3b의 (B)에 나타내었고, 이를 동결 건조시킨 시료 사진을 도 3a에 나타내었다.Longan meat hot water extract of Preparation Example 2 was prepared as a comparative example. A photograph of its liquid phase is shown in (B) of 3b, and a photograph of a sample obtained by freeze-drying it is shown in FIG. 3a.
비교예 3Comparative Example 3
준비예 3의 아로니아 열수 추출물을 비교예로 준비하였다. 이의 액상 사진을 3b의 (C)에 나타내었고, 이를 동결 건조시킨 시료 사진을 도 3a에 나타내었다.Aronia hot water extract of Preparation Example 3 was prepared as a comparative example. A photograph of its liquid phase is shown in (C) of 3b, and a photograph of a sample obtained by freeze-drying it is shown in FIG. 3a.
비교예 4Comparative Example 4
준비예 2의 용안육 추출물, 준비예 3의 아로니아 추출물 및 준비예 4의 복합 열수 추출물을 하기 표 1의 조성으로 혼합 및 교반하여 황칠 발효물을 포함하지 않는 복합 추출물을 제조하였다. 이의 액상 사진을 3b의 (D)에 나타내었고, 이를 동결 건조시킨 시료 사진을 도 3a에 나타내었다.The longan meat extract of Preparation Example 2, the aronia extract of Preparation Example 3, and the complex hot water extract of Preparation Example 4 were mixed and stirred in the composition shown in Table 1 below to prepare a complex extract not containing fermented hwangchil. A photograph of its liquid phase is shown in (D) of 3b, and a photograph of a sample obtained by freeze-drying it is shown in FIG. 3A.
(중량비)division
(weight ratio)
(준비예 1)Hwangchil Fermented Product
(Preparation example 1)
(준비예 2)Longan Meat Hot Water Extract
(Preparation Example 2)
(준비예 3)Aronia hot water extract
(Preparation example 3)
(준비예 4)Complex hot water extract
(Preparation Example 4)
실험예 1 : 간 세포 독성 및 간 세포 보호 효과 측정Experimental Example 1: Measurement of liver cytotoxicity and liver cytoprotective effect
(1) 세포 배양(1) cell culture
HepG2(hepatoma cell, human)를 한국세포주은행(Seoul, Korea)에서 구입하여 사용하였다. HepG2 세포는 37℃. 5% CO2 조건에서 10% FBS, 페니실린(penicillin, 100 μg/ml), 스트렙토마이신(streptomycin, 100 μg/ml)이 첨가된 배지에 배양하여 실험에 사용하였다.HepG2 (hepatoma cell, human) was purchased from the Korea Cell Line Bank (Seoul, Korea) and used. HepG2 cells were stored at 37°C. In 5% CO 2 condition, 10% FBS, penicillin (penicillin, 100 μg/ml), and streptomycin (streptomycin, 100 μg/ml) were used for the experiment by culturing in a medium.
(2) 세포 독성 실험(2) Cytotoxicity test
(2-1) HepG2세포의 산화적 손상 유도를 위한 CCl(2-1) CCl for oxidative damage induction of HepG2 cells 44 적정 농도 확인 Titration check
시료가 HepG2세포의 증식에 미치는 효과를 알아보기 위해 다음과 같이 MTT assay를 수행하였다. 24-well 세포배양접시(cell culture plate)에 3 x 104 cells/ml로 HepG2세포를 접종(seeding)한 후, 24시간 동안 CO2 인큐베이터(incubator)에서 배양하였다. 그리고, 24시간 후, CCl4 (0.1%, 0.2%, 0.3% 0.4%)와 0.25% DMSO가 혼합된 EMEM 배지로 HepG2세포의 산화적 손상을 유도하였다. 24시간 배양 후, 5 mg/ml의 MTT 시약(reagent) 20 μl를 처리하고 4시간 반응 후, ELISA 리더기(550 nm)로 측정하였다.In order to examine the effect of the sample on the proliferation of HepG2 cells, MTT assay was performed as follows. HepG2 cells were inoculated (seeding) in a 24-well cell culture plate at 3 x 10 4 cells/ml, and then cultured in a CO 2 incubator for 24 hours. And, 24 hours later, CCl 4 (0.1%, 0.2%, 0.3% 0.4%) and 0.25% DMSO mixed EMEM medium to induce oxidative damage to HepG2 cells. After 24 hours of incubation, 20 μl of 5 mg/ml of MTT reagent was treated, and after 4 hours of reaction, it was measured with an ELISA reader (550 nm).
도 4a는 CCl4 농도에 따른 HepG2세포 생존율 측정 결과이다. HepG2 세포에 각 농도의 CCl4를 처리 한 결과, CCl4 0.1% 처리한 HepG2 세포에서는 산화적 손상에 의한 세포독성이 나타나지 않았으며, CCl4 0.2% 처리하였을 경우 62.05%, CCl4 0.3% 처리하였을 경우 41.28%, CCl4 0.4%를 처리하였을 경우에 세포생존율이 28.63%로 확인하여 산화적 손상이 나타남을 확인할 수 있었으며, 0.4%의 CCl4 처리를 실험조건으로 선정하였다.4a is a result of measuring HepG2 cell viability according to the concentration of CCl 4 . As a result of treating HepG2 cells with CCl 4 at each concentration, cytotoxicity due to oxidative damage did not appear in HepG2 cells treated with CCl 4 0.1%, and when CCl 4 was treated with 0.2%, 62.05% and CCl 4 were treated with 0.3%. Case 41.28%, when treated with CCl 4 0.4%, the cell viability was confirmed to be 28.63%, it was confirmed that oxidative damage appeared, and 0.4% CCl 4 treatment was selected as the experimental condition.
(2-2) CCl(2-2) CCl 44 로 산화적 손상이 유도된 HepG2 세포에 대한 양성군 설정Setting a positive group for HepG2 cells induced by oxidative damage with
양성군(positive control)으로 선정한 실리마린(silymarin)이 HepG2 세포의 증식에 미치는 효과를 알아보기 위해 MTT assay를 수행하였다. MTT assay was performed to examine the effect of silymarin selected as a positive control on the proliferation of HepG2 cells.
24-well 세포배양접시에 3 x 104 cells/ml로 HepG2세포를 접종(seeding)한 후, 24시간 동안 CO2 인큐베이터에서 배양하였다. 24시간 후, 각 농도의 실리마린(50, 100, 200, 500 μg/ml)을 처리하고 0.4% CCl4와 0.25% DMSO가 혼합된 EMEM 배지로 산화적 손상을 유도하였다. HepG2 cells were inoculated (seeding) at 3 x 10 4 cells/ml in a 24-well cell culture dish, and then cultured in a CO 2 incubator for 24 hours. After 24 hours, each concentration of silymarin (50, 100, 200, 500 μg/ml) was treated and oxidative damage was induced with EMEM medium mixed with 0.4% CCl 4 and 0.25% DMSO.
24시간 배양 후, 5 mg/ml의 MTT reagent 20 μl를 처리하고 4시간 반응 후, ELISA 리더기기 (550 nm)로 측정하였으며, 측정 결과를 도 4b에 나타내었다.After incubation for 24 hours, 20 μl of 5 mg/ml MTT reagent was treated, and after 4 hours of reaction, it was measured with an ELISA reader (550 nm), and the measurement results are shown in FIG. 4B .
CCl4를 처리하여 산화적 손상을 유도한 HepG2 세포에 각 농도의 실리마린을 처리한 결과, CCl4를 처리한 HepG2 세포에서는 24.51%로 세포생존율이 감소하였으며, 50 μg/ml의 농도에서는 26.85%로 증진하며, 100 μg/ml에서는 28.12%, 200 μg/ml에서는 31.26%, 그리고 500 μg/ml의 농도에서는 84.96% 세포생존율이 증가함을 확인하였다. 이를 통해, 실리마린이 CCl4처리에 의한 산화적 손상이 유도된 HepG2 세포의 보호효과가 있음을 확인할 수 있었다. 따라서, 500 μg/ml의 실리마린 처리군을 이하 실험의 양성군(positive control)로 설정하였다.As a result of treatment with silymarin at each concentration of HepG2 cells treated with CCl 4 to induce oxidative damage, the cell viability decreased to 24.51% in HepG2 cells treated with CCl 4 , and to 26.85% at a concentration of 50 μg/ml. It was confirmed that the cell viability increased by 28.12% at 100 μg/ml, by 31.26% at 200 μg/ml, and by 84.96% at the concentration of 500 μg/ml. Through this, it was confirmed that silymarin had a protective effect on HepG2 cells induced by oxidative damage by CCl 4 treatment. Therefore, the silymarin treatment group of 500 μg/ml was set as a positive control group for the following experiments.
(2-3) 실시예 1 및 비교예 1 ~ 4 추출물의 HepG2 세포에 대한 독성 평가(2-3) Toxicity evaluation on HepG2 cells of Example 1 and Comparative Examples 1 to 4 extracts
24-well 세포배양접시에 3 x 104 cells/ml로 HepG2세포를 접종(seeding)한 후, 24시간 동안 CO2 인큐베이터에서 배양하였다. 24시간 후, 각 농도의 실시예 1, 비교예 1 ~ 4 각각을 처리하고 0.25% DMSO가 혼합된 EMEM 배지로 산화적 손상을 유도하였다. 이때, 비교예 1 ~ 비교예 3은 1.3 ~ 500μg/ml의 농도 범위로, 비교예 4 및 실시예 1은 10 ~ 500 μg/ml의 농도 범위에서 다양하게 농도를 달리하여 측정하였고, 그 결과를 도 4c에 나타내었다.HepG2 cells were inoculated (seeding) at 3 x 10 4 cells/ml in a 24-well cell culture dish, and then cultured in a CO 2 incubator for 24 hours. After 24 hours, each concentration of Examples 1 and Comparative Examples 1 to 4 was treated, and oxidative damage was induced with EMEM medium mixed with 0.25% DMSO. At this time, Comparative Examples 1 to 3 were measured at different concentrations in a concentration range of 1.3 to 500 μg/ml, and Comparative Examples 4 and 1 were measured at different concentrations in a concentration range of 10 to 500 μg/ml, and the results were measured It is shown in Figure 4c.
도 4c를 살펴보면, 비교예 2는 65 μg/ml 이상 농도에서, 비교예 4는 100 μg/ml 이상 농도에서부터 세포 독성이 나타나는 결과를 보였다. 이에 반해, 비교예 1, 비교예 3 및 실시예 1은 농도 증가에 따른 독성을 나타내지 않았으며, 양성군인 실리마린(500 μg/ml) 역시 세포 독성을 나타내지 않았다.Referring to FIG. 4c , Comparative Example 2 showed cytotoxicity at a concentration of 65 μg/ml or higher, and Comparative Example 4 showed cytotoxicity at a concentration of 100 μg/ml or higher. On the other hand, Comparative Example 1, Comparative Example 3, and Example 1 did not show toxicity according to the increase in concentration, and the positive group silymarin (500 μg/ml) also did not show cytotoxicity.
(3) 실시예 1 및 비교예 1 ~ 4 추출물의 HepG2 세포에 대한 세포 보호 효과 측정(3) Example 1 and Comparative Examples 1-4 Measurement of the cytoprotective effect on HepG2 cells of the extract
24-well 세포배양접시에 3 x 104 cells/ml로 HepG2세포를 접종(seeding)한 후, 24시간 동안 CO2 인큐베이터에서 배양하였다. 24시간 후, 각 농도의 실시예 1, 비교예 1 ~ 4 각각을 처리하고 0.4% CCl4와 0.25% DMSO가 혼합된 EMEM 배지로 산화적 손상을 유도하였다. 이때, 비교예 1 ~ 비교예 3은 1.3 ~ 500μg/ml의 농도 범위로, 비교예 4 및 실시예 1은 10 ~ 500 μg/ml의 농도 범위에서 다양하게 농도를 달리하여 측정하였고, 그 결과를 도 4d에 나타내었다.HepG2 cells were inoculated (seeding) at 3 x 10 4 cells/ml in a 24-well cell culture dish, and then cultured in a CO 2 incubator for 24 hours. After 24 hours, each concentration of Examples 1 and Comparative Examples 1 to 4 was treated, and oxidative damage was induced with EMEM medium mixed with 0.4% CCl 4 and 0.25% DMSO. At this time, Comparative Examples 1 to 3 were measured at different concentrations in a concentration range of 1.3 to 500 μg/ml, and Comparative Examples 4 and 1 were measured at different concentrations in a concentration range of 10 to 500 μg/ml, and the results were measured It is shown in Figure 4d.
CCl4로 산화적 손상이 유도된 HepG2 세포에 대한 비교예 1(황칠), 비교예 2(용안육), 비교예 3(아로니아), 비교예 4(복합물(황칠 제외)), 실시예 1의 세포 보호 효과 측정 결과, 비교예 4는 보호 효과가 나타나지 않았다. 그리고, 용안육 열수 추출물인 비교예 2의 경우, 65 μg/ml 이상에서 세포 생존율이 감소되는 경향을 보였는데, 이는 용안육 자체의 독성에 기인함을 알 수 있었다. Comparative Example 1 (Hwangchil), Comparative Example 2 (Yonganyuk), Comparative Example 3 (Aronia), Comparative Example 4 (composite (except Hwangchil)), Example 1 for HepG2 cells induced by oxidative damage with CCl 4 As a result of measuring the cytoprotective effect, Comparative Example 4 did not show a protective effect. And, in the case of Comparative Example 2, which is a hot-water extract of longan meat, the cell viability showed a tendency to decrease at 65 μg/ml or more, which was found to be due to the toxicity of the longan meat itself.
이에 반해, 비교예 1은 10 μg/ml 이상에서 세포 보호 효과가 나타났으며, 비교예 3은 100 μg/ml 이상에서 세포 보호 효과가 나타남을 확인알 수 있었으며, 실시예 1은 농도 의존적인 세포 보호 효과를 보였다. In contrast, Comparative Example 1 showed a cytoprotective effect at 10 μg/ml or more, Comparative Example 3 showed a cytoprotective effect at 100 μg/ml or more, and Example 1 showed a concentration-dependent cell showed a protective effect.
실험예 2 : ROS 증가 억제 측정을 통한 HepG2 세포에 대한 산화적 손상 예방 효과 측정Experimental Example 2: Measurement of the effect of preventing oxidative damage on HepG2 cells by measuring ROS increase inhibition
HepG2 세포 자체(control), 상기 실험예 1에서 실험한 상기 (2-1) ~ (2-4)의 0.4% CCl4 처리된 HepG2 세포, 500 μg/ml의 실리마린 처리된 HepG2 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따른 HepG2 세포 각각에 대한 형광현미경 측정 이미지를 도 5a(비교예 1), 도 5b(비교예 2), 도 5c(비교예 3), 도 5d(비교예 4) 및 도 5d(실시예 1)에 각각 나타내었다.HepG2 cells themselves (control), 0.4% CCl 4 treated HepG2 cells of (2-1) to (2-4) tested in Experimental Example 1, 500 μg/ml silymarin-treated HepG2 cells, Comparative Example 1 - 4 and fluorescence microscopy images for each of the HepG2 cells according to the concentration of Example 1, Figure 5a (Comparative Example 1), Figure 5b (Comparative Example 2), Figure 5c (Comparative Example 3), Figure 5D (Comparative Example 4) ) and FIG. 5d (Example 1), respectively.
측정방법은 상기 실험예 1과 동일한 방법으로 HepG2 세포를 배양 및 산화적 손상시킨 후, 24시간 뒤, 디클로로플오렛시디아세테이트(DCF-DA, Sigma, USA) 용액을 처리하여 30분간 37℃에서 배양하고, 배지를 제거 후 PBS(phosphate buffer saline)를 넣어 형광현미경으로 관찰하였다.In the measurement method, HepG2 cells were cultured and oxidatively damaged in the same manner as in Experimental Example 1, 24 hours later, treated with a dichlorofluoroethidiacetate (DCF-DA, Sigma, USA) solution and cultured at 37°C for 30 minutes. After removing the medium, PBS (phosphate buffer saline) was added and observed with a fluorescence microscope.
도 5a ~ 도 5e를 살펴보면, 비교예 1(황칠), 비교예 3(아로니아)은 100 μg/ml 이상에서 활성산소의 감소가 나타남을 확인할 수 있으며, 비교예 4 및 실시예 1은 10 μg/ml의 농도에서 활성 산소의 감소하는 결과를 보였다. 이에 반해, 도 5b를 살펴보면, 비교예 2(용안육)은 모든 농도에서 활성 산고 감소 효과가 없음을 확인할 수 있었다.5a to 5e, it can be seen that Comparative Example 1 (Hwangchil) and Comparative Example 3 (Aronia) show a decrease in active oxygen at 100 μg/ml or more, Comparative Example 4 and Example 1 are 10 μg At a concentration of /ml, it showed a decrease in active oxygen. In contrast, referring to FIG. 5B , it was confirmed that Comparative Example 2 (longan meat) had no effect on reducing active birth weight at all concentrations.
실험예 3 : HepG2 세포에 대한 ALT(Alanine transaminase) 및 AST(aspartate transaminase) 측정Experimental Example 3: ALT (Alanine transaminase) and AST (aspartate transaminase) measurement for HepG2 cells
간세포 기능 관련 효소의 작용을 확인하기 위해 CCl4로 산화적 손상이 유도된 HepG2 세포의 ALT 및 AST를 측정하였다. 측정은 HepG2 세포 자체(control), 상기 실험예 1에서 실험한 상기 (2-1) ~ (2-4)의 0.4% CCl4 처리된 HepG2 세포, 500 μg/ml의 실리마린 처리된 HepG2 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따라 HepG2 세포를 처리한 후, 상층액을 원심분리하여 불순물을 제거한 상등액을 이용하여 IFCC(International Ferderation of Clinical Chemistry and Laboratory Medicine) 방법을 기반으로 아가페 다이아그노틱스(Agappe Diagnostics)에서 제작된 키트로 매뉴얼을 참조하여 340 nm의 파장에서의 ALT, AST 활성도(activity)를 측정하였으며, 그 결과를 도 6a(ALT 측정) 및 도 6b(AST 측정)에 나타내었다.To confirm the action of enzymes related to hepatocyte function, ALT and AST of HepG2 cells induced by oxidative damage with CCl 4 were measured. Measurement is HepG2 cells themselves (control), 0.4% CCl 4 treated HepG2 cells of (2-1) to (2-4) tested in Experimental Example 1, 500 μg/ml of silymarin-treated HepG2 cells, comparison After treating the HepG2 cells according to the concentrations of Examples 1 to 4 and Example 1, the supernatant was centrifuged to remove impurities, and based on the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) method, Agape Diagno ALT and AST activity at a wavelength of 340 nm were measured with reference to the manual with a kit manufactured by Agappe Diagnostics, and the results are shown in FIGS. 6a (ALT measurement) and FIG. 6b (AST measurement) .
도 6a 및 도 6b를 살펴보면, CCl4로 산화적 손상이 유도된 HepG2 세포에 대한 ALT, AST 측정 결과, 비교예 1(황칠)은 10 μg/ml의 농도에서 감소하였으며, 비교예 2(용안육)은 변화가 없었다. 6a and 6b, ALT and AST measurement results for HepG2 cells induced by CCl 4 oxidative damage, Comparative Example 1 (hwangchil) was reduced at a concentration of 10 μg / ml, Comparative Example 2 (Yonganyuk) was no change.
그리고, 비교예 3(아로니아)는 100 μg/ml의 농도에서 ALT, AST의 감소를 보였으며, 실시예 1은 50 μg/ml의 농도에서부터 농도의존적으로 ALT, AST감소하는 경향을 보였다. And, Comparative Example 3 (Aronia) showed a decrease in ALT and AST at a concentration of 100 μg/ml, and Example 1 showed a tendency to decrease ALT and AST in a concentration-dependent manner from a concentration of 50 μg/ml.
그리고, 비교예 4의 경우, 실시예 1과 비교할 때, ALT, AST 감소율이 매우 적은 결과를 보였다.And, in the case of Comparative Example 4, as compared with Example 1, the ALT, AST reduction rate was very small.
실험예 4 : HepG2 세포에 대한 GSH(glutathione level) 측정Experimental Example 4: Measurement of glutathione level (GSH) for HepG2 cells
간세포의 비효소적 항산화 체계를 확인하기 위해 환원형 글루타치온을 측정하였다. 측정은 HepG2 세포 자체(control), 상기 실험예 1에서 실험한 상기 (2-1) ~ (2-4)의 0.4% CCl4 처리된 HepG2 세포, 500 μg/ml의 실리마린 처리된 HepG2 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따라 HepG2 세포를 처리한 후, HepG2 세포를 트립신(trypsin)으로 처리하여 포집하였다. 다음으로, 0.5 ml의 PBS와 0.5 ml의 10% TCA 혼합액을 HepG2 세포에 현탁시키고 원심분리하였다. 다음으로, 상등액을 buffer A(0.1 M sodium phosphate, 5 μM EDTA, 0.6 mM 5,5’-dithio-bis-2-nitrobenzoic acid, 0.2 mM NADPH)와 buffer B(10 U/ml, glutathione reductase in 0.1 M sodium phosphate buffer)로 혼합하고 412 nm의 흡광도를 측정하였고, 그 결과를 도 7에 나타내었다. Reduced glutathione was measured to confirm the non-enzymatic antioxidant system of hepatocytes. Measurements are HepG2 cells themselves (control), 0.4% CCl 4 treated HepG2 cells of (2-1) to (2-4) tested in Experimental Example 1, 500 μg/ml silymarin-treated HepG2 cells, comparison After treating the HepG2 cells according to the concentrations of Examples 1 to 4 and Example 1, the HepG2 cells were collected by treatment with trypsin. Next, a mixture of 0.5 ml of PBS and 0.5 ml of 10% TCA was suspended in HepG2 cells and centrifuged. Next, the supernatant was mixed with buffer A (0.1 M sodium phosphate, 5 μM EDTA, 0.6 mM 5,5'-dithio-bis-2-nitrobenzoic acid, 0.2 mM NADPH) and buffer B (10 U/ml, glutathione reductase in 0.1). M sodium phosphate buffer) and absorbance at 412 nm was measured, and the results are shown in FIG. 7 .
도 7을 살펴보면, CCl4를 처리한 군에서 GSH가 60%의 감소를 보였으며, 비교예 1(황칠)은 50 μg/ml 이상에서 환원형 글루타치온 레벨이 증가하는 것을 나타냈다. 이에 반해, 비교예 2(용안육)은 모든 농도에서 GSH변화가 나타나지 않았다. 그리고, 비교예 3(아로니아)는 65 μg/ml의 농도에서 GSH이 농도의존적으로 증가하는 것을 확인할 수 있었으며, 비교예 4 및 실시예 1은 10 μg/ml의 농도에서 GSH가 농도의존적으로 증가하는 것을 확인할 수 있었다.Referring to FIG. 7 , in the group treated with CCl 4 , GSH was reduced by 60%, and Comparative Example 1 (hwangchil) showed an increase in reduced glutathione levels at 50 μg/ml or more. On the other hand, in Comparative Example 2 (longan meat), there was no change in GSH at any concentration. And, in Comparative Example 3 (Aronia), it was confirmed that the concentration-dependent increase of GSH at the concentration of 65 μg / ml, Comparative Examples 4 and 1, GSH at the concentration of 10 μg / ml concentration-dependently increased could confirm that
실험예 5 : HepG2 세포에 대한 GGT 활성도(Gamma-glutamyl transpeptidase activity) 측정Experimental Example 5: Measurement of GGT activity (Gamma-glutamyl transpeptidase activity) for HepG2 cells
GGT는 간세포의 손상이 유도되면 방어적으로 발생되는 인자 중 하나로써, 간 손상의 지표로 사용된다. 측정은 HepG2 세포 자체(control), 상기 실험예 1에서 실험한 상기 (2-1) ~ (2-4)의 0.4% CCl4 처리된 HepG2 세포, 500 μg/ml의 실리마린 처리된 HepG2 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따라 HepG2 세포를 처리한 후, 상등액을 원심분리하여 불순물을 제거하였다. GGT is one of the factors that occur defensively when hepatocyte damage is induced, and is used as an indicator of liver damage. Measurements are HepG2 cells themselves (control), 0.4% CCl 4 treated HepG2 cells of (2-1) to (2-4) tested in Experimental Example 1, 500 μg/ml silymarin-treated HepG2 cells, comparison After treating the HepG2 cells according to the concentrations of Examples 1 to 4 and Example 1, the supernatant was centrifuged to remove impurities.
다음으로, 20 μl의 상등액과 180 μl의 반응 시약(185 mM Tris-HCl (pH 8.2), 2 mM gamma-glutamyl-ρ-nitroanilide, 20 mM glycylglycine, 0.8 mM NaNO2, 3.2 mM naphthyl ethylenediamine)을 섞어 반응시켰다. 그리고. 37℃, 1시간 반응 후, 40 μl의 1.0 N HCl로 반응을 종결한 후, 520 nm의 파장에서의 흡광도를 측정하여 비교 분석하였으며, 그 결과를 도 8에 나타내었다.Next, mix 20 μl of supernatant with 180 μl of reaction reagent (185 mM Tris-HCl (pH 8.2), 2 mM gamma-glutamyl-ρ-nitroanilide, 20 mM glycylglycine, 0.8 mM NaNO 2 , 3.2 mM naphthyl ethylenediamine). reacted. and. After the reaction at 37°C for 1 hour, the reaction was terminated with 40 μl of 1.0 N HCl, and the absorbance at a wavelength of 520 nm was measured and analyzed for comparison, and the results are shown in FIG. 8 .
도 8을 살펴보면, CCl4가 처리된 군에서 약 5.5배의 GGT 활성 증가를 보였으며, 비교예 1(황칠)은 50 μg/ml 이상에서 GGT의 활성이 농도의존적으로 감소됨을 보였다. 그리고, 비교예 2(용안육)은 100 μg/ml의 농도에서 감소를 나타내었으며, 비교예 2는 100 μg/ml의 농도에서 세포 독성이 나타나며, 세포 보호가 나타나지 않았으므로 이의 영향인 것으로 판단된다.Referring to FIG. 8, the CCl 4 treated group showed an increase in GGT activity of about 5.5 times, and Comparative Example 1 (hwangchil) showed that the activity of GGT was reduced in a concentration-dependent manner at 50 μg/ml or more. And, Comparative Example 2 (longan meat) showed a decrease at a concentration of 100 μg/ml, and Comparative Example 2 showed cytotoxicity at a concentration of 100 μg/ml, and cell protection did not appear, so it is judged to be an effect thereof.
그리고, 비교예 3(아로니아)는 100 μg/ml 이상에서 GGT의 활성 감소가 나타났으며, 비교예 4 및 실시예 1은 10 μg/ml의 농도에서부터 GGT 활성 감소를 보였는데, 특히 50 μg/ml의 농도 이상부터는 비교예 4에 비해 실시예 1이 월등하게 높은 GGT 활성 감소를 보였다.And, Comparative Example 3 (Aronia) showed a decrease in GGT activity at 100 μg/ml or more, and Comparative Example 4 and Example 1 showed a decrease in GGT activity from a concentration of 10 μg/ml, particularly 50 μg From a concentration of /ml or more, Example 1 showed a significantly higher decrease in GGT activity compared to Comparative Example 4.
실험예 6 : HepG2 세포에 대한 MDA(Malondialdehyde) 측정Experimental Example 6: Measurement of MDA (Malondialdehyde) for HepG2 cells
간세포의 MDA 함량 증가는 CCl4에 의한 산화적 스트레스에 따른 활성 산소와 밀접한 관계가 있으며, 활성 산소가 증가함에 따라 간세포의 미토콘드리아 내 MDA 함량이 증가된다. 지질의 과산화물인 MDA를 Emerit 등의 방법을 이용하여 실험하였다. The increase in the MDA content of hepatocytes is closely related to the active oxygen caused by oxidative stress caused by CCl 4 , and as the active oxygen increases, the MDA content in the mitochondria of the hepatocytes increases. MDA, a lipid peroxide, was tested using the method of Emerit et al.
HepG2 세포 자체(control), 상기 실험예 1에서 실험한 상기 (2-1) ~ (2-4)의 0.4% CCl4 처리된 HepG2 세포, 500 μg/ml의 실리마린 처리된 HepG2 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따라 HepG2 세포를 처리한 후, HepG2 세포 를 10% 균질액 0.2 ml와 0.2 N HCl 0.1 ml를 혼합하여 60℃에서 80분간 가열하여 시료를 가수분해시켰다. 다음으로, 가수분해한 시료에 0.4 mM 1-methyl-2-phenylindole 0.65 ml와 37% HCl 0.15 ml를 넣어 혼합시키고 45℃에서 40분 동안 반응시킨 다음 원심분리하여 얻은 상등액의 흡광도를 586 nm에서 측정하였으며, 그 결과를 도 9에 나타내었다. HepG2 cells themselves (control), 0.4% CCl 4 treated HepG2 cells of (2-1) to (2-4) tested in Experimental Example 1, 500 μg/ml silymarin-treated HepG2 cells, Comparative Example 1 After treating the HepG2 cells according to the concentrations of ~ 4 and Example 1, the HepG2 cells were mixed with 0.2 ml of a 10% homogenate and 0.1 ml of 0.2 N HCl and heated at 60° C. for 80 minutes to hydrolyze the sample. Next, 0.65 ml of 0.4 mM 1-methyl-2-phenylindole and 0.15 ml of 37% HCl were added to the hydrolyzed sample, mixed, reacted at 45° C. for 40 minutes, and then centrifuged. The absorbance of the obtained supernatant was measured at 586 nm. and the results are shown in FIG. 9 .
도 9를 살펴보면, CCl4를 처리하여 과생성된 MDA(2.3배)를 비교예 1(황칠), 비교예 3(아로니아)은 50 μg/ml의 농도에서부터 MDA 함량 감소를 나타냈으며, 비교예 2(용안육)은 100 μg/ml의 농도에서부터 MDA 함량 감소를 나타냈는데, 이는 용안육 추출물의 세포 독성에 기인한 것으로 판단된다.Referring to FIG. 9, Comparative Example 1 (Hwangchil) and Comparative Example 3 (Aronia) showed a decrease in the MDA content from a concentration of 50 μg/ml to the over-produced MDA (2.3 times) by treatment with CCl 4 , Comparative Example 2 (yongan meat) showed a decrease in MDA content from a concentration of 100 μg/ml, which is considered to be due to the cytotoxicity of the longan meat extract.
그리고, 비교예 4와 실시예 1은 10 μg/ml의 농도에서부터 MDA 함량 감소를 나타냈으며, 동일 농도에서 실시예 1이 비교예 4보다 상대적으로 높은 감소율을 보였다.And, Comparative Example 4 and Example 1 showed a decrease in the MDA content from a concentration of 10 μg/ml, and Example 1 showed a relatively higher reduction rate than Comparative Example 4 at the same concentration.
실험예 7 : HepG2 세포에 대한 oil red O 염색 관찰Experimental Example 7: Observation of oil red O staining for HepG2 cells
간세포의 지방 수준을 확인하기 위해 oil red O 염색을 수행하였다. Oil red O staining was performed to determine the fat level of hepatocytes.
HepG2 세포 자체(control), 상기 실험예 1에서 실험한 상기 (2-1) ~ (2-4)의 0.4% CCl4 처리된 HepG2 세포, 500 μg/ml의 실리마린 처리된 HepG2 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따라 HepG2 세포를 처리한 후, HepG2 세포를 2회 세척하고 Oil Red O 염색 용액을 10분간 20℃에서 반응시키고 헤마톡실린(haematoxyline)을 20초 동안 염색한 후, 염색된 HepG2 세포를 현미경으로 관찰하였고, 그 결과를 도 10에 나타내었다.HepG2 cells themselves (control), 0.4% CCl 4 treated HepG2 cells of (2-1) to (2-4) tested in Experimental Example 1, 500 μg/ml silymarin-treated HepG2 cells, Comparative Example 1 After treating the HepG2 cells according to the concentrations of ~ 4 and Example 1, the HepG2 cells were washed twice, the Oil Red O staining solution was reacted at 20° C. for 10 minutes, and hematoxyline was stained for 20 seconds. , Stained HepG2 cells were observed under a microscope, and the results are shown in FIG. 10 .
도 10의 oil red O 염색 결과를 살펴보면, CCl4를 처리하였을 때 과생성된 지질이 비교예 1, 비교예 3의 추출물을 처리하였을 때 oil red O 염색이 감소되는 것을 확인할 수 있었다.Looking at the oil red O staining results of FIG. 10 , it was confirmed that the oil red O staining was reduced when the extracts of Comparative Examples 1 and 3 were treated with the overproduced lipids when treated with CCl 4 .
실험예 8 : 신장 세포 독성 및 간 세포 보호 효과 측정Experimental Example 8: Measurement of renal cytotoxicity and hepatocytoprotective effect
(1) 세포 배양(1) cell culture
신장 세포인 LLC-PK1 세포주(CL-101, ATCC)를 37℃에서 95% 산소와 5% CO2를 공급해주며 10% FBS(fetal bovine serum, Hyclone, USA), 100 μg/ml 스트렙토마이신(streptomycin)과 100 units/ml 페니실린(penicillin)이 포함된 RPMI 배지에서 배양하였다.Renal cells, LLC-PK1 cell line (CL-101, ATCC) were supplied with 95% oxygen and 5% CO 2 at 37°C, and 10% FBS (fetal bovine serum, Hyclone, USA), 100 μg/ml streptomycin (streptomycin) ) and cultured in RPMI medium containing 100 units/ml penicillin.
(2) 라디칼로 인한 산화적 손상에 대한 신장세포의 독성 측정(2) Measurement of toxicity of kidney cells against oxidative damage caused by radicals
96-well 세포배양접시에 1x104 cells/ml로 LLC-PK1 세포주를 접종한 후, 2시간 안정화시켰다. 그 후, 각 농도의 시료를 처리하고 24시간 배양한 후, MTT 시약을 이용하여 세포 생존율을 ELISA reader (550 nm)로 측정하였으며, 그 결과를 도 11a에 나타내었다.After inoculating the LLC-PK1 cell line at 1x10 4 cells/ml in a 96-well cell culture dish, it was stabilized for 2 hours. After that, samples of each concentration were treated and cultured for 24 hours, and cell viability was measured with an ELISA reader (550 nm) using MTT reagent, and the results are shown in FIG. 11a .
도 11a에서 control은 LLC-PK1 세포주 자체이며, EGCG는 에피갈로카테킨 갈레이트(Epigallocatechin gallate)를 100 ng/ml를 LLC-PK1 세포주에 처리한 것이다. 그리고, 비교예 1 ~ 4 및 실시예 1은 도 11a에 나타낸 농도로 비교예 1 ~ 4 및 실시예 1의 추출물을 10 mM AAPH 함께 처리한 것이다.11a, the control is the LLC-PK1 cell line itself, and EGCG is epigallocatechin gallate treated with 100 ng/ml of the LLC-PK1 cell line. And, Comparative Examples 1 to 4 and Example 1 is a treatment with 10 mM AAPH extracts of Comparative Examples 1 to 4 and Example 1 at the concentrations shown in Figure 11a.
도 11a를 살펴보면, 비교예 2(용안육)을 제외한 비교예 1, 비교예 3 ~ 4 및 실시예 1에서는 독성을 나타내지 않았다. 비교예 2의 경우, 50 μg/ml의 농도에서부터 세포 독성이 나타나 500 μg/ml의 농도에서 약 42%의 세포 생존율을 보였다.Referring to FIG. 11a, Comparative Example 1, Comparative Examples 3 to 4, and Example 1 except for Comparative Example 2 (longan meat) did not show toxicity. In Comparative Example 2, cytotoxicity was observed at a concentration of 50 μg/ml, and a cell viability of about 42% was exhibited at a concentration of 500 μg/ml.
(3)(3) 라디칼로 인한 산화적 손상에 대한 신장세포의 보호능 측정Measurement of protective ability of kidney cells against oxidative damage caused by radicals
96-well 세포배양접시에 1x104 cells/ml로 LLC-PK1 세포주를 접종한 후, 2시간 안정화시켰다. 그 후, 각 농도의 시료와 라디칼 생성제인 10 mM AAPH (2,2-azobis(1-aminopropane)dihydrochloride)를 동시에 처리하고 24시간 배양한 후, MTT 시약을 이용하여 세포 생존율을 ELISA reader (550 nm)로 측정하였으며, 그 결과를 도 11b에 나타내었다.After inoculating the LLC-PK1 cell line at 1x10 4 cells/ml in a 96-well cell culture dish, it was stabilized for 2 hours. After that, samples of each concentration and 10 mM AAPH (2,2-azobis(1-aminopropane)dihydrochloride), a radical generating agent, were simultaneously treated and incubated for 24 hours, then cell viability was measured using MTT reagent with an ELISA reader (550 nm ), and the results are shown in FIG. 11B.
도 11b에서 control은 LLC-PK1 세포주 자체이며, EGCG는 에피갈로카테킨 갈레이트(Epigallocatechin gallate)를 100 ng/ml 및 10 mM AAPH를 LLC-PK1 세포주에 처리한 것이다. 그리고, 비교예 1 ~ 4 및 실시예 1은 도 11b에 나타낸 농도로 비교예 1 ~ 4 및 실시예 1의 추출물을 10 mM AAPH 함께 처리한 것이다.11B, the control is the LLC-PK1 cell line itself, and EGCG is 100 ng/ml of epigallocatechin gallate and 10 mM AAPH treated with the LLC-PK1 cell line. And, Comparative Examples 1 to 4 and Example 1 is a treatment with 10 mM AAPH extracts of Comparative Examples 1 to 4 and Example 1 at the concentrations shown in Figure 11b.
AAPH로 산화적 손상을 유도한 LLC-PK1 신장 세포의 추출물에 대한 세포 보호 결과를 확인해보면, 비교예 1(황칠)과 비교예 3(아로니아)의 경우, 각각 50 μg/ml, 250 μg/ml의 농도에서부터 세포 보호능이 증가하는 경향을 보였다. 이에 반해, 비교예 2(용안육)의 경우 100 μg/ml의 농도에서부터 농도의존적으로 세포 독성을 나타냈는데, 이는 용안육 추출물 자체 독성에 기인하는 것으로 판단된다. Checking the cytoprotective results for the extract of LLC-PK1 kidney cells induced by AAPH oxidative damage, in the case of Comparative Example 1 (Hwangchil) and Comparative Example 3 (Aronia), 50 μg/ml and 250 μg/ From the concentration of ml, the cytoprotective ability showed a tendency to increase. On the other hand, Comparative Example 2 (yongan meat) showed a concentration-dependent cytotoxicity from a concentration of 100 μg/ml, which is considered to be due to the toxicity of the longan meat extract itself.
그리고, 비교예 4 및 실시예 1 경우 농도의존적으로 신장 세포 보호능이 증가하는 것을 확인할 수 있었다.And, in Comparative Examples 4 and 1, it was confirmed that the renal cell protective ability was increased in a concentration-dependent manner.
실험예 9 : ROS 증가 억제 측정을 통한 LLC-PK1 세포에 대한 산화적 손상 예방 효과 측정Experimental Example 9: Measurement of the effect of preventing oxidative damage on LLC-PK1 cells by measuring ROS increase inhibition
LLC-PK1 세포 자체(control), 상기 실험예 8의 (3)에서 실험한 AAPH 처리된 LLC-PK1 세포, 100 ng/ml 의 EGCG 처리된 LLC-PK1 세포, 비교예 1 ~ 4 및 실시예 1의 농도에 따른 LLC-PK1 세포 각각에 대한 형광현미경 측정 이미지를 도 12a(비교예 1), 도 12b(비교예 2), 도 12c(비교예 3), 도 12d(비교예 4) 및 도 12e(실시예 1)에 각각 나타내었다.LLC-PK1 cells themselves (control), AAPH-treated LLC-PK1 cells tested in (3) of Experimental Example 8, EGCG-treated LLC-PK1 cells of 100 ng/ml, Comparative Examples 1 to 4 and Example 1 Fig. 12a (Comparative Example 1), Fig. 12B (Comparative Example 2), Fig. 12C (Comparative Example 3), Fig. 12D (Comparative Example 4) and Figs. (Example 1), respectively.
측정방법은 상기 실험예 8과 동일한 방법으로 LLC-PK1 세포를 배양 및 산화적 손상시킨 후, 24시간 뒤, 디클로로플오렛시디아세테이트(DCF-DA, Sigma, USA) 용액을 처리하여 30분간 37℃에서 배양하고, 배지를 제거 후 PBS(phosphate buffer saline)를 넣어 형광현미경으로 관찰하였다.As for the measurement method, LLC-PK1 cells were cultured and oxidatively damaged in the same manner as in Experimental Example 8, and then 24 hours later, treated with a dichlorofluoroethydiacetate (DCF-DA, Sigma, USA) solution at 37°C for 30 minutes. , and after removing the medium, PBS (phosphate buffer saline) was added and observed with a fluorescence microscope.
도 12a ~ 도 12e를 살펴보면, 비교예 2(용안육)을 제외한 모든 추출물에서 농도의존적인 활성 산소 억제능을 나타내었다.12A to 12E, all extracts except Comparative Example 2 (longan meat) showed concentration-dependent active oxygen inhibition ability.
Claims (9)
상기 복합 열수 추출물은 건무화과, 갈근, 대추, 비수리, 산수유, 복분자, 진피, 호로파 및 노니를 포함하는 혼합물의 열수 추출물을 포함하는 것을 특징으로 하는 간 및 신장 기능 개선용 황칠계 복합 추출물Including Hwangchil fermented product, longan meat extract, aronia extract and complex hot water extract,
The complex hot water extract is Hwangchil-based complex extract for improving liver and kidney function, characterized in that it comprises a hot water extract of a mixture containing dried figs, garlic root, jujube, bisuri, cornflower oil, bokbunja, dermis, fenugreek and noni
황칠나무 줄기 건조물 및 황칠나무 잎 건조물의 혼합물을 염수 처리한 후, 균주로 발효시킨 발효물을 포함하는 것을 특징으로 하는 간 및 신장 기능 개선용 황칠계 복합 추출물.
According to claim 1, wherein the fermented hwangchil
Hwangchil-based complex extract for improving liver and kidney function, comprising a fermented product fermented with a strain after brine treatment of a mixture of dried hwangchil tree stems and dried hwangchil tree leaves.
The complex extract of claim 1, wherein the longan meat extract and aronia extract are hot water extracts.
[Claim 2] The hwangchil system for improving liver and kidney function according to claim 1, wherein the fermented hwangchil, longan meat extract, aronia extract, and complex hot water extract are included in a weight ratio of 1: 0.5 to 1.2: 0.5 to 1.5: 3.5 to 6.5 by weight. complex extract.
상기 황칠 발효 추출물, 용안육 추출물, 아로니아 추출물 및 복합 열수 추출물을 혼합하는 2단계;를 포함하며,
상기 황칠 발효 추출물은
황칠나무 줄기 건조물 및 황칠나무 잎 건조물의 혼합물을 0.8 ~ 2 부피% 농도의 염수에 8 ~ 16시간 동안 침적시켜서 염수 처리하는 단계;
염수 처리된 혼합물을 수세한 후, 설탕 1.0 ~ 3.5 중량%, 소금 0.001 ~ 0.04 중량% 및 잔량의 수세한 혼합물을 혼합하는 단계; 및
혼합물에 균주를 접종시킨 후, 35 ~ 37℃ 하에서 5 ~ 8시간 동안 발효 및 정제하여 황칠 발효물을 제조하는 단계;를 포함하는 공정을 수행하여 제조하되,
상기 복합 열수 추출물은 건무화과, 갈근, 대추, 비수리, 산수유, 복분자, 진피, 호로파 및 노니를 혼합한 혼합물을 제조하는 1단계;
상기 혼합물 100 중량부 및 물 650 ~ 900 중량부를 혼합한 후, 혼합용액을 제조하는 2단계;
상기 혼합용액을 20 ~ 35℃ 하에서 20분 ~ 1시간 동안 침적시키는 3단계; 및
침적된 용액을 감압농축 및 정제하여 추출액을 수득하는 4단계;
를 포함하는 공정을 수행하여 제조한 것을 특징으로 하는 간 및 신장 기능 개선용 황칠계 복합 추출물의 제조방법.Step 1 of preparing each of the fermented hwangchil, longan meat extract, aronia extract and complex hot water extract; and
A second step of mixing the hwangchil ferment extract, longan meat extract, aronia extract and complex hot water extract;
The Hwangchil fermented extract is
A brine treatment step by immersing a mixture of dried hwangchil tree stems and dried hwangchil leaves in brine at a concentration of 0.8 to 2% by volume for 8 to 16 hours;
After washing the brine-treated mixture with water, mixing 1.0 to 3.5% by weight of sugar, 0.001 to 0.04% by weight of salt, and the remaining amount of the washed mixture; and
After inoculating the strain in the mixture, fermenting and purifying for 5 to 8 hours at 35 ~ 37 ℃ to prepare a hwangchil fermented product; but prepared by performing a process comprising,
The complex hot water extract is prepared in the first step of preparing a mixture of dried figs, garlic root, jujube, bisuri, cornflower oil, bokbunja, dermis, fenugreek and noni;
After mixing 100 parts by weight of the mixture and 650 to 900 parts by weight of water, a second step of preparing a mixed solution;
Step 3 of immersing the mixed solution at 20 to 35° C. for 20 minutes to 1 hour; and
Step 4 to obtain an extract by concentrating and purifying the deposited solution under reduced pressure;
A method for producing a Hwangchil-based complex extract for improving liver and kidney function, characterized in that it was prepared by performing a process comprising a.
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