KR101338210B1 - The evaluation method of astringent activity using protein quantification - Google Patents
The evaluation method of astringent activity using protein quantification Download PDFInfo
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Abstract
본 발명은 피부에 대한 수렴활성을 측정하는 방법에 관한 것이다. 탄닌-헤모글로빈 결합반응에서 단백질 정량 시험을 추가하여 만든 시험법으로 오차가 적고 재현성이 나타나는 것을 장점으로 한다.The present invention relates to a method for measuring astringent activity on the skin. This test is made by adding protein quantitative test in tannin-hemoglobin binding reaction. It has the advantage of small error and reproducibility.
Description
본 발명은 수렴활성 평가방법에 관한 것으로, 더욱 상세하게는 탄닌과 헤모글로빈 반응에서 단백질 정량을 이용한 수렴활성 평가법에 관한 것이다. 본 발명의 평가방법은, 시험관 내(in vitro) 수렴활성 측정으로 시험 조작이 간단하여 피부에 대한 수렴활성을 빠르게 확인할 수 있으며 원료 스크리닝 작업에서 시간과 비용을 절감할 수 있고, 생체 내(in vivo) 시험법으로 측정하기 전에 미리 활성을 예견할 수 있다.
The present invention relates to a method for assessing astringent activity, and more particularly, to a method for assessing astringent activity using protein quantification in tannin and hemoglobin reactions. In the evaluation method of the present invention, it is possible to quickly confirm the astringent activity on the skin by simple in-vitro convergence activity measurement, reduce the time and cost in the raw material screening operation, and in vivo The activity can be predicted in advance before measuring by the assay.
수렴작용의 원리는 피부 단백질이 고분자 플라보노이드와 결합하여 교차결합을 형성하여 피부가 수축되는 현상이다. 즉 주름짐이나 수축이다. 수렴활성이 있는 물질은 대개 단백질과 결합하는 성질이 있다. 수축작용은 피부와 점막 혈관 등을 수축시키고 세포 간극 및 림프 간극의 점액의 분비를 억제하며 피부와 점막의 표면에 난용성 피막을 형성하여 국소를 보호하고 조직을 조밀하게 하여 탄력을 유지하게 해 주는 역할을 한다. 또한 세포막 투과성을 감소시키는 기능도 한다. The principle of astringent action is the phenomenon that the skin protein is combined with the polymer flavonoids to form a cross-linking and shrink the skin. That is, wrinkles or shrinkage. Materials with astringent activity usually have a property of binding to proteins. Contractile action contracts skin and mucous membranes, inhibits the secretion of mucus in cell and lymph gaps, and forms sparingly soluble coatings on the skin and mucous membranes to protect local areas and keep tissues tight and elastic. Play a role. It also functions to reduce cell membrane permeability.
이러한 두드러진 특정을 측정하기 위해 탄닌-단백질 복합체 개념을 이해하고 단백질 정량법을 도입함으로써 안정성을 가지고 재현성을 가지는 시험법이 필요하다.To measure this prominent specificity, we need a test that is stable and reproducible by understanding the concept of tannin-protein complexes and introducing protein quantitation.
시험의 오차를 줄이고 재현성을 유도하고 통계적 유의 수준을 확인하기 위해 양성대조군으로 탄닌과 헤모글로빈의 농도 증가에 따른 수렴활성을 구하였고 시료 3종을 이용하여 수렴활성을 측정하여 수렴 활성 시험법으로서의 가능성을 검토하였다.In order to reduce the error of the test, induce reproducibility, and confirm the statistical significance level, the convergence activity was determined by increasing the concentration of tannin and hemoglobin as a positive control group, and the convergence activity was measured using three samples. Reviewed.
즉, 본 발명에서는 In vitro 수렴 활성의 문제점을 개선하고자 시험법에 단백질 정량법을 도입하였다.That is, in the present invention, protein quantification was introduced to the test method to improve the problem of in vitro astringent activity.
본 시험법은 신뢰 범위 내의 광학 밀도(optical density)를 측정할 수 있고, 모든 농도에서 시험 가능하다. 또한 재현성이 확인되어 시험 결과에 대한 신뢰도가 있었으며 통계적 유의 수준에 있음을 확인하였다.
The test method can measure optical density within the confidence range and can be tested at all concentrations. In addition, the reproducibility was confirmed, and there was a confidence in the test result and the statistical significance level was confirmed.
도 1은 본 발명의 시험법의 탄닌-헤모글로빈 복합물의 수렴활성 그래프(**p<0.01, ***p<0.001).
도 2는 본 발명의 시험법의 탄닌-시료 복합물의 수렴활성 그래프(MTFE( fruit of Maximowiczia typica extract): 오미자 추출물, ACE(Artemisia capillaries extract): 인진쑥 추출물, AP(acorn powder): 도토리 분말, TA( tannic acid): 탄닌산. 탄닌산의 EC50은 26 uM. **p<0.01, ***p<0.001).
도 3은 기존 시험법의 탄닌-헤모글로빈 복합물의 수렴활성 그래프(*p<0.05, ***p<0.001).1 is a graph of the convergence activity of the tannin-hemoglobin complex of the test method of the present invention (** p <0.01, *** p <0.001).
Figure 2 is a graph of the astringent activity of the tannin-sample complex of the present invention (MTFE (fruit of Maximowiczia typica extract): Schisandra chinensis extract, ACE (Artemisia capillaries extract): jinjin mugwort extract, AP (acorn powder): acorn powder, TA tannic acid: Tannin acid, EC 50 of tannic acid is 26 uM. ** p <0.01, *** p <0.001).
Figure 3 is a graph of the convergence activity of the tannin-hemoglobin complex of the conventional test method (* p <0.05, *** p <0.001).
수렴효과는 모공과 땀구멍 수축에 의한 것으로 탄력과 유연성을 제공해 준다. 수렴은 일시적인 수축작용이며, 탄닌과 같이 응고 단백질에 의한 화학적 작용과 차가운 물이나 에탄올 휘발에 의해서 피부 온도를 낮추는 물리적 작용으로 구분할 수 있다. 떫은 감을 먹을 때 느끼는 것이 수렴인데, 피부에서의 작용은 수렴물질이 단백질과 결합하여 조직을 수축시키는 것이다. 탄닌은 섬유 사이사이의 수분을 제거해 주고 섬유결합을 촉진시킨다. 일반적으로 탄닌 유래 물질과 효소의 결합은 단백질-탄닌 복합체를 형성하며 이는 아미노 결합과 수소결합에 의해 만들어진다. 탄닌의 페놀기가 순서대로 단백질과 교차 결합하는데 이들 교차 결합된 단백질이 수렴 활성인자로 작용하게 된다.The convergence effect is caused by shrinkage of pores and pores, providing elasticity and flexibility. Convergence is a temporary contraction action, which can be divided into chemical action by coagulation protein such as tannin and physical action by lowering skin temperature by cold water or ethanol volatilization. When persimmons eat persimmons, convergence is the effect on the skin. Converging substances bind proteins and constrict tissue. Tannins remove moisture between the fibers and promote fiber binding. In general, the combination of tannin-derived substances and enzymes forms protein-tannin complexes, which are made by amino and hydrogen bonds. The phenolic groups of tannins cross-link with proteins in order, and these cross-linked proteins act as convergent activators.
탄닌과 헤모글로빈의 결합을 이용하여 수렴활성을 측정할 수 있는데 드물지만 현재까지 사용되고 있는 시험법도 두 물질 사이에서 일어나는 반응 차를 UV 분광광도계로 측정하여 활성을 계산하고 있다. 하지만 이 방법은 문제점이 있다. 첫 째, 광학 밀도가 신뢰 범위 이상인 1.5이상으로 나오는데, 신뢰 범위 이상이기 때문에 결과에 대한 신뢰도가 없다. 둘 째는 첫 번째의 문제로 인해 시료의 고농도 시험이 불가하다는 점이다. 저농도에서는 시료의 일부가 가능하지만 농도를 증가시키면 다시 광학 밀도가 높아져서 정확한 활성을 얻을 수 없다. 세 번째의 문제점은 측정 시마다 결과값의 오차가 높다는 점이다. 재현성이 없으므로 결과에 대한 신뢰도가 미약하다. Convergence activity can be measured using a combination of tannins and hemoglobin. However, the test method used up to now calculates the activity by measuring the difference in reaction between the two substances with a UV spectrophotometer. However, this method has a problem. First, the optical density comes out above 1.5, which is above the confidence range, and there is no confidence in the result because it is above the confidence range. Second, the first problem prevents high concentration testing of the sample. At low concentrations, a portion of the sample is possible, but increasing the concentration again increases the optical density, resulting in inaccurate activity. The third problem is that the error of the result is high at every measurement. Since there is no reproducibility, the reliability of the result is weak.
이러한 문제점을 보완하기 위해 단백질 정량법을 도입하였다. 수렴반응이 일어나면 침전물이 생기는데 이 침전물을 제외한 상층액, 즉 비반응 단백질을 정량하여 수렴활성을 얻을 수 있다. 방법은 다음과 같다.
To quantify this problem, protein quantitation was introduced. When a convergence reaction occurs, a precipitate is formed. The supernatant except this precipitate, that is, non-reacted protein can be quantified to obtain astringent activity. The method is as follows.
실험방법Experimental Method
1. 0.1%의 탄닌과 0.05%의 헤모글로빈을 동량으로 혼합한 후 2500 rpm에서 5초간 vortex한다. 1. Mix equal amounts of 0.1% tannin and 0.05% hemoglobin and vortex for 5 seconds at 2500 rpm.
2. 20 ℃에서 5분간 정치시킨 후 4 ℃에서 10분간 원심 분리하여 상층액을 얻는다. 이 상층액은 헤모글로빈과 탄닌이 반응하지 않은 부분이다. 2. After standing at 20 ° C. for 5 minutes, centrifugation at 4 ° C. for 10 minutes gives a supernatant. This supernatant is the part where hemoglobin and tannin did not react.
3. 총 단백질과 헤모글로빈양에서 비반응, 즉 상층액을 빼주면 반응한 단백질이 계산되고 이 결과가 수렴활성이 된다. 수식은 다음과 같다.
3. The total amount of protein and hemoglobin is unreacted, that is, subtracting the supernatant, the reacted protein is calculated and the result is astringent activity. The formula is as follows.
[ 1- [ One- 비반응Non-response 단백질 / (헤모글로빈 + 전체 단백질) ] x 100 = 수렴활성 (%) Protein / (hemoglobin + total protein)] x 100 = astringent activity (%)
[ ] 부분은 반응한 단백질로 단백질 침전물을 형성하게 된다.
The [] part is the reacted protein, which forms a protein precipitate.
도 1은 본 발명의 시험법의 탄닌-헤모글로빈 복합물의 수렴활성 그래프를 나타낸 것이고, 도 2는 본 발명의 시험법의 탄닌-시료 복합물의 수렴활성 그래프를 나타낸 것이다. 그리고, 도 3은 기존 시험법의 탄닌-헤모글로빈 복합물의 수렴활성 그래프를 나타낸 것이다. 탄닌 농도에 따른 수렴활성의 오차값이 크고, 광학밀도가 높아서 계산 되어진 수렴활성 값이 100 %이상으로 나타나는데, 실제 이 값은 광학밀도가 1.5이상으로 의미가 없다고 할 수 있다.
1 shows a graph of the convergence activity of the tannin-hemoglobin complex of the test method of the present invention, Figure 2 shows a graph of the convergence activity of the tannin-sample complex of the test method of the present invention. And, Figure 3 shows a graph of the convergence activity of the tannin-hemoglobin complex of the conventional test method. The error value of convergence activity according to the tannin concentration is high and the optical density is high, and the calculated value of convergence activity appears to be 100% or more. Actually, this value is meaningless as 1.5 or more.
본 시험법은 신뢰 범위 내의 광학 밀도(optical density)를 측정할 수 있고, 모든 농도에서 시험 가능하다. 또한 재현성이 확인되어 시험 결과에 대한 신뢰도가 있었으며 통계적 유의 수준에 있음을 확인하였다.The test method can measure optical density within the confidence range and can be tested at all concentrations. In addition, the reproducibility was confirmed, and there was a confidence in the test result and the statistical significance level was confirmed.
Claims (4)
0.1% tannin and 0.05% hemoglobin were mixed in the same amount, rotated at 2500 rpm for 5 seconds, left at 20 ° C for 5 minutes, centrifuged at 4 ° C for 10 minutes, and the ratio of the total protein and the reacted protein in hemoglobin Converging activity evaluation method characterized by calculating the convergence activity by.
[ 1- 비반응 단백질 / (헤모글로빈 + 전체 단백질) ] x 100 = 수렴활성 (%)
The method of evaluating convergence activity according to claim 1, wherein the convergence activity is obtained by the following formula:
[1- unreacted protein / (hemoglobin + total protein)] x 100 = astringent activity (%)
4. The method of claim 3, wherein the unreacted protein is the amount of supernatant centrifuged after the binding reaction.
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| JP2003083962A (en) * | 2001-09-12 | 2003-03-19 | T Hasegawa Co Ltd | Method for screening skin tightening substance |
| EP1990617A1 (en) * | 2007-05-09 | 2008-11-12 | FOSS Analytical A/S | Determination of tannins in vinefication products |
| US7785889B2 (en) * | 2001-03-27 | 2010-08-31 | Sapporo Breweries Limited | Food astringency evaluating method using lipid membrane sensors with immobilized peptides |
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| US7785889B2 (en) * | 2001-03-27 | 2010-08-31 | Sapporo Breweries Limited | Food astringency evaluating method using lipid membrane sensors with immobilized peptides |
| JP2003083962A (en) * | 2001-09-12 | 2003-03-19 | T Hasegawa Co Ltd | Method for screening skin tightening substance |
| EP1990617A1 (en) * | 2007-05-09 | 2008-11-12 | FOSS Analytical A/S | Determination of tannins in vinefication products |
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