KR20090035274A - Composition comprising paeonia lactiflora and gastrodia elata extracts as an effective component - Google Patents
Composition comprising paeonia lactiflora and gastrodia elata extracts as an effective component Download PDFInfo
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Abstract
Description
본 발명은 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물 및 이를 포함하여 구성되는 인지개선용 기능성 식품과 주름개선용 기능성 화장품에 관한 것이다.The present invention relates to a composite composition comprising peony and cheonma extract as an active ingredient, and functional food for cognitive improvement and functional cosmetic for wrinkle improvement comprising the same.
최근 인구구조가 고령화되면서 세포의 노화와 연관된 퇴행성 신경질환의 하나로 노인성 치매의 발병이 증가하고 있다. 그러나 이를 치료하기 위하여 판매되고 있는 뇌신경 치료제의 경우 대부분 합성품 유래로서, 효능이 낮거나 부작용이 일어나는 등 뚜렷한 효과를 얻지 못하여 일정 규모 이상의 시장 규모가 형성되지 않는 경우가 많다. 이에 반하여 퇴행성 신경질병에 대한 치료제 개발은 국민 보건 복지상 시급한 과제라고 할 수 있다.With the recent aging of the population, the development of senile dementia is one of the degenerative neurological diseases associated with cell aging. However, most of the neurosurgery drugs sold for the treatment are derived from synthetic products, and there are many cases in which the market size is not formed more than a certain size because the effect is low and the side effects are not obtained. On the other hand, the development of treatment for neurodegenerative diseases is an urgent task for the public health welfare.
한편, 피부 노화는 크게 내인성 노화와 광노화(photoaging)로 나눌 수 있는 데, 이중 광노화 현상은 자외선의 노출을 피하면 예방할 수 있는 피부 노화 현상이다. 그러나 현재 자외선을 차단하여 피부노화를 억제하는 기능성 화장품의 대부분은 그 원료를 수입에 의존하고 있어, 이로 인한 비용 지출이 큰 부분을 차지하고 있다.On the other hand, skin aging can be largely divided into endogenous aging and photoaging, double photoaging is a skin aging that can be prevented by avoiding exposure to ultraviolet light. However, at present, most of the functional cosmetics that block ultraviolet rays to prevent skin aging depend on imports of the raw materials, and the cost is large.
그러나 천연한방 식물인 작약은 고부가가치의 약용식물로써, 그 주성분인 페오니플로린(paeoniflorin)은 중추억제 작용, 혈관확장 작용, 항산화 작용 등의 효능이 있는 것으로 알려져 있다. 아울러 천마의 경우 풍증의 치료 또는 어혈 증상의 개선을 목적으로 널리 사용되어 온 생약재로서, 근래에 재배방법이 개발되어 대량으로 생산할 수 있는 기반이 마련되어 있는 실정이다.However, peony, a natural herbaceous plant, is a high-value medicinal plant, and its main ingredient, peoniflorin, is known to have a central inhibitory effect, vasodilation, and antioxidant activity. In addition, in the case of cheonma as a herbal medicine that has been widely used for the treatment of bouts of affairs or to improve the blood symptoms, the cultivation method has been developed in recent years has a foundation that can be produced in large quantities.
상기와 같은 문제점을 해결하기 위하여 창작된 본 발명의 목적은 다음과 같다.An object of the present invention created to solve the above problems is as follows.
첫째, 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물의 항산화 효과와 작용 기전을 규명하고, 이를 이용하여 부작용의 위험이 없는 인지개선용 기능성 식품을 제공하고자 한다. First, the antioxidant effect and the mechanism of action of the composite composition comprising peony and cheonma extract as active ingredients, and to use this to provide a functional food for cognitive improvement without the risk of side effects.
둘째, 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물을 이용하여 자외선 차단 및 노화 방지 등의 기능성이 부여된 주름개선용 기능성 화장품을 제공하고자 한다. Second, to provide a functional cosmetics for improving wrinkles, such as UV protection and anti-aging, using a composite composition comprising peony and cheonma extract as an active ingredient.
셋째, 국내 천연소재를 이용한 생리활성 신소재의 연구를 통하여 천연자원에 대한 경제적 효용가치 증가시키고, 이들 자원의 고부가가치화를 실현하고자 한다.Third, through the study of physiologically active new materials using natural materials in Korea, it is intended to increase the economic utility value of natural resources and to realize high value added of these resources.
상기와 같은 과제를 해결하기 위하여 본 발명은 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물 및 이를 포함하여 구성되는 인지개선용 기능성 식품과 주름개선용 기능성 화장품을 제공한다.In order to solve the above problems, the present invention provides a composite composition comprising peony and cheonma extract as an active ingredient, and functional food for cognitive improvement and functional cosmetics for wrinkle improvement comprising the same.
또한 본 발명의 작약 추출물 및 천마추출물은 각각 (a)0.5~1.0cm 크기로 절단된 작약근 및 천마건조분말을 준비하는 단계; (b)상기 작약근 및 천마건조분말에 8~12배의 증류수를 가하고, 이를 90~97℃의 온도에서 210~270분 및 450~510분 동안 열수 추출한 다음 여과액으로 여과시키는 단계; (c)상기 여과액을 50~55℃의 온도에서 감압 농축한 다음, 실온까지 냉각하는 단계; (d)상기 (c)단계에서 생성된 물질이 70~80% 범위의 에탄올 용액이 되도록 에탄올을 가하는 단계; (e)상기 (d)단계에서 생성된 물질을 3~7℃ 범위에서 210~270분 동안 방치한 다음, 여과액을 동결건조하는 단계; 를 통하여 제조된다. In addition, the peony extract and cheonma extract of the present invention are (a) preparing peony root and cheonma dry powder cut into sizes of 0.5 ~ 1.0cm, respectively; (b) adding 8-12 times distilled water to the peony root and cheonma dry powder, extracting the hydrothermal extract for 210 to 270 minutes and 450 to 510 minutes at a temperature of 90 to 97 ° C., and then filtering the filtrate with filtrate; (c) concentrating the filtrate under reduced pressure at a temperature of 50-55 ° C. and then cooling to room temperature; (d) adding ethanol such that the material produced in step (c) becomes an ethanol solution in a range of 70 to 80%; (e) leaving the material produced in step (d) in a range of 3 to 7 ° C. for 210 to 270 minutes, and then lyophilizing the filtrate; It is manufactured through.
여기에서 인지개선용 기능성 식품은 활성산소종(reactive oxygen species;ROS)의 활성도 및 신경세포의 지질과산화(Lipid peroxidation)의 감소효과, 글루타치온(glutathione;GSH)의 함량 증가 효과를 갖는 것으로, 실험을 통하여 이를 효과를 확인하였다. 아울러 주름개선용 기능성 화장품의 경우, 콜라겐(collagen)의 합성 효과 및 콜라게나아제(collagenase)의 활성 저해 효과를 실험을 통하여 확인하였다.Here, the functional food for cognitive improvement has the effect of reducing the activity of reactive oxygen species (ROS), lipid peroxidation (Lipid peroxidation) of neurons, and increasing the content of glutathione (GSH). This confirmed the effect through. In addition, in the case of functional cosmetics for wrinkle improvement, the synthetic effect of collagen (collagen) and the inhibitory effect of collagenase (collagenase) activity was confirmed through experiments.
이상과 같은 본 발명에 따르면 다음과 같은 효과가 기대된다.According to the present invention as described above is expected the following effects.
첫째, 본 발명의 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물은 천연 추출물을 이용함으로써, 부작용의 위험이 없는 인지개선용 기능성 식품의 제공이 가능하다. First, the composite composition comprising the peony and cheonma extract of the present invention as an active ingredient, by using a natural extract, it is possible to provide a functional food for cognitive improvement without the risk of side effects.
둘째, 본 발명의 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물을 이용함으로써, 자외선 차단 및 노화 방지 등의 기능성이 부여된 주름개선용 기능성 화장품의 제공이 가능하다. Second, by using the composite composition of the peony and cheonma extract of the present invention as an active ingredient, it is possible to provide a functional cosmetics for wrinkle improvement imparted functionality such as UV protection and anti-aging.
셋째, 본 발명의 작약 및 천마추출물을 유효 성분으로 하는 복합 조성물을 이용함으로써, 국내 천연자원에 대한 경제적 효용가치 증가와 함께 이들 자원의 고부가가치화를 실현한다.Third, by using the composite composition comprising the peony and cheonma extract of the present invention as an active ingredient, it is possible to increase the economic utility value of domestic natural resources and to add high value to these resources.
본 발명은 작약추출물 및 천마추출물을 유효성분으로 포함하는 복합조성물인바, 작약 및 천마추출물의 추출 방법 및 실시예에 대하여 살펴보고, 이를 포함하여 구성되는 인지개선용 기능성 식품 및 주름개선용 기능성 화장품의 효능에 대하여 실험예를 통하여 검증하고자 한다.The present invention is a composite composition containing a peony extract and a cheonma extract as an active ingredient, and looks at the extraction method and embodiment of the peony and cheonma extract, the functional food for cognitive improvement and functional cosmetics for wrinkles including the same Efficacy is to be verified through experimental examples.
■ 작약 및 천마추출물의 제조 ■ Manufacture of Peony and Cinnamon Extract
작약은 천궁, 당귀, 지황, 황기와 함께 국내 5대 기본 생약재 중의 하나로서, 작약의 품질을 평가하는 지표물질로는 작약근의 주요 생리활성물질인 페오니플로린(paeoniflorin)이 사용된다. 작약을 식품이나 화장품의 소재로 사용할 경우, 안전성과 추출설비 및 제조 단가 등에 장점이 많은 열수 추출을 이용하도록 한다. Peony is one of the five basic herbal medicines in Korea, along with Cheongung, Angelica, Chihwang, and Astragalus. Peonyniflorin, the main bioactive substance of peony root, is used as an indicator for evaluating the quality of peony. If peony is used as a material for food or cosmetics, hot water extraction should be used, which has many advantages in safety, extraction equipment, and manufacturing cost.
천마의 경우 주요생리활성물질인 게스트로딘(gastrodin), 베닐릴알콜(benzyl alcohol), 바닐린(vanillin) 등은 물에서 추출이 용이하므로, 이러한 특성을 이용하여 열수 추출하였다.In the case of Chunma, gastrodin, benzyl alcohol, vanillin, etc., which are the main bioactive substances, are easily extracted from water, and thus hot water was extracted using these properties.
여기에서 작약 추출물 및 천마추출물은 각각 (a)0.5~1.0cm 크기로 절단된 작약근 및 천마건조분말을 준비하는 단계; (b)상기 작약근 및 천마건조분말에 8~12배 의 증류수를 가하고, 이를 90~97℃의 온도에서 210~270분 및 450~510분 동안 열수 추출한 다음 여과액으로 여과시키는 단계; (c)상기 여과액을 50~55℃의 온도에서 감압 농축한 다음, 실온까지 냉각하는 단계; (d)상기 (c)단계에서 생성된 물질이 70~80% 범위의 에탄올 용액이 되도록 에탄올을 가하는 단계; (e)상기 (d)단계에서 생성된 물질을 3~7℃ 범위에서 210~270분 동안 방치한 다음, 여과액을 동결건조하는 단계; 를 통하여 제조되는 것을 특징으로 한다. Here, peony extract and cheonma extract are prepared by (a) preparing peony root and cheonma dry powder cut into 0.5 ~ 1.0cm size; (b) adding 8-12 times distilled water to the peony root and cheonma dry powder, extracting the hydrothermal extract for 210-270 minutes and 450-510 minutes at a temperature of 90-97 ° C., and then filtering the filtrate with filtrate; (c) concentrating the filtrate under reduced pressure at a temperature of 50-55 ° C. and then cooling to room temperature; (d) adding ethanol such that the material produced in step (c) becomes an ethanol solution in a range of 70 to 80%; (e) leaving the material produced in step (d) in a range of 3 to 7 ° C. for 210 to 270 minutes, and then lyophilizing the filtrate; It is characterized in that it is manufactured through.
[[ 실시예Example ]]
1. 작약추출물의 제조1. Preparation of Peony Extract
작약추출물을 생산하기 위하여, 본 발명의 실시예로 작약근 300g을 0.5㎝~1㎝ 크기로 절단한 시료에 증류수 3000㎖을 가한 다음, 환류냉각기를 부착하여 95℃에서 4시간 열수추출하고 이를 여과한다. 그리고 이 여과액을 50~55℃에서 전량이 200㎖될 때까지 감압농축기로 농축하여 실온까지 냉각한 다음, 에탄올을 가하여 75%에탄올 용액이 되게 한다. 다음으로 이를 교반하여 5℃에서 24시간 방치하고 생성되는 침전물을 제거한다. 이렇게 획득된 여과액을 동결건조함으로써 작약의 유용물질이 함유된 작약추출물을 생산한다. (도1).In order to produce a peony extract, 3000 ml of distilled water was added to a sample cut into 300 g of peony root in a size of 0.5 cm to 1 cm according to an embodiment of the present invention, followed by attaching a reflux condenser and extracting hot water at 95 ° C. for 4 hours and filtering it. do. The filtrate is concentrated in a vacuum condenser until the total amount is 200ml at 50 ~ 55 ° C, cooled to room temperature, and ethanol is added to make 75% ethanol solution. Next, it is stirred and left for 24 hours at 5 ° C. to remove the resulting precipitate. By freeze-drying the filtrate thus obtained to produce a peony extract containing the useful material of the peony. (Figure 1).
2. 천마추출물의 제조2. Preparation of Cheonma Extract
천마추출물은 작약추출물과 마찬가지로 도1과 같은 순서를 따라 생산하였다. 여기에서는 천마건조분말을 사용하였으며, 천마가 작약에 비하여 열에 안정적인 특 성을 이용하여 8시간 동안 열수 추출하였다.Chunma extract was produced in the same order as in Fig. 1 as peony extract. Here, cheonma dry powder was used, and hot water was extracted for 8 hours using heat-stable properties compared to peony.
3. 작약 및 천마추출물의 추출 수율3. Extraction yield of peony and cheonma extract
상기 실시예를 통하여 획득된 작약 및 천마추출물의 추출 수율은 표1과 같다. 이때 작약 및 천마추출물의 최종 수득률은 각각 26.5% 및 52.5%이다.Extraction yield of peony and cheonma extract obtained through the above embodiment is shown in Table 1. The final yield of peony and cheonma extract is 26.5% and 52.5%, respectively.
[표1]Table 1
■ 인지개선용 기능성 식품의 신경세포 보호효과 및 기전 ■ Neuroprotective effect and mechanism of functional food for cognitive improvement
작약추출물 및 천마추출물을 유효성분으로 하는 복합조성물을 포함하여 구성되는 인지개선용 기능성 식품의 효능을 확인하기 위하여 실험예1 내지 실험예4의 실험을 실시하였다.In order to confirm the efficacy of the functional food for cognitive improvement comprising a composite composition containing the peony extract and cheonma extract as an active ingredient, the experiments of Experimental Examples 1 to 4.
우선 실험의 실시 이전에 PC12 세포를 배양하였다. 그리고 세포가 일정 농도가 되면 세포를 유리시킨 다음 이를 원심분리 하여 세척한 후 RPMI 1640 배지에 세포를 분주하여 이용하였다. 이렇게 획득된 PC12 세포에 작약 및 천마추출물 시료를 각각 다양한 농도로 처리하였다. 처리된 시료는 모두 디메틸술폭시화물(Dimethyl sulfoxide;DMSO)의 최종 농도가 0.2%를 넘지 않도록 디메틸술폭시화물에 용해시켰다.First, PC12 cells were cultured before the experiment. After the cells were released to a certain concentration, the cells were released, washed by centrifugation, and the cells were used by dispensing the cells in RPMI 1640 medium. The thus obtained PC12 cells were treated with various concentrations of peony and cheonma extract samples, respectively. All treated samples were dissolved in dimethyl sulfoxide so that the final concentration of dimethyl sulfoxide (DMSO) did not exceed 0.2%.
이하, 인지개선용 기능성 식품의 신경세포 보호효과 및 기전과 관련된 실험 에 대하여 살펴본다. Hereinafter, the experiments related to neuroprotective effect and mechanism of functional foods for cognitive improvement.
[[ 실험예1Experimental Example 1 ] 작약 및 천마추출물의 세포독성 측정Cytotoxicity of Peony and Cinnamon Extracts
1. 실험방법1. Experimental method
작약 및 천마추출물의 세포독성을 측정하기 위하여 MTT 방법을 이용하였다. 즉 PC12 세포의 농도를 5×105cells/㎖로 조절하여 마이크로타이터 플레이트(microtiter plate)에 100㎕씩 넣은 다음, 작약 및 천마추출물을 농도별로 처리하여 24~72시간 동안 배양하였다. 그리고 각 well당 MTT 라벨 시약(MTT labeling reagent)에 일렉트론 커플링 시약(electron coupling reagent)을 첨가하여 형성된 MTT 라벨 혼합물(MTT labeling mixture)을 최종농도가 0.5㎎/㎖가 되도록 4시간 동안 처리하고, 550nm 파장에서 흡광도를 이용하여 세포 독성을 조사하였다.MTT method was used to measure the cytotoxicity of peony and sperm extract. That is, by adjusting the concentration of PC12 cells to 5 × 10 5 cells / ㎖ 100μl to the microtiter plate (microtiter plate), and treated with the concentration of peony and cheonma extract by incubation for 24 to 72 hours. Then, the MTT labeling mixture formed by adding an electron coupling reagent to the MTT labeling reagent for each well was treated for 4 hours so that the final concentration was 0.5 mg / ml, Cytotoxicity was investigated using absorbance at 550 nm wavelength.
2. 실험결과2. Experimental Results
도2에서와 같이, 작약 및 천마추출물은 100㎍/㎖까지의 농도에서도 아무런 독성을 나타내지 않았다.As shown in Figure 2, peony and cheonma extract showed no toxicity even at concentrations up to 100 µg / ㎖.
[[ 실험예2Experimental Example 2 ] 작약 및 천마추출물이 세포 내 Peony and Chunma Extracts in Cells ROSROS 생산량에 미치는 효과 Effect on yield
1. 실험방법1. Experimental method
형광시약의 일종인 DCFH-DA(2',7'-Dichlorofluorescein diacetate) 시약을 세포 속에 넣어 발생하는 형광을 측정하여 세포 내의 활성산소종(reactive oxygen species;ROS)을 측정할 수 있다. 따라서 ROS 활성도를 측정하기 위해 DCFH-DA를 라벨링시킨 후 산화적 스트레스를 주고, 이 스트레스에 대한 작약 및 천마추출물의 보호 및 회복효과를 측정하였다. 즉 PC12 세포를 5×105cells/㎖의 농도로 분주한 다음, 배양액에 DCFH-DA를 well당 25μM이 되도록 15분간 처리하였다. 그리고 여기에 작약 및 천마추출물들과 신경손상을 유발시키는 물질인 과산화수소(Hydrogen peroxide, H2O2)를 처리하였다. 반응 후 형성된 세포 내 ROS는 각각 여기 파장 485nm, 방출 파장 530nm에서 형광 정도(fluorescence)를 이용하여 측정하였다. Reactive oxygen species (ROS) in cells can be measured by measuring the fluorescence generated by placing a DCFH-DA (2 ', 7'-Dichlorofluorescein diacetate) reagent, which is a kind of fluorescent reagent, into the cells. Therefore, DCFH-DA was labeled to measure ROS activity, followed by oxidative stress, and the protection and recovery effects of peony and sperm extract were measured. In other words, PC12 cells were dispensed at a concentration of 5 × 10 5 cells / ml, and then cultured for 15 minutes so that DCFH-DA was 25 μM per well. In addition, peony and cheonma extracts were treated with hydrogen peroxide (Hydrogen peroxide, H 2 O 2 ), a substance causing nerve damage. Intracellular ROS formed after the reaction were measured using fluorescence at excitation wavelength 485 nm and emission wavelength 530 nm, respectively.
2. 실험결과2. Experimental Results
실험결과 도3에서 보는 바와 같이 과산화수소만을 처리하여 증가된 양성 대조군에 비해 작약 및 천마추출물 처리군의 소거능 (scavenging activity)이 농도 의존적으로 증가함을 알 수 있다. ROS 활성도는 작약의 최고 농도인 100㎍/㎖에서는 0.51배, 천마의 최고농도인 100㎍/㎖에서는 0.56배 감소하였다. As shown in FIG. 3, it can be seen that the scavenging activity of the peony and cheonma extract treatment groups was increased in a concentration-dependent manner compared to the positive control group treated with hydrogen peroxide only. The ROS activity decreased 0.51 fold at 100 μg / ml, the highest concentration of Peony, and 0.56 fold at 100 μg / ml, the highest concentration of cheonma.
[[ 실험예3Experimental Example 3 ] 작약 및 천마추출물의 지질과산화 정도 측정Determination of Lipid Peroxidation in Peony and Chunma Extracts
1. 실험방법1. Experimental method
PC12 세포를 배양하여 신경손상을 유발시키는 화학물질인 과산화수소(hydrogen peroxide, H2O2) 300μM과 작약 및 천마추출물을 4시간 처리하였다. 그 리고 이 배양액에 20% 아세트산(pH 3.5)을 최종 15%가 되게 처리한 후 고무폴리스맨(rubber policeman)으로 긁어모아 원심분리하였다. 원심분리 후 상등액 0.2㎖를 취하여 8% 라우릴황산나트륨(sodium dodecyl sulfate;SDS)을 0.15㎖ 넣고 0.8%의 TBA(2-thiobarbituric acid)를 가한 후, 95℃에서 15분간 가열하여 1㎖의 부탄올을 첨가하고 15분간 원심분리 하였다. 다음으로 원심분리 후 지질과산화의 지표로서 사용되는 말론다이알데하이드(Malondialdehyde;MDA)의 양을 여기파장 513nm, 방출파장 553nm에서 형광정도(fluorescence)를 측정하였다.PC12 cells were cultured and treated with 300 μM of hydrogen peroxide (H 2 O 2 ), a chemical that causes nerve damage, and a peony and cheonma extract for 4 hours. Then, the culture solution was treated with 20% acetic acid (pH 3.5) to a final 15%, and then scraped with a rubber policeman and centrifuged. After centrifugation, 0.2 ml of the supernatant was added, 0.15 ml of 8% sodium dodecyl sulfate (SDS) was added, and 0.8% TBA (2-thiobarbituric acid) was added. Added and centrifuged for 15 minutes. Next, after centrifugation, the amount of malondialdehyde (MDA) used as an indicator of lipid peroxidation was measured at an excitation wavelength of 513 nm and an emission wavelength of 553 nm.
2. 실험결과2. Experimental Results
실험결과 도4에서와 같이 과산화수소만을 처리해 증가된 양성 대조군에 비하여 작약 및 천마추출물의 지질과산화(lipid peroxidation) 정도가 농도 의존적으로 감소하였다. 특히 작약추출물의 최고 농도인 100㎍/㎖에서는 양성 대조군에 비하여 약 32%, 천마추출물의 최고농도인 100㎍/㎖에서는 약 40% 지질과산화 정도가 감소하였다.As a result, as shown in FIG. 4, the degree of lipid peroxidation of peony and spermatozoa extract was reduced in a concentration-dependent manner compared to the positive control group treated with hydrogen peroxide only. In particular, the highest concentration of peony extract, 100 µg / ml, decreased about 32% compared to the positive control, and the highest concentration of cheonma extract, 100 µg / ml, decreased about 40% lipid peroxidation.
[[ 실험예4Experimental Example 4 ] ] 글루타치온Glutathione 함량 측정 Content measurement
1. 실험방법1. Experimental method
PC12 세포에 작약 및 천마추출물을 30분 동안 전처리한 후 신경손상을 유발시키는 화학물질인 과산화수소(hydrogen peroxide, H2O2)를 300μM 농도로 4시간 동 안 처리하였다. 그리고 이를 0.5㎖의 working buffer에 넣고 균질화 하였다. 이 상등액 0.25㎖에 동량의 4% 설포살리실산(sulfosalicylic acid)을 넣고 4℃에서 12시간 보관 후 원심분리하였다. 이 상등액 0.1㎖에 1㎖의 working buffer와 OPA(O-phthaldialdehyde)를 3.725mM이 되게 처리한 다음 실온에서 20분 동안 방치 후 여기파장 320nm, 방출파장 426nm에서 형광정도(fluorescence)를 측정하였다. Peony and cheonma extract were pretreated with PC12 cells for 30 minutes and then treated with hydrogen peroxide (H 2 O 2 ), a chemical that causes nerve damage, at 300 μM for 4 hours. And it was homogenized into 0.5ml working buffer. 0.25 ml of the supernatant was added with the same amount of 4% sulfosalicylic acid and stored at 4 ° C. for 12 hours, followed by centrifugation. 0.1 ml of the supernatant was treated with 1 ml of working buffer and OPA (O-phthaldialdehyde) to 3.725 mM, and then allowed to stand at room temperature for 20 minutes, followed by measurement of fluorescence at an excitation wavelength of 320 nm and an emission wavelength of 426 nm.
2. 실험결과2. Experimental Results
산화적 스트레스에 의한 세포 손상의 증가는 글루타치온(Glutathione;GSH) 레벨의 감소를 통해 나타나기 때문에 이를 측정함으로써 산화적 스트레스에 의한 세포 손상을 측정하였다. 그 결과 과산화수소에 의해 감소된 양성 대조군에 비해 작약 및 천마추출물에서 글루타치온 레벨이 농도 의존적으로 증가하였다. 특히 작약의 최고 농도인 100㎍/㎖ 에서는 1.48배, 천마의 최고 농도인 100㎍/㎖에서는 1.70배 증가하였다(도5).The increase in cellular damage caused by oxidative stress may be attributed to glutathione (GSH). Since it appears through a decrease in levels, it was measured to measure cell damage by oxidative stress. As a result, glutathione levels were increased in a dose-dependent manner in Peony and Chunma extract compared to the positive control group reduced by hydrogen peroxide. In particular, the highest concentration of
상기와 같은 실험예들을 통하여 본 발명의 상기 인지개선용 기능성 식품은 활성산소종(reactive oxygen species;ROS)의 활성도 및 신경세포의 지질과산화(Lipid peroxidation)를 감소시키고, 글루타치온(glutathione;GSH)의 함량을 증가시키는 것으로 판명되었다. 따라서 본 발명의 작약 및 천마추출물은 산화적 스트레스를 받았을 때 그 손상을 보호 및 회복시킬 수 있는 능력이 있음을 알 수 있다. Through the experimental examples as described above, the functional food for cognitive improvement of the present invention reduces the activity of reactive oxygen species (ROS) and lipid peroxidation (Lipid peroxidation) of neurons, and the glutathione (GSH) of It has been found to increase the content. Therefore, it can be seen that the peony and cheonma extract of the present invention have the ability to protect and recover the damage when subjected to oxidative stress.
■ 주름개선용 기능성 화장품의 피부노화 억제 효능 ■ Anti-aging effect of wrinkles functional cosmetics
피부노화는 엠엠피(matrix metalloproteinase;MMP)의 일종인 콜라게나아제를 활성화시켜 콜라겐(교원질) 또는 기질단백질(extracellula matrix;ECM)의 파괴를 통하여 세포벽을 파괴시킴으로써 유발된다. 특히 자외선은 콜라겐의 양을 감소시킴과 동시에 MMP-1의 양을 증가시켜 피부 주름을 형성한다(도6). 따라서 본 발명에서는 인체 상피세포(CCD986SK) 및 각질세포(HaCaT)를 이용하여 ECM의 가장 중요한 성분인 콜라겐의 합성 정도 등과 MMP-1(collagenase-1) 또는 MMP-13(collagenase-3)의 합성 정도 등을 조사하였으며, 아울러 UVA(장파장 자외선)를 조사 후 작약 및 천마추출물의 MMPs 활성 정도 등을 측정하였다.Skin aging is caused by activating collagenase, a type of matrix metalloproteinase (MMP), which destroys cell walls through the destruction of collagen (collagen) or extracellula matrix (ECM). In particular, ultraviolet rays reduce the amount of collagen and at the same time increase the amount of MMP-1 to form skin wrinkles (Fig. 6). Therefore, in the present invention, the degree of synthesis of collagen, which is the most important component of ECM, and the degree of synthesis of MMP-1 (collagenase-1) or MMP-13 (collagenase-3) using human epithelial cells (CCD986SK) and keratinocytes (HaCaT). In addition, after irradiation of UVA (long wavelength UV), the degree of MMPs activity of peony and cheonma extract were measured.
실험의 진행이전에 인체 상피세포인 CCD986SK와 각질세포인 HaCaT를 일정 조건하에서 배양하였다. Before the experiment, human epithelial cells, CCD986SK and keratinocytes, HaCaT, were cultured under certain conditions.
[[ 실험예5Experimental Example 5 ] 작약 및 천마추출물의 콜라겐 합성 촉진 효능Promoting Collagen Synthesis of Peony and Chunma Extract
1. 실험방법1. Experimental method
ECM의 가장 중요한 성분인 콜라겐의 합성 촉진효과를 조사하기 위하여 CCD986SK 세포 및 HaCaT 세포를 배양 후 작약 및 천마추출물을 처리하고 24시간 후에 상층액을 회수하여 그 중 200㎕를 Sircol red 용액 1㎖와 혼합하였다. 그리고 이를 상온에서 30분간 반응시키면서 5분마다 한 번씩 섞은 다음 10,000 rpm에서 10분간 원심분리 하였다. 이 상층액을 버리고 침전물을 0.4N NaOH 1㎖에 녹여서 흡광도 550nm에서 측정하였다.In order to investigate the synergistic effect of collagen, the most important component of ECM, after treatment with CCD986SK cells and HaCaT cells, the peony and cheonma extract were treated, and after 24 hours, the supernatant was recovered and 200 μl was mixed with 1 ml of Sircol red solution. It was. The mixture was mixed once every 5 minutes while reacting at room temperature for 30 minutes and then centrifuged at 10,000 rpm for 10 minutes. This supernatant was discarded and the precipitate was dissolved in 1 ml of 0.4N NaOH and measured at an absorbance of 550 nm.
2. 실험결과2. Experimental Results
도7 및 도8에서 볼 수 있는 바와 같이, 작약 및 천마추출물을 처리했을 때 콜라겐의 합성이 촉진되는 것을 확인할 수 있다.As can be seen in Figures 7 and 8, it can be seen that the synthesis of collagen is accelerated when the peony and cheonma extract are treated.
[[ 실험예6Experimental Example 6 ] ] ELISAELISA 를 이용한 콜라겐 합성 촉진 효능Collagen synthesis promoting effect
1. 실험방법1. Experimental method
병소감염진단테스트(enzyme-linked immunosorbent assay;ELISA)를 통하여 콜라겐의 합성 촉진 효능을 측정하였다. 즉 CCD986SK 세포를 48 well plate에 계대 배양 후 작약 및 천마추출물을 처리하고 24시간 후에 상층액을 회수한 다음, 이를 96 well plate에 100㎕씩 분주하여 24시간 동안 항원을 부착시킨 후 세척하였다. 그리고 I형 콜라겐의 합성 지표로 사용되는 1차 항체(procollagen type I N-terminal propeptide;PICP)를 각 well당 100㎕ 넣어 상온에서 2시간 반응시키고 세척하였다. 그리고 2차 항체(anti-mouse)를 희석하고, 각 well당 이를 100㎕씩 넣어 실온에서 2시간 반응시킨 다음, 다시 이를 세척하여 티엠이(3,3',5,5'-Tetramethyl-benzidine;TMB)를 100㎕씩 각 well에 분주하였다. 그리고 이의 발색 반응 후 2N H2SO4 50㎕씩을 넣고 기질 반응을 정지시켰다. 흡광도는 ELISA 리더로 450nm에서 측정하였다. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of promoting collagen synthesis. That is, the CCD986SK cells were passaged in 48 well plates, treated with peony and cheonma extract, and after 24 hours, the supernatant was collected, and then, 100 µl was dispensed into 96 well plates, and the antigens were attached and washed for 24 hours. In addition, 100 μl of a primary antibody (procollagen type I N-terminal propeptide; PICP), which is used as an index of synthesis of type I collagen, was added to each well for 2 hours at room temperature and washed. After diluting the secondary antibody (anti-mouse), 100 μl of each well was reacted at room temperature for 2 hours, and then washed again to make TEM (3,3 ', 5,5'-Tetramethyl-benzidine; TMB) was aliquoted into each well. After the color reaction, 50N of 2N H 2 SO 4 was added thereto to stop the substrate reaction. Absorbance was measured at 450 nm with ELISA reader.
2. 실험결과2. Experimental Results
CCD986SK 세포에 작약 및 천마추출물을 처리하고 PICP 항체를 이용하여 콜라겐의 합성 촉진 효과를 ELISA로 수행한 결과, 작약 및 천마추출물에서 콜라겐의 합성이 촉진되는 것을 확인할 수 있다(도9).Treatment of peony and cheonma extracts on CCD986SK cells and the effect of promoting collagen synthesis by ELISA using PICP antibodies were confirmed that the synthesis of collagen is promoted in peony and cheonma extract (FIG. 9).
[[ 실험예7Experimental Example 7 ] 형질전환Transformation
1. 실험방법1. Experimental method
작약 및 천마추출물의 교원질 촉진자 유전자(collagen alpha2(I) gene;COL1A2) 전사 활성도에 대한 효과를 측정하기 위하여 COL1A2의 프로모터(promoter)를 루시페라아제 (luciferase)를 포함하는 벡터 업스트림(vector(pGL3-promoter) upstream)에 삽입시켜 재조합 플라스미드를 만들고, COL1A2 전사촉진제의 활성도에 의존적으로 루시페라아제가 발현되도록 하였다. 그리고 CCD986SK 세포에 Lipofectamine를 이용하여 pGL3-COL1A2를 형질전환시킨 후, CCD986SK 세포를 48 well plate에 계대 배양하고, 여기에 작약 및 천마추출물을 처리하여 24 시간 동안 배양하였다. 다음으로 이의 융해물을 만들어 상층액만 취하여 리포터 유전자 에세이(reporter gene assay)를 수행하여 루시페라아제의 활성도를 측정하였다.In order to measure the effect of collagen alpha2 (I) gene (COL1A2) transcriptional activity of peony and spermatozoa extracts, the promoter of COL1A2 was vectored upstream (luciferase) vector (pGL3-promoter) containing luciferase. upstream) to generate recombinant plasmids and allow luciferase to be expressed depending on the activity of the COL1A2 promoter. After transforming pGL3-COL1A2 using Lipofectamine to CCD986SK cells, the CCD986SK cells were passaged in 48 well plates, and then treated with peony and cheonma extract, and incubated for 24 hours. Next, the lysate was made, and only the supernatant was taken to carry out a reporter gene assay to measure the activity of luciferase.
2. 실험결과2. Experimental Results
천마추출물과 작약 추출물 모두에서 COL1A2 promoter의 전사 활성도가 증가 된 것을 확인할 수 있다.(도10). It can be seen that the transcriptional activity of the COL1A2 promoter was increased in both the extract and the peony extract (FIG. 10).
[[ 실험예8Experimental Example 8 ] 작약 및 천마추출물의 Peony and Chunma Extract 콜라게나아제Collagenase 활성 저해 효능 Activity inhibition effect
1. 실험방법1. Experimental method
ECM의 가장 중요한 성분인 콜라게나아제 활성 중 MMP-1 및 MMP-13의 활성 억제 효과를 조사하기 위하여 CCD986SK 세포 및 HaCaT 세포를 48 well plate에 계대 배양 후 작약 및 천마추출물을 12시간 전 처리하고 PBS로 1회 세척 후 UVA를 조사하였다. 그리고 24시간 후에 상층액을 취하여 10,000rpm에서 5분간 원심분리 하였다. 다음으로 96 well plate에 상층액 50㎕과 농도 1mM의 에이피엠에이(4-aminophenylmercuric acetate;APMA)를 넣어 잘 섞어준 후, MMP-1 효소 활성은 37℃에서 6시간 반응시키고, MMP-13 효소 활성은 37℃에서 40분간 반응시켰다. 마지막으로 MMP-1과 MMP-13에 해당하는 기질 용액을 첨가하여 섞어준 후 여기파장 340nm, 방출파장 490nm에서 1시간 동안 5분 간격으로 형광정도를 측정하였다.To investigate the inhibitory effect of MMP-1 and MMP-13 on collagenase activity, which is the most important component of ECM, after passage of CCD986SK cells and HaCaT cells in 48 well plates, peony and sperm extracts were treated 12 hours before PBS. UVA was irradiated after washing once. After 24 hours, the supernatant was taken and centrifuged for 5 minutes at 10,000 rpm. Next, add 50 µl of the supernatant and 1 mM AMP (4-aminophenylmercuric acetate; APMA) to a 96 well plate, and then mix well. The MMP-1 enzyme activity was reacted at 37 ° C. for 6 hours and the MMP-13 enzyme activity. Reacted for 40 minutes at 37 ° C. Finally, after adding and mixing the substrate solutions corresponding to MMP-1 and MMP-13, the fluorescence was measured at an interval of 5 minutes for 1 hour at an excitation wavelength of 340 nm and an emission wavelength of 490 nm.
2. 실험결과2. Experimental Results
도11 및 도12에서와 같이, MMP-1과 MMP-13의 활성이 작약 및 천마추출물의 농도 의존적으로 저해됨을 확인할 수 있다(도 11, 도12).As shown in Figures 11 and 12, it can be seen that the activity of MMP-1 and MMP-13 is inhibited in a concentration-dependent manner of peony and cheonma extract (Figs. 11 and 12).
즉, 본 발명의 주름개선용 기능성 화장품은 콜라겐(collagen)의 합성 촉진 효능 및 콜라게나아제(collagenase)의 활성 저해 효능을 통하여 노화를 예방할 수 있다.That is, the functional cosmetics for wrinkle improvement of the present invention can prevent aging through the effect of promoting the synthesis of collagen (collagen) and the inhibitory effect of the activity of collagenase (collagenase).
본 발명은 상기에서 언급한 바와 같이 바람직한 실시예와 관련하여 설명되었으나, 본 발명의 요지를 벗어남이 없는 범위 내에서 다양한 수정 및 변형이 가능하다. 따라서 본 발명의 청구범위는 이건 발명의 진정한 범위 내에 속하는 수정 및 변형을 포함한다.While the invention has been described in connection with the preferred embodiment as mentioned above, various modifications and variations are possible without departing from the spirit of the invention. Therefore, the claims of the present invention include modifications and variations that fall within the true scope of the invention.
도1은 작약 및 천마추출물 제조의 실시예를 나타내는 순서도.1 is a flow chart showing an embodiment of the preparation of peony and cheonma extract.
도2는 작약 및 천마추출물이 세포 생존능력에 미치는 영향을 나타내는 그래프.Figure 2 is a graph showing the effect of peony and cheonma extract on cell viability.
도3은 작약 및 천마추출물이 활성산소종 생산에 미치는 영향을 나타내는 그래프.Figure 3 is a graph showing the effect of peony and cheonma extract on the production of reactive oxygen species.
도4는 작약 및 천마추출물이 지질과산화 정도에 미치는 영향을 나타내는 그래프.Figure 4 is a graph showing the effect of peony and cheonma extract on the degree of lipid peroxidation.
도5는 작약 및 천마추출물이 글루타치온 농도에 미치는 영향을 나타내는 그래프.5 is a graph showing the effect of peony and cheonma extract on glutathione concentration.
도6은 피부 노화 과정을 나타내는 개념도.6 is a conceptual diagram showing the skin aging process.
도7 및 도8은 각각 CCD986SK 세포 및 HaCaT 세포를 이용한 작약 및 천마추출물의 콜라겐 합성 촉진 효능을 나타내는 그래프.7 and 8 are graphs showing collagen synthesis promoting efficacy of peony and cheonma extract using CCD986SK cells and HaCaT cells, respectively.
도9는 ELISA에 의한 작약 및 천마추출물의 콜라겐 합성 촉진 효능을 나타내는 그래프.Figure 9 is a graph showing the collagen synthesis promoting efficacy of peony and cheonma extract by ELISA.
도10은 작약 및 천마추출물이 루시페라아제 활성도에 미치는 영향을 나타내는 그래프.10 is a graph showing the effect of peony and cheonma extract on luciferase activity.
도11 및 도12는 CCD986SK 세포 및 HaCaT 세포를 이용한 작약 및 천마추출물의 콜라게나아제 활성 저해 효능을 나타내는 그래프.11 and 12 are graphs showing the collagenase activity inhibitory effect of peony and cheonma extract using CCD986SK cells and HaCaT cells.
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| KR20190006127A (en) * | 2017-07-07 | 2019-01-17 | (주)에스디생명공학 | Composition for Preventing or Improving Skin Wrinkle Comprising Enzyme Treated Extract of Gastrodia as Active Ingredient |
| KR20190006128A (en) * | 2017-07-07 | 2019-01-17 | (주)에스디생명공학 | Composition for Improving Skin Conditions Comprising Enzyme Treated Extract of Gastrodia as Active Ingredient |
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| KR20070019120A (en) * | 2005-08-11 | 2007-02-15 | 주식회사 코리아나화장품 | Anti-Wrinkle Cosmetic Composition Comprising the Extract of Paeonia lactiflora as Active Ingredient |
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