KR102594018B1 - Detoxification composition for removing toxins and wastes accumulated in the body. - Google Patents
Detoxification composition for removing toxins and wastes accumulated in the body. Download PDFInfo
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- KR102594018B1 KR102594018B1 KR1020230061467A KR20230061467A KR102594018B1 KR 102594018 B1 KR102594018 B1 KR 102594018B1 KR 1020230061467 A KR1020230061467 A KR 1020230061467A KR 20230061467 A KR20230061467 A KR 20230061467A KR 102594018 B1 KR102594018 B1 KR 102594018B1
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- liver
- alcohol
- composition
- toxins
- plant
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- A23V2200/334—Foods, ingredients or supplements having a functional effect on health treating the effects of consuming alcohol, narcotics or other addictive behavior, e.g. treating hangover or reducing blood alcohol levels
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/14—Extraction
Abstract
본 발명의 식물유래 복합물을 유효성분으로 포함하는 체내 독소 제거 및 독소 축적에 인한 증상 개선용 건강기능식품 조성물 및 이를 이용한 건강기능식품은 우수한 항산화 효과 (DPPH 자유라디칼 소거능)과 당 독소 생성 억제와 당 독소 축적으로 인한 당뇨 및 당뇨합병증 원인 물질 제거 (알파 글루코시다아제 활성 억제) 효과가 있고, 알코올에 의해 손상된 간 세포 보호 뿐만 아니라 간 독소 제거 (Aldehyde Dehydrogenase, Alcohol Dehydrogenase 효소 활성 증가)효과가 있다.The health functional food composition for removing toxins from the body and improving symptoms caused by accumulation of toxins containing the plant-derived complex of the present invention as an active ingredient and the health functional food using the same have excellent antioxidant effects (DPPH free radical scavenging ability), inhibition of sugar toxin production and sugar absorption. It is effective in removing substances that cause diabetes and diabetic complications caused by toxin accumulation (inhibiting alpha glucosidase activity), protecting liver cells damaged by alcohol, and removing liver toxins (increasing the activity of Aldehyde Dehydrogenase and Alcohol Dehydrogenase enzymes).
Description
본 발명은 흰 전나무 추출물 등의 포함하는 식물유래 복합물의 체내 독소 제거 효과를 가지고 독소 축적으로 발생될 수 있는 여러 증상 개선할 수 있는 흰 전나무를 포함하는 식물유래 복합물을 유효성분으로 포함하는 알코올에 의한 간 독소 축적 억제 및 제거용 조성물 및 이를 이용한 건강기능식품에 관한 것이다.The present invention is a plant-derived complex containing white fir extract, etc., which has the effect of removing toxins from the body and can improve various symptoms that may occur due to toxin accumulation. Alcohol containing a plant-derived complex containing white fir as an active ingredient It relates to a composition for inhibiting and removing liver toxin accumulation and a health functional food using the same.
현대사회를 살아가는 사람들은 화학첨가물, 미세먼지, 환경호르몬, 공해물질, 의약품에 의한 독소, 비위생적 환경에 의한 독소 등 수많은 유해물질에 노출된 삶을 살아가고 있다. 이러한 유해물질이 체내 독소 형태로 배출되지 않고 지속적으로 축적될 경우 면역력을 떨어뜨리고 균형을 무너뜨려 신체기능을 망가뜨리고 삶의 질을 저하시키는 만성질환을 일으키게 된다.People living in modern society are exposed to numerous harmful substances, such as chemical additives, fine dust, environmental hormones, pollutants, toxins from pharmaceuticals, and toxins from unsanitary environments. If these harmful substances are not excreted in the form of toxins in the body and continue to accumulate, they lower immunity and break balance, causing chronic diseases that destroy body functions and reduce quality of life.
독소 중에서 가장 핵심적인 산소독성, 즉 활성산소는 생명유지 과정에서 산소가 체내 여러 반응에 의해 생성되며 제거가 잘 이루어지지 않는 경우 체내 필수 성분인 단백질, 당, DNA 등에 손상을 일으켜 암, 심혈관 질환, 노화와 같은 증상을 유발한다. 체내에는 활성산소를 제거하기 위한 자연적인 장치로 과산화물제거효소(Superoxide dismutase, SOD), 글루타티온과산화효소(glutathione peroxidase,GPX), 카탈라아제(catalase, CAT), 글루타티온환원효소(glutathione reductase), 글루타티온-S-전달효소 (glutathione-S-transferase) 등이 존재하지만 체내 축적되는 독소를 모두 분해하기엔 한계가 있다. 최근에는 이를 보완하기 위해 항산화효과에 도움을 주는 음식들과 천연물 유래 황산화제에 대한 연구가 증가하고 있다.Among toxins, the most important oxygen toxicity, namely free radicals, is produced by oxygen in various reactions in the body during the process of maintaining life. If it is not removed properly, it causes damage to proteins, sugars, and DNA, which are essential components of the body, leading to cancer, cardiovascular disease, and other causes. It causes aging-like symptoms. The body's natural mechanisms for removing oxygen radicals include superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), glutathione reductase, and glutathione-S. -Transfer enzymes (glutathione-S-transferase) exist, but they have limitations in decomposing all toxins accumulated in the body. Recently, to complement this, research on foods and natural product-derived sulfating agents that help with antioxidant effects is increasing.
간은 우리 몸에서 해독을 담당하는 중요한 기관으로서 간이 독소에 노출되고 축적되면 다른 기관에까지 큰 문제를 일으킨다. 간 독소의 직접적인 원인 중 하나는 알코올이다. 알코올 섭취가 지속되었을 때 나타나는 대표적인 증상은 지방간이다. 지방간은 관리정도에 따라 얼마든지 회복 가능한 가역적인 특징이 있지만 이에 대한 마땅한 치료없이 음주가 이어진다면 간으로 염증 유발 세포들이 침투해와서 간염(hepatitis)이 발생하고 간이 점차 손상되며 비가역적 질병으로 전환된다. 간염이 반복되다 보면 간이 딱딱하게 굳는 간섬유화(fibrosis), 섬유화가 심해져 작은 덩어리인 결절이 발생하는 간경변증(cirrhosis), 더 이상 간이 제 기능을 못하는 간부전(liver failure)까지 순서대로 진행된다. The liver is an important organ responsible for detoxification in our body. When the liver is exposed to toxins and accumulates them, it causes major problems in other organs. One of the direct causes of liver toxins is alcohol. A typical symptom that appears when alcohol consumption continues is fatty liver. Fatty liver has the characteristic of being reversible and can be recovered depending on the level of management, but if drinking continues without proper treatment, inflammatory cells infiltrate the liver, causing hepatitis, gradually damaging the liver, and turning into an irreversible disease. . If hepatitis is repeated, it progresses in order from fibrosis, in which the liver hardens, to cirrhosis, in which the fibrosis worsens and small lumps form nodules, and liver failure, in which the liver no longer functions properly.
특히, 간은 손상 시 자각 증상이 없기 때문에 초기에 미리 관리하는 것이 가장 중요하다. 또한, 알코올은 간 뿐아니라 중추 신경계를 억제하고 점진적으로 뇌 기능을 저해하는 물질로서 뇌 활동에 영향을 미치는 중독 증상을 유발하고 신체의 조정력 결함과 행동 변화를 유발하기에 간의 해독능력이 떨어지면 신체 전반에 걸친 문제점이 발생한다. In particular, since there are no noticeable symptoms when the liver is damaged, it is most important to manage it early. In addition, alcohol is a substance that suppresses not only the liver but also the central nervous system and gradually impairs brain function, causing addiction symptoms that affect brain activity, defects in body coordination, and behavioral changes. Therefore, when the liver's detoxification ability is reduced, the entire body is affected. Problems arise across the range.
간의 해독능력을 평가하기 가장 좋은 지표는 알코올 분해효소인 알코올 탈수소효소(Aldehyde Dehydrogenase, ADH)와 알데하이드 탈수소효소 (Alcohol Dehydrogenase, ALDH)이다. ADH는 간세포에 존재하며 NAD+의존적으로 에탄올을 아세트알데하이드로 전환시키는 역할을 한다. 이 때 발생되는 아세트알데하이드가 숙취를 유발하는 원인물질로 알려져있으며 이를 분해하는 효소가 바로 ALDH이다. ALDH는 아세트알데하이드가 아세트산염으로 전환되는 과정을 촉매하며 알코올로부터 유래되는 독소 분해에 핵심적인 역할을 한다.The best indicators to evaluate the liver's detoxification ability are the alcohol-decomposing enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH exists in hepatocytes and plays a role in converting ethanol to acetaldehyde in a NAD+-dependent manner. Acetaldehyde generated at this time is known to be the causative agent of hangovers, and the enzyme that breaks it down is ALDH. ALDH catalyzes the conversion of acetaldehyde to acetate and plays a key role in the decomposition of toxins derived from alcohol.
이에 본 발명은 부작용이 거의 없는 천연물인 흰 전나무 또는 흰 전나무를 포함하는 식물유래 복합물의 항산화 효과와 당 독소, 간 독소 제거효과를 확인함으로써 흰 전나무 또는 흰 전나무를 포함하는 식물유래 복합물의 알코올에 의한 간 독소 축적 억제 및 제거용 조성물을 제공하고자 한다.Accordingly, the present invention confirms the antioxidant effect and the sugar toxin and liver toxin removal effect of white fir or a plant-derived complex containing white fir, which are natural products with almost no side effects, thereby reducing the alcohol-related effects of white fir or a plant-derived complex containing white fir. The object is to provide a composition for inhibiting and removing liver toxin accumulation.
본 발명은 현대사회에서 발생하는 만성질환인 당독소와 간 독소 유래의 질병을 예방할 수 있도록 흰 전나무 또는 흰 전나무를 포함하는 식물유래 복합물의 간 독소 제거 및 간 건강 건강기능식품 조성물 및 이의 건강기능식품을 제공하고자 한다.The present invention provides a health functional food composition for liver toxin removal and liver health of a plant-derived complex containing white fir or white fir to prevent diseases caused by sugar toxins and liver toxins, which are chronic diseases occurring in modern society, and health functional foods thereof. We would like to provide.
본 발명의 실시예에 따르면 흰 전나무 추출물을 유효성분으로 함유하는 알코올에 의한 간 독소 축적 억제 및 제거용 조성물일 수 있다.According to an embodiment of the present invention, it may be a composition for inhibiting and removing liver toxin accumulation due to alcohol containing white fir extract as an active ingredient.
본 발명의 또 다른 실시예에 따르면, 흰 전나무 추출물, 드레곤 헤드 추출물, 오크라 추출물로 이루어진 군에서 선택되는 어느 하나의 추출물을 함유하는 것을 특징으로 하는 알코올에 의한 간 독소 축적 억제 및 제거용 조성물일 수 있다.According to another embodiment of the present invention, it may be a composition for inhibiting and removing liver toxin accumulation due to alcohol, characterized in that it contains any one extract selected from the group consisting of white fir extract, dragon head extract, and okra extract. there is.
본 발명의 또 다른 실시예에 따르면, 흰 전나무 추출물, 드레곤 헤드 추출물, 오크라 추출물로 구성되는 건강식품 조성물에 있어서, 상기 조성물은 드레곤헤드 0.5~1 중량부, 흰 전나무 1~3중량부, 오크라 1~6중량부를 포함하여 구성되는 것일 수 있다.According to another embodiment of the present invention, in a health food composition consisting of white fir extract, dragon head extract, and okra extract, the composition contains 0.5 to 1 part by weight of dragon head, 1 to 3 parts by weight of white fir, and 1 part by weight of okra. It may be comprised of ~6 parts by weight.
또한, 상기 조성물은 활성산소 독성, 당 독소,당뇨 합병증, 알코올에 의한 간 독소를 제거하고, 당뇨성 망막증(diabetic retinopathy), 당뇨성 백내장(diabetic cataract), 당뇨성 신증 (diabetic nephropathy), 당뇨성 신경병증(diabetic neuropathy) 또는 당뇨성 혈관합병증 중 하나 이상 선택되는 당뇨 합병증을 예방할 수 있는 것일 수 있다.In addition, the composition removes free radical toxicity, sugar toxins, diabetes complications, liver toxins caused by alcohol, and treats diabetic retinopathy, diabetic cataracts, diabetic nephropathy, and diabetic nephropathy. It may be possible to prevent one or more diabetic complications selected from diabetic neuropathy or diabetic vascular complications.
본 발명의 또 다른 실시예에 따르면, 상기 조성물을 포함하는 알코올에 의한 간 독소 축적 억제 및 제거용 건강기능 식품일 수 있다. 상기 건강기능식품은 산제, 과립제, 정제, 캡슐제, 환제, 현탁액, 에멀젼, 시럽의 약학 투여형태 또는 티백제, 침출차, 건강 음료의 형태인 것일 수 있다.According to another embodiment of the present invention, the composition may be a health functional food for suppressing and eliminating liver toxin accumulation caused by alcohol. The health functional food may be in the form of pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, and syrups, or in the form of tea bags, leached teas, and health drinks.
본 발명에 따른 조성물은 실험예에 따르면 우수한 항산화 효과 (DPPH 자유라디칼 소거능)과 당 독소 생성 억제와 당 독소 축적으로 인한 당뇨 및 당뇨합병증 원인 물질 제거 (알파 글루코시다아제 활성 억제) 효과가 있고, 알코올에 의해 손상된 간 세포 보호 뿐만 아니라 간 독소 제거 (Aldehyde Dehydrogenase, Alcohol Dehydrogenase 효소 활성 증가)효과가 있음을 확인하였다.According to experimental examples, the composition according to the present invention has an excellent antioxidant effect (DPPH free radical scavenging ability), inhibits the production of sugar toxins, removes substances that cause diabetes and diabetic complications due to accumulation of sugar toxins (inhibits alpha glucosidase activity), and has the effect of suppressing alcohol It was confirmed that it not only protects damaged liver cells, but also has the effect of removing liver toxins (increasing the activity of Aldehyde Dehydrogenase and Alcohol Dehydrogenase enzymes).
도 1은 본 발명 식물 유래 복합물의 최종 당화산물 생성 억제 효과를 나타낸다
도 2는 본발명 식물유래 복합물의 알파 글루코시다아제 활성 억제효과를 나타낸다.
도 3은 본 발명에 따른 식물유래 복합물이 간세포 생존율을 나타낸다.
도 4는 본 발명에 따른 식물유래 복합물이 알코올에 의해 손상이 유도된 간세포 보호 효과를 나타낸다.
도 5는 본 발명에 따른 식물유래 복합물의 ALDH 효소 활성 촉진 효과를 나타낸다.
도 6은 본 발명의 실험예에서 사용된 세 가지 추출물의 성상을 나타낸다.
도 7은 본 발명의 실험예에서 사용된 추출물로 제조한 제조예이다.Figure 1 shows the effect of inhibiting the production of advanced glycation products of the plant-derived complex of the present invention.
Figure 2 shows the alpha-glucosidase activity inhibitory effect of the plant-derived complex of the present invention.
Figure 3 shows the hepatocyte survival rate of the plant-derived complex according to the present invention.
Figure 4 shows the protective effect of the plant-derived complex according to the present invention on liver cells damaged by alcohol.
Figure 5 shows the effect of promoting ALDH enzyme activity of the plant-derived complex according to the present invention.
Figure 6 shows the properties of the three extracts used in the experimental examples of the present invention.
Figure 7 is a production example prepared from the extract used in the experimental example of the present invention.
본 발명은 DPPH 라디칼 소거능, 체내 최종당화산물 생성 억제, 알파 글루코시다아제 활성억제, 알코올에 의해 손상된 간세포 보호, 알코올 분해효소 활성 테스트를 통해 흰 전나무 추출물 및 흰 전나무 추출물을 포함하는 식물유래 복합물의 체내독소 축적 억제 및 제거 효과를 확인하였다. 이를 토대로 천연물질 유래의 부작용이 적은 디톡스 효능을 갖는 건강기능식품 조성물 및 이를 활용한 건강기능식품을 제공하고자 한다.The present invention is a plant-derived complex containing white fir extract and white fir extract that can be used in the body through tests for DPPH radical scavenging ability, inhibition of advanced glycation end product production in the body, inhibition of alpha-glucosidase activity, protection of liver cells damaged by alcohol, and alcohol degrading enzyme activity. The effect of inhibiting and removing toxins was confirmed. Based on this, we aim to provide a health functional food composition with a detox effect with fewer side effects derived from natural substances and a health functional food using the same.
이하, 본 발명의 식물유래 복합물을 유효성분으로 포함하는 체내 독소 제거 및 독소 축적으로 인한 증상 개선용 건강기능식품 조성물 및 이를 이용한 건강기능식품과 관련한 실험예를 들어 설명하면 다음과 같다.Hereinafter, a health functional food composition for removing toxins from the body and improving symptoms caused by toxin accumulation containing the plant-derived complex of the present invention as an active ingredient and an experimental example related to a health functional food using the same will be described as follows.
본 발명의 실험예는 흰 전나무를 포함하는 식물 유래 복합물의 디톡스 효능 및 체내 독소 축적으로 인한 증상 개선효과를 확인하기 위해 효소반응 실험 및 세포실험을 진행하였다.In an experimental example of the present invention, enzyme reaction experiments and cell experiments were conducted to confirm the detox effect of a plant-derived complex containing white fir and the symptom improvement effect due to accumulation of toxins in the body.
<실험예 1> 흰 전나무 추출물 및 식물유래 복합물 추출물 제조<Experimental Example 1> Preparation of white fir extract and plant-derived complex extract
1-1. 흰 전나무 추출물 제조방법1-1. White fir extract manufacturing method
깨끗이 씻고 건조한 흰전나무 껍질에 증류수를 가하고, 100℃에서 가열하여 추출액을 획득하였다.추출된 용액은 5um 포어사이즈의 필터를 이용하여 여과한 다음 감압회전 농축기로 농축하였다. 여과 후 남은 잔사에 다시 동량의 증류수를 사용하여 동일 과정으로 2번 더 추출, 여과 및 감압 농축하였다. 농축된 열수 추출물을 분무건조장치 (Spray dryer)를 이용하여 분무 건조하였다. 건조된 분말은 농도에 맞게 물에 희석하여 실험의 최종 원료로 사용되었다. Distilled water was added to cleanly washed and dried white fir bark, and the extract was obtained by heating at 100°C. The extracted solution was filtered using a 5um pore size filter and then concentrated using a reduced pressure rotary concentrator. The residue remaining after filtration was extracted twice more using the same amount of distilled water, filtered, and concentrated under reduced pressure. The concentrated hot water extract was spray dried using a spray dryer. The dried powder was diluted in water according to concentration and used as the final raw material for the experiment.
1-2. 드레곤헤드 추출물 제조방법1-2. Dragon head extract manufacturing method
깨끗이 씻고 건조한 드레곤 헤드 잎에 30% 에탄올을 가하여 추출액을 획득하였다. 추출된 용액 외 잔여물은 제거하였고 제거 후 다시 한번 여과한 다음 유동층 과립기를 사용하여 건조하였다. 건조된 분말은 농도에 맞게 물에 희석하여 실험의 최종 원료로 사용되었다. An extract was obtained by adding 30% ethanol to thoroughly washed and dried dragon head leaves. Residues other than the extracted solution were removed, filtered once again, and then dried using a fluidized bed granulator. The dried powder was diluted in water according to concentration and used as the final raw material for the experiment.
1-3. 오크라 추출물 제조방법1-3. Okra extract manufacturing method
깨끗이 씻고 건조한 오크라 열매에 증류수를 가하고 100℃에서 가열하여 추출액을 획득하였다. 추출된 용액은 5um 포어사이즈의 필터를 이용하여 여과한 다음 감압회전 농축기로 농축하였다. 여과 후 남은 잔사에 다시 동량의 증류수를 사용하여 동일 과정으로 2번 더 추출, 여과 및 감압 농축하였다. 농축된 열수추출물을 분무건조장치 (Spray dryer)를 이용하여 분무 건조하였다. 건조된 분말은 농도에 맞게 물에 희석하여 실험의 최종 원료로 사용되었다. Distilled water was added to thoroughly washed and dried okra fruits and heated at 100°C to obtain an extract. The extracted solution was filtered using a 5um pore size filter and then concentrated using a reduced pressure rotary concentrator. The residue remaining after filtration was extracted twice more using the same amount of distilled water, filtered, and concentrated under reduced pressure. The concentrated hot water extract was spray dried using a spray dryer. The dried powder was diluted in water according to concentration and used as the final raw material for the experiment.
1-4. 다양한 조합비의 흰 전나무를 포함하는 식물 유래 복합물 제조1-4. Preparation of plant-derived complexes containing white fir in various combination ratios
후술할 실험을 위해 비교예를 1 내지 4로 설정하였고 흰 전나무 및 식물유래 복합물의 최적 혼합비율을 도출하기 위한 실시예를 1 내지 9로 설정하여 실험을 실시하였다. 하기의 표 1은 드레곤헤드, 흰전나무, 오크라의 비교예를 나타내고, 표 2는 상기 식물유래 추출물의 최적 혼합비를 도출하기 위한 혼합 조성비를 나타낸다.For the experiment to be described later, the comparative examples were set to 1 to 4, and the examples were set to 1 to 9 to derive the optimal mixing ratio of white fir and plant-derived complexes and experiments were conducted. Table 1 below shows comparative examples of dragonhead, white fir, and okra, and Table 2 shows the mixing ratio for deriving the optimal mixing ratio of the plant-derived extract.
본 발명의 흰 전나무 추출물, 드레곤 헤드 추출물, 오크라 추출물을 포함하는 식물 유래 복합 조성물의 조성비는 드레곤헤드 0.5~1 중량부, 흰 전나무 1~3중량부, 오크라 1~6중량부로 이루어지는 것일 수 있다.The composition ratio of the plant-derived complex composition containing the white fir extract, dragon head extract, and okra extract of the present invention may be 0.5 to 1 part by weight of dragon head, 1 to 3 parts by weight of white fir, and 1 to 6 parts by weight of okra.
1-5. 통계처리1-5. Statistical processing
본 발명의 실험 결과는 SPSS package program(version 20.0, SPSS Inc., Chicago, IL, USA)을 이용하여 평균과 표준편차 를 구하였으며 실험군 간 차이의 유의성은 Tne-way ANOVA Multiple comparisons, Tukey test에 의하여 P<0.05 수준에서 검증하였다.For the experimental results of the present invention, the mean and standard deviation were calculated using the SPSS package program (version 20.0, SPSS Inc., Chicago, IL, USA), and the significance of the differences between experimental groups was determined by Tne-way ANOVA Multiple comparisons and Tukey test. It was verified at the P<0.05 level.
<실험예 2> 항산화 효과 확인<Experimental Example 2> Confirmation of antioxidant effect
상기 실험예 1에 따라 설정된 비교예 및 실시예의 식물유래 복합물의 항산화 활성을 확인하기 위해 DPPH 자유라디칼 소거 활성을 확인하는 실험을 수행하였다.To confirm the antioxidant activity of the plant-derived complexes of Comparative Examples and Examples set according to Experimental Example 1, an experiment was performed to confirm DPPH free radical scavenging activity.
상기 배합비에 따른 샘플은 여러농도 (31, 62, 125, 250, 500, 1000㎍/㎖)로 제조하여 실험에 사용되었으며 DPPH solution과 1:5 비율로 반응시켰다. 잘 섞어준 후 차광하여 실온에서 30분 반응시키고 microplate를 이용하여 517nm에서 흡광도를 측정하였다. 소거효과의 비교를 위한 positive control는 EGCG를 사용하였으며 계산식1에 대입하여 실시예의 산화억제 효과를 평가하였다.Samples according to the above mixing ratio were prepared at various concentrations (31, 62, 125, 250, 500, 1000㎍/㎖) and used in the experiment, and were reacted with DPPH solution in a 1:5 ratio. After mixing well, the mixture was shielded from light and reacted at room temperature for 30 minutes, and the absorbance was measured at 517 nm using a microplate. EGCG was used as a positive control for comparison of the scavenging effect, and the oxidation inhibition effect of the example was evaluated by substituting it into Calculation Equation 1.
[계산식 1][Calculation Formula 1]
Inhibition ratio of sample (%) :{(Acs-As)/Acs}*100Inhibition ratio of sample (%) :{(Acs-As)/Acs}*100
Acs: 샘플이 첨가되지 않은 DPPH 용액의 흡광도 As: 샘플과 DPPH가 반응한 흡광도Acs: Absorbance of DPPH solution without sample addition As: Absorbance of DPPH reaction with sample
하기의 표 3은 비교예 1 내지 3의 식물유래 복합물의 각 농도에 대한 DPPH 라디칼 소거능을 나타내고 표 4는 비교예 4 및 실시예 1 내지 9의 식물유래 복합물의 각 농도에 대한 DPPH 라디칼 소거능을 나타낸다.Table 3 below shows the DPPH radical scavenging ability for each concentration of the plant-derived complexes of Comparative Examples 1 to 3, and Table 4 shows the DPPH radical scavenging activity for each concentration of the plant-derived complexes of Comparative Example 4 and Examples 1 to 9. .
실험결과, 여러 농도의 세 가지 추출물 단독 실험 시 (비교예 1~3) 세 가지 군 모두 항산화 능력을 확인하였으며 비교예 2에서 가장 우수한 효능이 확인되었다. 이를 토대로 세 추출물의 시너지 효과가 가장 잘 나타날 수 있는 최적의 배합비 (실시예 1~9)를 연구하였으며 그 결과를 통상적인 배합비 (비교예 1)와 비교하였다. As a result of the experiment, when the three extracts at various concentrations were tested alone (Comparative Examples 1 to 3), the antioxidant ability of all three groups was confirmed, and the best efficacy was confirmed in Comparative Example 2. Based on this, the optimal mixing ratio (Examples 1 to 9) that can best demonstrate the synergistic effect of the three extracts was studied, and the results were compared with the typical mixing ratio (Comparative Example 1).
표 4의 결과에 따르면 실시예 1~9는 통상적인 배합비인 1:1:1 (비교예 4)에 비해 DPPH 소거활성이 높게 나타나는 것을 확인할 수 있었으며 특히 1:3:1 비율에서 가장 우수한 효능이 나타났으며 양성대조군으로 사용된 EGCG의 70~87%에 해당하는 수치이다. 또한 가장 효과가 좋은 실시예 8에서 DPPH radical이 50% 소거되는 농도 (IC50)는 250㎍/㎖로 확인되었다.According to the results in Table 4, it was confirmed that Examples 1 to 9 showed higher DPPH scavenging activity compared to the typical mixing ratio of 1:1:1 (Comparative Example 4), and in particular, the best efficacy was achieved at the 1:3:1 ratio. This figure corresponds to 70-87% of EGCG used as a positive control. In addition, in Example 8, which was the most effective, the concentration (IC 50 ) at which 50% of DPPH radicals were eliminated was confirmed to be 250 μg/ml.
<실험예 3> 식물유래 복합물의 당 독소 제거 효과 확인<Experimental Example 3> Confirmation of sugar toxin removal effect of plant-derived complex
3-1. 최종당화산물 (AGEs) 생성 억제 효과 확인3-1. Confirmation of the effect of inhibiting the production of advanced glycation end products (AGEs)
본 발명의 실험군은 상기 실험예 2에서 우수한 항산화 효과를 보인 상위 4개 배합비 (실시예 4, 5, 8, 9)와 비교예 2, 4를 비교하는 방식으로 진행하였으며 IC50 농도를 기준으로 효과가 나타날 것이라 예상되는 125, 250, 500㎍/㎖ 농도에 대해 실험을 실시하였다.The experimental group of the present invention was conducted by comparing the top four mixing ratios (Examples 4, 5, 8, and 9) that showed excellent antioxidant effects in Experimental Example 2 with Comparative Examples 2 and 4, and the effect was determined based on IC 50 concentration. Experiments were conducted at concentrations of 125, 250, and 500㎍/㎖, which were expected to appear.
당 독소 축적으로 발생되는 최종당화산물은 체내에서 노화를 촉진하고 만성염증을 일으켜 당뇨병, 비만, 심혈관 질환, 암 등 여러 만성질환을 일으키는 원인 물질이다. 본 실험예에서는 다양한 배합비의 식물유래 복합물의 최종당화산물 생성 억제에 미치는 효과를 확인하였다.Advanced glycation end products (ADGs), which are generated from the accumulation of sugar toxins, are the causative agent of various chronic diseases such as diabetes, obesity, cardiovascular disease, and cancer by accelerating aging in the body and causing chronic inflammation. In this experimental example, the effect of various mixing ratios of plant-derived complexes on inhibiting the production of advanced glycation end products was confirmed.
최종당화산물 생성 억제 실험은 Vinson&Howar법을 변형하여 진행하였다. 10mg의 BSA를 0.2M Phosphate buffer (pH 7.4)에 용해하여 10mg/ml농도의 BSA를 준비하였으며 반응기간 동안 박테리아 등의 오염을 방지하기 위해 0.02%(w/v) 소듐 아자이드(sodium azide)를 넣어주었다. The experiment to inhibit the production of advanced glycation end products was conducted by modifying the Vinson & Howar method. 10mg of BSA was dissolved in 0.2M Phosphate buffer (pH 7.4) to prepare BSA at a concentration of 10mg/ml, and 0.02% (w/v) sodium azide was added to prevent contamination by bacteria during the reaction period. I put it in.
BSA와 소듐 아자이드가 섞인 용액에 0.2M의 Glucose와 125, 250, 500㎍/㎖농도의 샘플 (비교예 2,4와 실시예4,5,8,9를 1:1:1 비율로 혼합하여 60℃에서 3일간 반응시킨 후, fluorescence spectrophotometer를 이용하여 반응 전후의 발광 정도(Excitation: 350nm, Emission: 450nm)를 측정하였다. Samples of 0.2M Glucose and concentrations of 125, 250, and 500㎍/ml in a solution of BSA and sodium azide (Comparative Examples 2 and 4 and Examples 4, 5, 8, and 9 mixed in a 1:1:1 ratio) After reacting at 60°C for 3 days, the degree of luminescence (Excitation: 350 nm, Emission: 450 nm) before and after the reaction was measured using a fluorescence spectrophotometer.
또한, 최종당화산물에 억제효과가 있다고 알려진 양성대조군으로 아미노구아니딘 (aminoguanidine, AG)을 사용하여 동일한 농도에서 효능을 비교하였다. 최종당화산물 억제율은 계산식 2에 대입하여 계산하였다.In addition, aminoguanidine (AG), which is known to have an inhibitory effect on advanced glycation end products, was used as a positive control and its efficacy was compared at the same concentration. The inhibition rate of advanced glycation end products was calculated by substituting equation 2.
[계산식 2][Calculation Formula 2]
Inhibition ratio of sample (%) : 100 - (As/Acs) * 100Inhibition ratio of sample (%): 100 - (As/Acs) * 100
Acs : 샘플이 첨가되지 않은 시료의 발광도 As : 샘플이 첨가된 시료의 발광도Acs: Luminescence of the sample without the sample added As: Luminescence of the sample with the sample added
음성대조군으로는 샘플을 넣지 않은 상태에서 BSA (10mg/ml)와 글루코스 (0.2M)만 반응시킨 시료를 사용하였다.As a negative control, a sample reacted with only BSA (10 mg/ml) and glucose (0.2M) was used without adding any sample.
하기의 표 5는 실험 조성물별 최종 당화산물 생성 억제율을 나타내고, 도 1은 실험예 3에 따른 식물 유래 복합물의 최종당화산물 생성억제효과를 나타낸다. Table 5 below shows the inhibition rate of advanced glycation end products for each experimental composition, and Figure 1 shows the effect of inhibiting advanced glycation end products of the plant-derived complex according to Experimental Example 3.
실험결과, 비교예 2, 4 그리고 실시예 4, 5, 8, 9 모두 당화가 유도된 군에 비해 유의하게 (p<0.05) 최종당화산물의 생성을 억제하였다. 흰 전나무 단독군인 비교예 2는 농도 의존적인 최종당화산물 생성 억제효과를 나타내었으며 3종 복합물은 비교예 2보다 탁월한 억제능을 가짐이 확인되었다. As a result of the experiment, Comparative Examples 2 and 4 and Examples 4, 5, 8, and 9 all significantly ( p <0.05) suppressed the production of advanced glycation products compared to the glycation-induced group. Comparative Example 2, which was a single white fir group, showed a concentration-dependent inhibitory effect on the production of advanced glycation end products, and the three-type composite was confirmed to have a superior inhibitory ability than Comparative Example 2.
이 수치는 비교예 2, 4에 비해 우수한 값으로 실시예 4, 5, 8, 9에서 시너지효과가 나타났음을 의미한다. 또한, 모든 실시예에서는 양성대조군으로 사용된 아미노구아니딘과 유사한 효과를 나타내었으며 특히 실시예 8은 양성대조군의 최종당화산물 억제율에 83~92%에 해당하는 우수한 효능을 보였다.This value is superior to Comparative Examples 2 and 4, meaning that a synergistic effect was observed in Examples 4, 5, 8, and 9. In addition, all examples showed similar effects to aminoguanidine used as the positive control, and in particular, Example 8 showed excellent efficacy of 83 to 92% in the inhibition rate of advanced glycation end products of the positive control.
3-2. α-글루코시데이즈 활성 효과 확인3-2. Confirmation of α-glucosidase activity effect
실험군은 상기 실험예 3-1과 동일하게 실시하였다. 당뇨병환자의 식후 급격한 혈당 상승을 억제하는 α-글루코시데이즈 억제제 작용기전은 인슐린 분비를 통하지않고 이당류 분해효소를 가역적으로 억제하여 장에서의 탄수화물 흡수를 지연시켜 식후 혈당을 감소시키고 인슐린 비의존성 당뇨병의 고혈당으로 인한 인슐린 분비 지연을 개선시킨다. 따라서, α-글루코시데이즈 억제 활성에 의하여 당뇨병 치료 활성을 시험할 수 있다. The experimental group was conducted in the same manner as in Experimental Example 3-1. The mechanism of action of α-glucosidase inhibitors, which suppress the rapid rise in blood sugar level after a meal in diabetic patients, is to reversibly inhibit disaccharide degrading enzymes rather than through insulin secretion, thereby delaying carbohydrate absorption in the intestines, thereby reducing postprandial blood sugar levels and reducing the risk of non-insulin-dependent diabetes. Improves delayed insulin secretion caused by high blood sugar. Therefore, diabetes treatment activity can be tested by α-glucosidase inhibitory activity.
알파-글루코시다아제 활성을 평가하기 위해 효소-기질반응을 이용한 분광학적 방법으로 측정하였다. 먼저, 96well plate에 농도별 비교예 2, 4와 실시예 4, 5, 8, 9를 각각 10μL씩 분주한 후 0.1M phosphate buffer에 용해한 알파글루코시다아제 (1U/ml) 100μL을 분주하여 실온에서 20분간 1차 반응을 시켜주었다. To evaluate alpha-glucosidase activity, it was measured spectroscopically using an enzyme-substrate reaction. First, 10 μL each of Comparative Examples 2 and 4 and Examples 4, 5, 8, and 9 by concentration were dispensed into a 96-well plate, and then 100 μL of alpha-glucosidase (1 U/ml) dissolved in 0.1 M phosphate buffer was dispensed at room temperature. The first reaction was performed for 20 minutes.
이 때 억제능을 비교할 수 있는 양성대조군으로는 아카보스를 사용하였으며 실시예와 동일한 농도로 처리해주었다. 여기에 기질로 사용된 1 mM 농도의 pNPG(p-nitrophenol glucoside) 100 μL을 가하여 37℃에서 10분간 2차 효소반응을 시킨 후, 기질로부터 유리되어 나오는 p-nitrophenol을 405 nm에서 흡광도를 측정하였다. 알파글루코시다아제 활성 저해율은 계산식 3에 대입하여 계산하였다.At this time, acarbose was used as a positive control to compare the inhibitory ability and was treated at the same concentration as in the example. Here, 100 μL of 1 mM pNPG (p-nitrophenol glucoside) used as a substrate was added and a secondary enzyme reaction was performed at 37°C for 10 minutes. The absorbance of p-nitrophenol liberated from the substrate was measured at 405 nm. . The inhibition rate of alpha-glucosidase activity was calculated by substituting it into equation 3.
[계산식 3][Calculation Formula 3]
저해율 (%) = (시료 첨가군 -대조구 첨가군)/대조구 첨가군 * 100Inhibition rate (%) = (sample addition group - control addition group)/control addition group * 100
하기의 표 6은 실험 조성물별 알파 글루코시다아제 활성 억제율을 나타내고, 도 2는 실험예 3에 따른 식물유래 복합물의 알파 글루코시다아제 활성 억제효과를 나타낸다.Table 6 below shows the inhibition rate of alpha-glucosidase activity by experimental composition, and Figure 2 shows the alpha-glucosidase activity inhibition effect of the plant-derived complex according to Experimental Example 3.
아무것도 처리하지 않은 음성대조군에서의 효소활성을 100%로 기준잡아 실험 샘플의 억제능을 평가하였다. 실험결과, 양성대조군인 아카보스를 투여한 군에서는 농도의존적으로 71~92%의 억제효과가 나타났으며 비교예 2, 4와 실시예 4, 5, 8, 9에서도 유의한 (p<0.05) 억제 효과가 관찰되었다. The inhibitory ability of the experimental samples was evaluated by setting the enzyme activity in the untreated negative control group to 100%. As a result of the experiment, the group administered acarbose, which was the positive control group, showed an inhibitory effect of 71 to 92% in a concentration-dependent manner, and significant ( p < 0.05) inhibition was also observed in Comparative Examples 2 and 4 and Examples 4, 5, 8, and 9. The effect was observed.
비교예 2 125㎍/㎖ 우수한 효과를 보이진 못했지만 농도 의존적으로 억제효과가 증가하였으며 실시예 4, 5, 8, 9는 통상적인 배합비인 비교예 4에 비해 우수한 효능을 보였다. 다른 실험예에서와 마찬가지로 실시예 3에서의 효능이 가장 탁월하였으며 그 외 배합비에서도 시너지 효과가 일어났음을 유추할 수 있다. 다양한 배합비의 식물유래 복합물이 치료제로 사용되는 아카보스와 유사한 효능을 갖는만큼 천연물 유래의 혈당강하제 소재로 활용될 수 있을 것으로 예상된다.Comparative Example 2 Although 125㎍/㎖ did not show an excellent effect, the inhibitory effect increased in a concentration-dependent manner, and Examples 4, 5, 8, and 9 showed excellent efficacy compared to Comparative Example 4, which was a typical mixing ratio. As in other experimental examples, the efficacy in Example 3 was the most excellent, and it can be inferred that a synergistic effect occurred in other mixing ratios. As plant-derived complexes of various mixing ratios have similar efficacy to acarbose, which is used as a treatment, it is expected that they can be used as a material for hypoglycemic agents derived from natural products.
<실험예 4> 식물유래 복합물의 알코올에 의해 유도된 간 독소 제거 효과 확인<Experimental Example 4> Confirmation of the effect of plant-derived complexes on removing liver toxins induced by alcohol
4-1. 식물유래 복합물이 간세포 생존율에 미치는 영향4-1. Effect of plant-derived compounds on hepatocyte survival rate
실험 조성물은 상기 실험예 3과 동일하게 실시하였다. 흰 전나무를 포함하는 식물유래 복합물이 간세포에 미치는 영향을 확인하기 위해 인간간암 세포주인 HepG2를 이용하였다. HepG2 세포는 10% FBS와 1%의 항생제(100 U/ml penicillin streptomycin)가 포함된 DMEM 배지(Dulbecco's Modified Eagle Medium)에서 5% CO2, 37℃ 조건에서 배양하였다. The experimental composition was performed in the same manner as in Experimental Example 3 above. To confirm the effect of plant-derived compounds containing white fir on hepatocytes, HepG2, a human liver cancer cell line, was used. HepG2 cells were cultured in DMEM medium (Dulbecco's Modified Eagle Medium) containing 10% FBS and 1% antibiotic (100 U/ml penicillin streptomycin) at 5% CO 2 and 37°C.
96well에 HepG2를 5 * 103 cells/well 농도로 분주한 후 24시간 배양하여 세포를 부착하였다. 이후 농도별 식물유래 복합물을 처리하여 24시간 동안 배양하였고 정확한 생존율 측정을 위해 PBS로 3번 세척하였다. 그 후 0.5mg/ml의 MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) 용액을 100㎕ 분주하여 2시간 동안 배양한 후 DMSO를 분주하여 결정화된 formazan crystal을 용해하였다. 용해된 formazan의 흡광도는 570nm에서 측정하였다. 각 군의 세포 생존율은 아무것도 처리하지 않은 대조군을 100%로 간주하여 샘플 처리군의 생존율(viability)를 퍼센트로 표시하였다.HepG2 was dispensed into 96 wells at a concentration of 5 * 10 3 cells/well and then cultured for 24 hours to attach the cells. Afterwards, plant-derived complexes of various concentrations were treated, cultured for 24 hours, and washed three times with PBS to accurately measure survival rate. After that, 100㎕ of 0.5mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution was dispensed and incubated for 2 hours. DMSO was then added to form the crystallized formazan crystal. was dissolved. The absorbance of dissolved formazan was measured at 570 nm. The cell viability of each group was expressed as a percentage, assuming that the untreated control group was 100%.
도 3은 본 발명의 실험예 4에 따른 식물유래 복합물이 간세포 생존율을 나타낸다. 실험결과, 다양한 농도의 식물유래 복합물은 간세포 생존율에 영향을 미치지 않는 안전한 농도임을 확인하였다.Figure 3 shows the hepatocyte survival rate of the plant-derived complex according to Experimental Example 4 of the present invention. As a result of the experiment, it was confirmed that various concentrations of plant-derived complexes were safe concentrations that did not affect the survival rate of hepatocytes.
4-2. 식물유래 복합물이의 알코올 처리된 간세포 생존율에 미치는 영향4-2. Effect of plant-derived complex on survival rate of alcohol-treated hepatocytes
흰 전나무를 포함하는 식물유래 복합물이 간세포에 미치는 영향을 확인하기 위해 인간간암 세포주인 HepG2를 이용하였다. HepG2 세포는 10% FBS와 1%의 항생제(100 U/ml penicillin streptomycin)가 포함된 DMEM 배지(Dulbecco's Modified Eagle Medium)에서 5% CO2, 37℃ 조건에서 배양하였다. To confirm the effect of plant-derived compounds containing white fir on hepatocytes, HepG2, a human liver cancer cell line, was used. HepG2 cells were cultured in DMEM medium (Dulbecco's Modified Eagle Medium) containing 10% FBS and 1% antibiotic (100 U/ml penicillin streptomycin) at 5% CO 2 and 37°C.
알코올성 간 손상 유도를 위해 24시간 동안 96well에 배양된 HepG2 세포에 0.4M의 ethyl alchol을 첨가하였고 식물유래 복합물의 간 세포 보호 효과를 확인하기 위해 농도별 샘플을 함께 처리하였다. To induce alcoholic liver damage, 0.4M ethyl alcohol was added to HepG2 cells cultured in 96 wells for 24 hours, and samples of each concentration were processed together to confirm the liver cell protection effect of the plant-derived complex.
효과를 비교하기 위한 간 세포 손상 유도군은 샘플대신 DW를 분주하였고 효과를 비교하기 위한 양성대조군으로는 일반적으로 시판되는 밀크시슬 원료를 사용하였다. 24시간 배양 후 0.5mg/ml의 MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)용액을 100㎕분주하여 2시간 동안 배양하였으며 DMSO를 분주하여 결정화된 formazan crystal을 용해하였다. 용해된 formazan의 흡광도는 570nm에서 측정하였다. 간 세포 손상이 유도되지 않은 군의 생존율을 100%로 간주하고 각 군의 세포 생존율(viability)를 퍼센트로 표시하였다.For the liver cell damage inducing group to compare the effect, DW was dispensed instead of the sample, and for the positive control group to compare the effect, commonly commercially available milk thistle raw materials were used. After 24 hours of incubation, 100 ㎕ of 0.5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution was dispensed and incubated for 2 hours. DMSO was added to crystallize the formazan. The crystal was dissolved. The absorbance of dissolved formazan was measured at 570 nm. The survival rate of the group in which liver cell damage was not induced was considered 100%, and the cell viability of each group was expressed as a percentage.
도 4는 본 발명의 실험예 4에 따른 식물유래 복합물이 알코올에 의해 손상이 유도된 간세포 보호 효과를 나타낸다. 실험결과, HepG2 세포에 0.4M의 알코올을 처리하였을 경우, 아무것도 처리하지 않은 대조군의 대비 약 60%의 생존율을 보였지만 농도별 식물유래 복합물을 처리하여 함께 배양할 경우 농도의존적으로 손상이 줄어 생존율이 증가함을 확인할 수 있었다. Figure 4 shows the protective effect of the plant-derived complex according to Experimental Example 4 of the present invention on liver cells damaged by alcohol. As a result of the experiment, when HepG2 cells were treated with 0.4M alcohol, the survival rate was about 60% compared to the untreated control group. However, when treated with plant-derived complexes at different concentrations and cultured together, the damage was reduced in a concentration-dependent manner and the survival rate increased. It was confirmed that this was the case.
또한 고농도의 식물유래 복합물은 동일 농도의 양성대조군과 유사한 세포 보호효과를 가짐이 확인되었다. 이를 통해, 식물유래 복합물은 알코올로 인한 독성으로부터 HepG2 세포를 보호한다는 것을 확인할 수 있다.In addition, it was confirmed that high concentrations of plant-derived complexes had a cell protection effect similar to that of the positive control group at the same concentration. Through this, it can be confirmed that the plant-derived complex protects HepG2 cells from toxicity caused by alcohol.
4-3. Aldehyde Dehydrogenase 활성 촉진 효과 확인4-3. Confirmation of the effect of promoting Aldehyde Dehydrogenase activity
알코올에 의한 간 독소 제거에 필수적인 알데하이드 탈수소 효소 (Aldehyde Dehydrogenase, ALDH)는 아세트알데하이드를 아세트산으로 산화시켜주는 역할을 한다. 이 때 NAD+ 한 분자를 환원시켜 NADH양이 증가하게 되는데 시중에 판매되는 ALDH 활성을 측정하는 키트들은 이 원리를 이용한다.Aldehyde dehydrogenase (ALDH), which is essential for removing liver toxins caused by alcohol, oxidizes acetaldehyde to acetic acid. At this time, the amount of NADH increases by reducing one molecule of NAD+, and commercially available kits that measure ALDH activity use this principle.
본 실험에서는 ALDH 활성을 측정하기 위해 sigma사의 Aldehyde Dehydrogenase activity colorimetric assay kit을 활용하였다. 우선, HepG2 세포는 10% FBS와 1%의 항생제(100 U/ml penicillin/streptomycin)가 포함된 DMEM 배지(Dulbecco's Modified Eagle Medium)에서 5% CO2, 37℃ 조건에서 배양하였다. 배양 시 다양한 농도의 비교예, 실험예을 첨가하였으며 효과를 비교하기 위한 음성대조군으로는 동량의 DW를, 양성대조군으로는 동량의 시판되는 밀크시슬 원료를 이용하였다. In this experiment, Sigma's Aldehyde Dehydrogenase activity colorimetric assay kit was used to measure ALDH activity. First, HepG2 cells were cultured in DMEM medium (Dulbecco's Modified Eagle Medium) containing 10% FBS and 1% antibiotics (100 U/ml penicillin/streptomycin) at 5% CO 2 and 37°C. During cultivation, various concentrations of comparative and experimental examples were added, and the same amount of DW was used as a negative control group to compare the effects, and the same amount of commercially available milk thistle raw material was used as a positive control group.
배양 후 kit에서 제공되는 Assay buffer를 통해 회수 및 균질화 과정을 거쳤으며 13,000rpm에서 5분간 원심분리하여 실험에 적용할 샘플을 회수하였다. kit제조사에서 제공한 배합리로 혼합한 assay buffer, ALDH 기질, 아세트알데하이드 mix 50㎕에 샘플 50㎕을 혼합하여 실온에서 5분 반응 후 매2~3분간 450nm에서 흡광도를 측정하였다. ALDH 활성은 계산식 4에 대입하여 계산하였으며 대조군의 활성을 100으로 기준잡아 %로 환산하여 표7, 도5에 나타내었다.After incubation, recovery and homogenization were performed using the assay buffer provided in the kit, and samples to be applied to the experiment were recovered by centrifugation at 13,000 rpm for 5 minutes. 50 μl of the sample was mixed with 50 μl of the assay buffer, ALDH substrate, and acetaldehyde mix provided by the kit manufacturer, reacted for 5 minutes at room temperature, and then absorbance was measured at 450 nm every 2 to 3 minutes. ALDH activity was calculated by substituting equation 4, and the activity of the control group was converted to % by taking 100 as the standard and shown in Table 7 and Figure 5.
[계산식 4][Calculation Equation 4]
ALDH activity (%) = B / △T * V * 희석배율 = nmol/min/ml = mU/mlALDH activity (%) = B / △T * V * dilution ratio = nmol/min/ml = mU/ml
B: 실험군에서 발생한 NADH 양(nmol) △T: 반응 시간(분)B: Amount of NADH generated in the experimental group (nmol) △T: Reaction time (minutes)
V: 반응 웰에서 사용된 실험군 부피(ml)V: experimental group volume (ml) used in reaction wells
하기의 표 7은 실험 조성물별 ALDH 효소 활성율을 나타내었고, 표 8은 각각의 실험군간 통계결과를 나타내었다. 실험군간 유의한 차이가 있으면 공란으로, 유의한 차이가 없다면 Not significant (NS)로 표시하였다. 도 5는 식물유래 복합물의 ALDH 활성효과를 나타낸다.Table 7 below shows the ALDH enzyme activity rate for each experimental composition, and Table 8 shows statistical results between each experimental group. If there was a significant difference between the experimental groups, it was marked as blank, and if there was no significant difference, it was marked as Not significant (NS). Figure 5 shows the ALDH activity effect of plant-derived complexes.
실험결과, 비교예 2, 4에서는 음성대조군 대비 38~62%의 활성 촉진 효과를확인하였으며 양성대조군 역시 64~79%의 우수한 활성 촉진능을 보였다. 실시예 4,5,8,9의 경우, 모두 음성대조군 대비 농도의존적 유의성 (p<0.05)을 보였으며 실시예 8의 경우, 모든 실시예 중 가장 우수한 효능을 보이며 양성대조군에 웃도는 효과를 확인하였다. 이는 흰전나무 추출물을 포함하는 식물유래 복합물이 간 독성 개선 효과가 있음을 시사하며 시판되는 타 원료대비 우수한 효능을 가짐을 확인하였다. As a result of the experiment, Comparative Examples 2 and 4 confirmed an activity-promoting effect of 38-62% compared to the negative control group, and the positive control group also showed an excellent activity-promoting effect of 64-79%. In the case of Examples 4, 5, 8, and 9, all showed concentration-dependent significance ( p < 0.05) compared to the negative control group, and in the case of Example 8, the efficacy was confirmed to be superior to that of the positive control group, showing the best efficacy among all examples. . This suggests that the plant-derived complex containing white fir extract has an effect on improving liver toxicity, and was confirmed to have superior efficacy compared to other commercially available raw materials.
본 발명에서는 DPPH 라디칼 소거능, 체내 최종당화산물 생성 억제, 알파 글루코시다아제 활성억제, 알코올에 의해 손상된 간세포 보호, 알코올 분해효소 활성 테스트를 통해 흰 전나무 추출물 및 흰 전나무 추출물을 포함하는 식물유래 복합물의 체내독소 축적 억제 및 제거 효과를 확인하였다. 이를 토대로 천연물질 유래의 부작용이 적은 디톡스 효능을 갖는 건강기능식품 조성물 및 이를 활용한 건강기능식품의 제공이 가능하다.In the present invention, white fir extract and plant-derived complexes containing white fir extract are tested for DPPH radical scavenging ability, inhibition of advanced glycation end product production in the body, inhibition of alpha-glucosidase activity, protection of liver cells damaged by alcohol, and alcohol degrading enzyme activity in the body. The effect of suppressing and removing toxins was confirmed. Based on this, it is possible to provide a health functional food composition with a detox effect with fewer side effects derived from natural substances and a health functional food using the same.
5. 흰 전나무 추출물을 포함하는 건강기능성 식품제형물 제조5. Manufacture of health functional food formulation containing white fir extract
흰 전나무 추출물을 유효성분으로 함유하는 알코올에 의한 간 독소 축적 억제 및 제거를 위한 해독작용을 갖는 조성물은 정제 및 캡슐제, 연질 캡슐제, 과립제, 액제 형태로 제조될 수 있다. 도 7은 흰 전나무 추출물을 이용한 건강기능성 식품 제형물로 제조된 정제형을 나타낸다. A composition containing white fir extract as an active ingredient and having a detoxifying effect for suppressing and removing alcohol-induced liver toxin accumulation can be manufactured in the form of tablets, capsules, soft capsules, granules, and liquid. Figure 7 shows a tablet manufactured as a health functional food formulation using white fir extract.
본 발명의 실시예에 따르면, 상기 조성물을 포함하는 체내 독소 축적억제 및 독소 제거용 건강기능성 식품일 수 있다. 상기 건강기능식품은 상기 조성물이 0.01 내지 99.9중량%로 포함되도록 산제, 과립제, 정제, 캡슐제, 환제, 현탁액, 에멀젼, 시럽의 약학 투여형태 또는 티백제, 침출차, 건강 음료의 형태인 것일 수 있다.According to an embodiment of the present invention, it may be a health functional food for inhibiting the accumulation of toxins in the body and removing toxins containing the composition. The health functional food may be in the form of a pharmaceutical dosage form of powder, granule, tablet, capsule, pill, suspension, emulsion, or syrup, or in the form of tea bag, leached tea, or health drink so that the composition contains 0.01 to 99.9% by weight. .
본 발명은 천연물을 원료로 하는 식물 유래 추출물 및 복합 추출물을 제공하여 부작용이 거의 없는 알코올에 의한 간 독소 축적 억제 및 제거용 건강기능 식품 조성물 및 건강기능 식품을 제공하여 건강에 관심도가 높은 현대인의 수요에 맞춰 경쟁력이 있으므로 산업상 이용가능성이 있다.The present invention provides plant-derived extracts and complex extracts made from natural products, and provides a health functional food composition and health functional food for suppressing and removing liver toxins caused by alcohol with almost no side effects, which is in demand by modern people with a high interest in health. Since it is competitive, it has the potential for industrial use.
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