KR20190032857A - Composition comprising Amla and Hibiscus extracts for preventing lose of hair or promoting of hair - Google Patents
Composition comprising Amla and Hibiscus extracts for preventing lose of hair or promoting of hair Download PDFInfo
- Publication number
- KR20190032857A KR20190032857A KR1020170121220A KR20170121220A KR20190032857A KR 20190032857 A KR20190032857 A KR 20190032857A KR 1020170121220 A KR1020170121220 A KR 1020170121220A KR 20170121220 A KR20170121220 A KR 20170121220A KR 20190032857 A KR20190032857 A KR 20190032857A
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- South Korea
- Prior art keywords
- hair
- hibiscus
- amla
- extract
- composition
- Prior art date
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- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
- A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
Abstract
Description
본 발명은 탈모방지 또는 발모촉진용 조성물에 관한 것으로, 더욱 구체적으로 암라 및 히비스커스의 혼합 추출물을 함유하는 탈모방지 또는 발모촉진용 조성물에 관한 것이다.The present invention relates to a composition for preventing hair loss or for promoting hair growth, and more particularly, to a hair loss preventing or hair growth promoting composition containing a mixed extract of Amara and Hibiscus.
현대사회로 가면서 탈모환자가 급격히 증가하고 있으며, 그에 대한 관심과 고민도 점점 더 심화되고 있다. 우리 몸의 두피에 있는 머리카락은 매일 0.3mm씩 자라 1년에 총 15cm가 자라며, 전체의 85% 정도가 성장하고 나머지 15%는 성장을 멈추는 것으로 알려져 있다. 빠지는 머리카락은 주로 성장을 멈춘 머리카락인데, 탈모는 빠지는 대상이 되는 성장을 멈춘 머리카락의 비율이 늘어남을 의미한다. 정상의 경우, 머리카락은 하루에 80~100개가 빠지지만, 그렇지 않은 경우 100개 이상이 빠지며, 모모세포의 힘이 약해지면서 성장기가 짧아지고 다음 성장기까지의 기간이 길어지면서 자라난 모발조차 제대로 성장하지 못하는 것을 의미한다.As we move into modern society, patients with hair loss are increasing rapidly, and interest and anxieties about it are getting worse. Hair on the scalp of our body grows by 0.3mm every day, growing 15cm in a year, 85% of the whole is growing, and the remaining 15% is said to stop growing. The missing hair is mainly hair that has stopped growing, which means that the percentage of hair that has stopped growing is increasing. In normal cases, the hair falls off 80 to 100 times a day, but if it does not, more than 100 are lost. When hair growth is shortened due to weakening of the hair cells and the growth period is extended, .
탈모증(脫毛症, Baldness)란, 신체의 털, 특히 머리카락이 부족한 상태를 말한다. 탈모의 가장 큰 원인으로는 유전적인 소인과 남성호르몬, 그리고 노화현상으로 알려져 있고, 그 밖에 혈액순환장애, 과다한 스트레스, 영양불균형, 지루성 피부염, 모발 관리제품의 잘못된 사용이나 과다한 염색 탈색 등의 미용관리도 복합적으로 작용하는 것으로 알려져 있다. 유전적인 소인이 있는 사람의 모낭에서 테스토스테론이 5-α reductase에 의해 5-α dihydrotestosterone(DHT)으로 되며, 이에 의해 모낭세포의 단백 합성이 지연되어 휴지기 모낭의 비율이 증가하며 나이가 들면서 탈모가 진행된다. 국소적으로 두피의 전두부 및 두정부의 모발이 연모로 변하여 점차 가늘어지고 길이가 짧아지며 모낭이 소형화되면서 미만성으로 소실되어 가는 것을 특징으로 한다.Hair baldness (毛毛 症, Baldness) refers to the hair of the body, especially the lack of hair. The most common cause of hair loss is known as genetic swelling, male hormone, and aging phenomenon. In addition, it is known that the hair loss is caused by the blood circulation disorder, excessive stress, nutritional imbalance, seborrheic dermatitis, Are also known to work in combination. In humans with genetic predisposition, testosterone is converted to 5-α dihydrotestosterone (DHT) by 5-α reductase, thereby delaying the protein synthesis of hair follicles and increasing the percentage of dormant hair follicles. do. The hair of the front part of the scalp and the hair of the two parts of the scalp are locally changed into a soft palate and become gradually thinner and shorter in length, and the hair follicle is miniaturized and disappears diffusely.
탈모에 대한 치료는 경구복용법, 국소도포, 모발이식술을 이용하고 있으며 FDA의 허가를 취득한 약물로 Minoxidil과 Finasteride가 있다. Minoxidil은 고혈압 치료를 위한 혈관 확장제로 개발되었으나 부작용으로 다모증이 보고되면서 발모제로 개발되었다. 내복약으로 사용되는 Finasteride는 5-α reductase type Ⅱ의 활성을 억제함으로써 안드로젠의 중간 대사체인 dihydrotestosterone(DHT)의 농도를 낮추는 기전을 통하여 남성형 탈모의 치료에 이용되고 있다. 그러나 Minoxidil과 Finasteride의 효능은 사람마다 일률적이지 않고, 부작용들이 보고되고 있다. 현재 탈모증에 대한 치료는 완벽하지 않으며, 효과적이고 안전한 치료법에 대한 수요는 줄지 않고 있다. 따라서 한약재를 이용한 효과적인 탈모 예방 및 치료 물질의 개발을 통한 차별화 된 연구가 필요한 실정이다.Treatment for hair loss includes oral dosing, topical application, hair grafting, and FDA approved medications Minoxidil and Finasteride. Minoxidil was developed as a vasodilator for the treatment of hypertension, but as a side effect, it was developed as a hair growth agent. Finasteride, which is used as an oral medicine, is used for the treatment of male pattern hair loss through a mechanism of lowering the concentration of dihydrotestosterone (DHT), an intermediate metabolite of androgen, by inhibiting the activity of 5-α reductase type II. However, the efficacy of Minoxidil and Finasteride is not uniform in every person, and side effects are reported. Currently, treatment for alopecia is not perfect, and the demand for effective and safe treatments is not decreasing. Therefore, there is a need for differentiated research through prevention of effective hair loss using herbal medicines and development of therapeutic substances.
암라(Amla)는 필렘블린(phyllembiln), 갈릭산(gallic acid), 지아틴(zeatin), 비타민C 등의 다양한 영양소가 함유되어 있으며 특히, 오렌지의 20배, 사과의 160배에 해당하는 천연 비타민 C를 함유 하고 있다.Amla contains a variety of nutrients such as phyllembiln, gallic acid, zeatin and vitamin C. Particularly, natural vitamins such as 20 times of orange and 160 times of apple C.
히비스커스(Hibiscus sabdariffa L.)은 아욱과에 속하는 식물로서 열대와 아열대 지방에서 주로 자라며, 두껍고 붉은 컵 모양의 꽃받침은 냉 음료뿐만 아니라 차로 음용되는데 고혈압, 발열, 간질환, 염증, 담석 및 비만에 효과가 있다. 히비스커스 추출물은 총 지질과 콜레스테롤, 중성지방의 감소 효과가 보고되었으며, 고지혈증 예방과 항산화 효과, 항암 효과가 탁월한 것으로 보고 되고 있다. Hibiscus (Hibiscus sabdariffa L.) is a plant belonging to the family Malvaceae which grows mainly in the tropics and subtropics. Thick red cup-shaped calyx is drunk not only in cold drinks but also in tea. It is effective for hypertension, fever, liver disease, inflammation, gallstone and obesity . Hibiscus extract has been reported to reduce total lipid, cholesterol, and triglyceride, and has been reported to have excellent antioxidant, antioxidant and anticancer effects.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구노력한 결과, 본원발명의 암라 및 히비스커스의 배합비에 따른 혼합 추출물의 경우, 기존의 암라 또는 히비스커스 단독 추출물보다 탈모방지 또는 발모촉진 효과가 더 우수하고 시너지 효과를 발휘하는 것을 확인하고, 본 발명을 완성하게 되었다.As a result, the present inventors have made intensive researches to overcome the problems of the prior art. As a result, the inventors of the present invention have found that the mixed extracts according to the blending ratio of Amaranth and Hibiscus of the present invention have a hair- Excellent synergistic effect was achieved, and the present invention was completed.
따라서, 본 발명의 주된 목적은 탈모방지 또는 발모촉진 효과가 우수한 암라 및 히비스커스 혼합 추출물을 함유하는 탈모방지 또는 발모촉진용 조성물을 제공하는 데 있다.Accordingly, it is a primary object of the present invention to provide a composition for preventing hair loss or for promoting hair growth, which contains Amara and Hibiscus mixed extract which is excellent in hair loss prevention or hair growth promoting effect.
본 발명의 한 양태에 따르면, 본 발명은 암라 및 히비스커스 혼합 추출물을 유효성분으로 하는 탈모방지 또는 발모촉진용 조성물을 제공한다.According to one aspect of the present invention, there is provided a composition for preventing hair loss or promoting hair growth comprising an extract of Amara and Hibiscus as an active ingredient.
현대사회로 가면서 탈모환자가 급격히 증가하면서 그에 대한 치료방법 또한 다양해지고 있다. 하지만, 완벽한 탈모증 치료방법에 대한 개발은 여전히 미미한 실정이다. 이에, 본 발명자들은 한약재를 이용한 효과적인 탈모 예방 및 치료물질을 연구하던 중, 암라 및 히비스커스 혼합 추출물이 각각의 단독 추출물에 비해 탈모방지 및 발모촉진에 있어서 시너지 효과를 발휘하는 것을 발견하고 본 발명을 완성하게 되었다.As the number of patients with hair loss increased rapidly in the modern society, the treatment methods for them have also been diversified. However, the development of a complete method for treating alopecia is still insignificant. Accordingly, the inventors of the present invention have found that, when researching effective hair loss preventive and therapeutic substances using herbal medicines, the combined extracts of Amaranth and Hibiscus have synergistic effects in preventing hair loss and accelerating hair growth compared with the respective single extracts, .
본 발명에 있어서, 추출물은 종래에 사용되어진 어떠한 용매로도 추출할 수 있으며, 바람직하게는 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매로 추출한 것을 특징으로 한다. 상기 저급알코올에는 주정도 포함한다.In the present invention, the extract can be extracted with any solvent conventionally used, and is preferably extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. Included in the lower alcohol is a major amount.
본 발명의 일 구현 예에 따른 제조방법에서, 상기 추출물은 에탄올 추출물일 수 있으나, 이에 제한되지 않는다. 추출하는 유기용매에 따라 약재의 유효성분의 추출 정도와 손실 정도가 차이날 수 있으므로, 알맞은 유기용매를 선택하여 사용하도록 한다. 추출방법은 특별히 제한되지 않고 예를 들어 열수 추출, 냉침 추출, 초음파 추출 및 환류 추출 등이 있다. 추출액을 농축할 경우에는 감압농축, 역삼투압 농축 등의 방법이 사용될 수 있다. 농축 후 건조 단계는 분무건조, 열풍건조, 동결건조, 진공건조, 감압건조, 포말건조, 고주파건조, 적외선건조 등을 포함하나 이에 제한되지 않는다.In the production method according to an embodiment of the present invention, the extract may be an ethanol extract, but is not limited thereto. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient in the medicinal product may differ. Therefore, an appropriate organic solvent should be selected and used. The extraction method is not particularly limited and includes, for example, hot water extraction, cold extraction, ultrasonic extraction and reflux extraction. When the extract is concentrated, methods such as concentration under reduced pressure and reverse osmosis can be used. The post-concentration drying step includes, but is not limited to, spray drying, hot air drying, freeze drying, vacuum drying, vacuum drying, foam drying, high frequency drying, infrared drying and the like.
본 발명에 있어서, 상기 혼합 추출물은 각 식물 추출물을 혼합하거나 식물 혼합물을 추출한 것을 특징으로 한다. In the present invention, the mixed extract is characterized in that each plant extract is mixed or a plant mixture is extracted.
본 발명에 있어서, 상기 혼합 추출물은 암라 및 히비스커스가 9 : 1 내지 1 : 9의 중량비, 바람직하게는 9 : 1 내지 5 : 5의 중량비, 더욱 바람직하게는 8 : 2 내지 6 : 4의 중량비로 혼합된 것을 특징으로 한다. 본 발명의 실험예에 따르면, 암라 및 히비스커스가 상기 중량비 범위내에서 암라 또는 히비스커스 단독에 비해 우수한 탈모 방지 및 발모 촉진 효과를 나타냄을 확인하였다. In the present invention, the mixed extract has a weight ratio of Amara and Hibiscus of 9: 1 to 1: 9, preferably 9: 1 to 5: 5, more preferably 8: 2 to 6: 4 . According to the experimental examples of the present invention, it was confirmed that amaranth and hibiscus exhibit superior hair loss prevention and hair growth promoting effect compared with amaranth or hibiscus alone within the above weight ratio range.
본 발명에 있어서, 상기 혼합 추출물은 10 mg/ml 내지 500 mg/ml의 농도로 사용될 수 있으나, 바람직하게는 50 mg/ml 내지 100 mg/ml의 농도로 사용되는 것을 특징으로 한다. 본 발명의 실험예에 따르면, 혼합 추출물의 농도를 50 mg/ml 내지 100 mg/ml의 농도로 사용했을 때 우수한 탈모 방지 및 발모 촉진 효과를 나타내었다. In the present invention, the mixed extract may be used at a concentration of 10 mg / ml to 500 mg / ml, but is preferably used at a concentration of 50 mg / ml to 100 mg / ml. According to the experimental example of the present invention, when the concentration of the mixed extract was used at a concentration of 50 mg / ml to 100 mg / ml, excellent hair loss prevention and hair growth promoting effect were shown.
본 발명에 있어서, 상기 추출물은 농축 분말화하여 기능성 티백차의 소재로 사용되는 것을 특징으로 한다. 본 발명의 추출물은 세포독성을 나타내지 않아 티백차 소재 및 화장품 소재로 안전하다. In the present invention, the extract is concentrated and powdered to be used as a material for a functional tea bag. The extract of the present invention does not show cytotoxicity and is safe as a tea bag material and cosmetic material.
본 발명에 있어서, 상기 추출물은 기능성 샴푸 및 트리트먼트의 소재로 사용되는 것을 특징으로 한다.In the present invention, the extract is used as a material for functional shampoos and treatments.
본 발명에 있어서, 상기 추출물은 조성물 총 중량 대비 0.01 내지 40중량%로 포함되는 것을 특징으로 한다. 이는 기능성 티백차 제품부터 기능성 샴푸 및 트리트먼트 제품으로 개발 시 각각의 기호도 및 제품 제형의 안정화를 고려하여 중량범위를 적절히 설정할 수 있다.In the present invention, the extract is contained in an amount of 0.01 to 40% by weight based on the total weight of the composition. It is possible to set the weight range appropriately considering the preference of each product and stabilization of the product formulation when developing the product from functional tea bag to functional shampoo and treatment product.
본 발명의 실험예 1(세포독성 및 증식효과 확인)에 따르면, 암라-히비스커스 복합물질의 세포독성 및 세포증식효과를 확인하기 위하여 인간섬유아세포에서 세포독성시험을 실시한 결과, 10, 50, 100 mg/ml 처리군에서 세포독성을 나타내지 않는 것으로 확인되었다. 또한 인간모유두세포에서의 세포증식능을 확인한 결과 50 mg/ml, 9:1~5:5 범위에서 세포증식효과가 나타나는 것을 확인하였다. 특히, 8:2~7:3에서 인간모유두세포의 세포증식효과가 가장 높게 나타나는 것으로 확인되었다. According to Experimental Example 1 (confirming cytotoxicity and proliferation effect) of the present invention, cytotoxicity test of human fibroblast cells was carried out in order to examine the cytotoxicity and cell proliferation effect of the amaranthus hibiscus complex material. As a result, 10, 50 and 100 mg / ml < / RTI > treated group. In addition, cell proliferative activity was confirmed in human dermal papilla cells, and cell proliferation effect was observed in the range of 50 mg / ml, 9: 1 to 5: 5. Especially, it was confirmed that the cell proliferation effect of human dermal papilla cells was the highest at 8: 2 ~ 7: 3.
본 발명의 실험예 2(인간모유두세포에서의 유효성 검증)에 따르면, 성장인자인 IGF-1, VEGF의 발현변화를 확인한 결과, 각각 50 mg/ml와 100 mg/ml의 농도에서 9:1~4:6범위 내에서의 배합 시 성장인자의 변화가 상승되는 것을 확인할 수 있었으며, 8:2~5:5의 범위내에서는 30 mg/ml의 농도에서도 효과가 나타나는 것을 확인할 수 있었다. 결과적으로, 8:2~6:4 비율에서 성장인자의 발현값이 가장 높게 증가되는 것을 확인하였다.According to Experimental Example 2 (validation in human dermal papilla cells) of the present invention, the expression of IGF-1 and VEGF, which are growth factors, were examined and found to be 9: 1 ~ It was confirmed that the change in the growth factor was increased during the mixing in the range of 4: 6, and the effect was observed even at the concentration of 30 mg / ml within the range of 8: 2 to 5: 5. As a result, it was confirmed that the expression level of the growth factor was the highest at the ratio of 8: 2 to 6: 4.
또한, 성장억제인자인 5α-reductase는 암라 단독 및 암라:히비스커스 9:1~4:6범위내에서의 배합 시 억제가 되는 것을 확인할 수 있었음. 9:1~6:4 범위에서 50 mg/ml의 농도에서는 Minoxidil의 30 mg/ml에서의 억제효과와 유사하게 나타나는 것이 확인되었다. 또한, TGF-β1의 발현변화를 확인한 결과 30 mg/ml의 농도에서 모든 시료에서 억제가 되는 것을 확인할 수 있었으며, 유의적인 효과를 확인하기 위해서는 5mg/ml 이상의 농도를 적용해야 할 것으로 확인되었다. 특히, 7:3~4:6의 배합 시 5mg/ml의 농도에서는 Minoxidil의 억제효과와 유사하게 나타나는 것이 확인되었다. 또한, DKK-1의 발현변화를 확인한 결과 암라:히비스커스 8:2~2:8 범위에서 억제가 되는 것을 확인할 수 있었으며, 7:3~5:5의 범위에서 억제효과가 가장 높게 나타나는 것을 확인하였다. 결과적으로, 7:3~5:5 비율에서 3종의 성장억제인자의 발현값이 가장 높게 억제되는 것을 확인하였다.In addition, it was confirmed that 5α-reductase, which is a growth inhibitory factor, is inhibited in combination with Amara alone and Amara: Hibiscus within the range of 9: 1 to 4: 6. The inhibitory effect of Minoxidil at 30 mg / ml was similar to that of 50 mg / ml in the range of 9: 1 to 6: 4. In addition, the expression of TGF-β1 was found to be inhibited in all samples at a concentration of 30 mg / ml, and it was confirmed that a concentration of 5 mg / ml or more should be applied to confirm the significant effect. Especially, the combination of 7: 3 ~ 4: 6 showed similar inhibitory effect to Minoxidil at the concentration of 5mg / ml. As a result of confirming the expression of DKK-1, the inhibition was observed in the range of 8: 2 to 2: 8 of the hibiscus, and it was confirmed that the inhibitory effect was the highest in the range of 7: 3 to 5: 5 . As a result, it was confirmed that the expression levels of the three growth inhibitory factors were most suppressed at the ratio of 7: 3 to 5: 5.
본 발명의 실험예 3(NIH3T3 세포를 이용한 모세혈관 확장 효과)에서는, 모세혈관 확장효과를 확인하기 위하여 세포증식효과와 성장인자를 분석하였으며, K+Channel억제환경에서 50 mg/ml, 8:2~6:4 비율에서 모세혈관 확장 효과 및 성장인자의 발현이 높게 나타난 것을 확인하였다.In Experimental Example 3 of the present invention (capillary vasodilation effect using NIH3T3 cells), the cell proliferation effect and growth factors were analyzed in order to examine capillary vasodilation effect. In the K + channel inhibition environment, 50 mg / ml, 8: ~ 6: 4 ratios showed that capillary vasodilation effect and expression of growth factors were high.
이상 설명한 바와 같이, 본 발명에 따르면, 본원발명 암라 추출물을 함유하는 조성물은 세포독성을 나타내지 않아 안전하며, 탈모 유발인자들의 발현을 억제하고, 발모 성장인자들의 발현을 촉진할 뿐만 아니라, 뛰어난 모세혈관 확장효과가 나타내므로, 탈모 개선 또는 발모촉진 효과가 우수하여 탈모개선 또는 발모촉진용 티백차 조성물 또는 기능성 헤어제품으로 이용될 수 있다. 또한, 본원발명의 암라 및 히비스커스의 혼합 추출물의 경우, 기존의 암라 또는 히비스커스의 단독 추출물보다 탈모방지 또는 발모촉진에 있어서 시너지 효과를 나타내는 장점이 있다. 특히, 히비스커스의 단독 추출물의 경우 탈모방지 또는 발모촉진 효과가 거의 없거나 매우 약한 반면, 암라와 조합하여 사용하는 경우 암라의 탈모방지 또는 발모촉진 효과를 효과적으로 상승시킬 수 있다.INDUSTRIAL APPLICABILITY As described above, according to the present invention, the composition containing the AQUA extract of the present invention is safe because it does not exhibit cytotoxicity, inhibits the expression of hair growth factors, promotes the expression of hair growth factors, It can be used as a tea bag composition for improving hair loss or promoting hair growth or as a functional hair product. In addition, the mixed extract of Amaranthus and Hibiscus of the present invention has an advantage of synergy effect in preventing hair loss or accelerating hair growth than a single extract of Amaranth or Hibiscus. Particularly, the extract of hibiscus alone has little or very little effect of preventing hair loss or promoting hair growth, but when used in combination with amaranth, the hair loss prevention effect or hair growth promoting effect of amaranth can be effectively increased.
도 1은 Human Fibroblast cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 세포독성을 확인한 그래프이다.
도 2a 및 2b는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 세포증식능을 확인한 그래프이다.
도 3은 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 5α-reductase 활성을 측정한 그래프이다.
도 4는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 TGF-β-1 활성을 측정한 그래프이다.
도 5는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 DKK-1 (Dickkopf-1) 활성을 측정한 그래프이다.
도 6는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 IGF-1 활성을 측정한 그래프이다.
도 7는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 VEGF 활성을 측정한 그래프이다.
도 8는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 FGF 활성을 측정한 그래프이다.
도 9는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 세포증식촉진 효과를 측정한 그래프이다.
도 10는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 IGF-1 활성을 측정한 그래프이다.
도 11는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 VEGF 활성을 측정한 그래프이다.
도 12는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 FGF 활성을 측정한 그래프이다.
도 13은 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 K channel opening effect를 확인한 그래프이다.FIG. 1 is a graph showing cytotoxicity according to the ratio and concentration of Amara-Hibiscus blend extracts using human fibroblast cells.
FIGS. 2A and 2B are graphs showing cell proliferative activity according to the ratio and concentration of Amara-Hibiscus blend extracts using Dermal Papilla cells. FIG.
FIG. 3 is a graph showing the activity of 5α-reductase according to the ratio and concentration of Amara-Hibiscus blend extract using Dermal Papilla cell.
FIG. 4 is a graph showing TGF-.beta.-1 activity according to the ratio and concentration of Amara-hibiscus blend extract using Dermal Papilla cell.
FIG. 5 is a graph showing DKK-1 (Dickkopf-1) activity according to the ratio and concentration of a mixture of Amara-Hibiscus extracts using Dermal Papilla cells.
FIG. 6 is a graph showing IGF-1 activity according to the ratio and concentration of Amara-Hibiscus blend extracts using Dermal Papilla cells.
FIG. 7 is a graph showing VEGF activity according to the ratio and concentration of Amara-Hibiscus blend extract using Dermal Papilla cell.
FIG. 8 is a graph showing FGF activity according to the ratio and concentration of Amara-Hibiscus blend extract using Dermal Papilla cell.
FIG. 9 is a graph showing the optimal mixing ratio of Amara-hibiscus blend extracts using the NIH3T3 cell line and promoting cell proliferation in the optimum concentration group.
FIG. 10 is a graph showing the optimum blending ratio of amla-hibiscus blended extracts using the NIH3T3 cell line and the IGF-1 activity in the optimum concentration group.
FIG. 11 is a graph showing the optimum mixing ratio of VHF and the optimum concentration of VHF activity in the mixture of Amara-Hibiscus using the NIH3T3 cell line.
FIG. 12 is a graph showing the optimum compounding ratio and the FGF activity in the optimum concentration group of the mixture of Amara-hibiscus using the NIH3T3 cell line.
FIG. 13 is a graph showing the optimum compounding ratio of Amla-Hibiscus blend extracts using the NIH3T3 cell line and the K channel opening effect in the optimum concentration group.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.
비교예 및 실시예: 암라 및 히비스커스의 혼합 추출물을 함유하는 조성물의 제조COMPARATIVE EXAMPLES AND EXAMPLES: Preparation of Compositions Containing Mixed Extracts of Amara and Hibiscus
실시예에서 사용된 암라 및 히비스커스는 건조된 원료를 사용하였고, 70% 에탄올에 3시간 동안 추출한 후, 60℃에서 25 brix가 될 때까지 농축하였다. 농축된 암라 및 히비스커스 추출물의 고형분 함량을 측정하고, 고형분 함량을 기준으로 희석하여 농도별(10 mg/ml, 50 mg/ml, 70 mg/ml, 100 mg/ml, 200 mg/ml, 250 mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml) 시료를 준비한 후, 하기 표 1과 같이 배합하여 세포독성 및 증식효과 확인, 인간모유두세포에서의 유효성 검증, 및 NIH3T3 세포를 이용한 모세혈관 확장 효과 실험에 사용하였다. Amla and hibiscus used in the examples were dried raw materials, extracted in 70% ethanol for 3 hours, and then concentrated to 25 brix at 60 ° C. The solids content of the concentrated Aramara and Hibiscus extracts was measured and diluted on the basis of the solids content. The concentrations of the extracts were 10 mg / ml, 50 mg / ml, 70 mg / ml, 100 mg / The results are shown in Table 1. The results are shown in Table 1 below. The results are shown in Table 1. The results are shown in Table 1 below. It was used for capillary vasodilation.
실험예 1: 세포독성 및 증식효과 확인Experimental Example 1: Identification of cytotoxicity and proliferation effect
1) Human Fibroblast cell을 이용한 세포독성 확인1) Identification of cytotoxicity using human fibroblast cell
암라:히비스커스 배합비 및 농도에 따른 세포독성을 확인하기 위하여 인간섬유아세포(Human Fibroblast, American Type Culture Collection, USA)를 이용하여 세포독성시험을 실시하였다. 세포는 12well 플레이트 기준 1x105/well으로 분주하며, 각각의 조건에 따른 물질을 처리하여 시험을 진행함. 물질처리 24시간 후 각 well에 MTT solution(5 mg/ml)을 20 μl씩 처리하고 1시간 동안 5% CO2, 37℃ 인큐베이터에서 반응을 진행하였다. 이후 용액 제거 후 1X PBS를 이용하여 세척한 후 DMSO 100 μl를 분주하여 540 nm에서 OD값을 측정하였다.A: Cytotoxicity test was carried out using human fibroblast (American Type Culture Collection, USA) to confirm the cytotoxicity according to the blend ratio and concentration of hibiscus. The cells are plated at 1 × 10 5 / well on a 12-well plate, and the material is treated according to the respective conditions to conduct the test. Twenty-four hours after the material treatment, 20 μl of MTT solution (5 mg / ml) was added to each well, and reaction was carried out in a 5% CO2 incubator at 37 ° C for 1 hour. After washing with 1X PBS, 100 μl of DMSO was added and the OD value was measured at 540 nm.
도 1은 Human Fibroblast cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 세포독성을 확인한 그래프이다. 시험에 앞서 암라-히비스커스 복합물질의 각 고형분 함량 퍼센트를 기준으로 1, 5, 10, 25, 50% (순서대로 10, 50, 100, 250, 500 mg/ml)의 물질을 제조하였으며, 이를 각각 비율 10부터 0까지 총 11개 군으로 나누어 처리하였다.FIG. 1 is a graph showing cytotoxicity according to the ratio and concentration of Amara-Hibiscus blend extracts using human fibroblast cells. Prior to the test, substances of 1, 5, 10, 25, 50% (10, 50, 100, 250, 500 mg / ml in order) were prepared based on the percentage of each solid content of the Amara-
인간유래 섬유아세포를 이용한 세포독성을 확인한 결과, 암라-히비스커스 1, 5, 10%(10, 50, 100 mg/ml)의 농도에서는 세포독성을 나타내지 않으며, 이외 고농도인 25, 50% (250 및 500 mg/ml)에서는 독성을 나타내었다. 따라서, 독성을 나타내지 않는 1, 5, 10%(10, 50, 100 mg/ml)의 농도를 사용하는 것이 바람직하다.As a result of cytotoxicity using human-derived fibroblasts, no cytotoxicity was observed at the concentrations of 1, 5 and 10% (10, 50 and 100 mg / ml) of Amara-Hibiscus and 25, 50% 500 mg / ml) showed toxicity. Therefore, it is preferable to use a concentration of 1, 5, 10% (10, 50, 100 mg / ml) which does not show toxicity.
2)Dermal papilla cell에서의 세포증식효과 확인2) Confirmation of cell proliferation effect in dermal papilla cell
암라:히비스커스 배합비 및 농도에 따른 세포증식 효과를 확인하기 위하여 인간모유두세포(Human Dermal papilla, CEFO, Korea)를 이용하여 세포증식 촉진효과 시험을 실시하였디. 세포는 12well 플레이트 기준 1x105/well으로 분주하며, testosterone 선처리에 따른 탈모조건 형성 후 각각의 조건에 따른 물질을 처리하여 시험을 진행함. 물질처리 24시간 후 각 well에 MTT solution(5 mg/ml)을 20 μl씩 처리하고 1시간 동안 5% CO2, 37℃ 인큐베이터에서 반응을 진행하였다. 이후 용액 제거 후 1X PBS를 이용하여 세척한 후 DMSO 100 μl를 분주하여 540 nm에서 OD값을 측정하였다.Amla: In order to confirm the effect of hibiscus blend ratio and concentration on cell proliferation, human dermal papilla cells (Human Dermal papilla, CEFO, Korea) were used for cell proliferation promoting effect test. Cells were plated at 1 × 10 5 / well on a 12-well plate. Testosterone was pretreated and the cells were treated with the substances according to their respective conditions. Twenty-four hours after the material treatment, 20 μl of MTT solution (5 mg / ml) was added to each well, and reaction was carried out in a 5% CO2 incubator at 37 ° C for 1 hour. After washing with 1X PBS, 100 μl of DMSO was added and the OD value was measured at 540 nm.
도 2a 및 2b는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 세포증식능을 확인한 그래프이다. Dermal papilla cell을 이용한 모유두세포 증식능을 확인하기 위하여 테스토스테론을 이용한 탈모유발 환경 조성 후 양성대조군인 미녹시딜과의 비교 분석을 실시하였다. 물질의 농도는 암라-히비스커스 고형분 함량기준 1, 3, 5, 7, 10, 20, 30, 40, 50% (순서대로 10, 30, 50, 70, 100, 200, 300, 400, 500 mg/ml) 이며, 이를 각각 비율 10부터 0까지 총 11개 군으로 나누어 시험을 진행하였다.FIGS. 2A and 2B are graphs showing cell proliferative activity according to the ratio and concentration of Amara-Hibiscus blend extracts using Dermal Papilla cells. FIG. To investigate the dermal papilla cell proliferative activity, we compared the positive control group, minoxidil, with testosterone - induced hair loss inducing environment. The concentrations of the substances were 1, 3, 5, 7, 10, 20, 30, 40 and 50% (in the order of 10, 30, 50, 70, 100, 200, 300, 400, 500 mg / ml), which were divided into 11 groups, ranging from 10 to 0, respectively.
시험 결과 암라-히비스커스 5% (50 mg/ml) 물질이 높은 유효성을 나타내었으며, 9:1 내지 5:5에서 높은 세포증식능, 7:3 및 8:2 비율에서 가장 높은 세포증식능을 보였다. As a result of the test, 5% (50 mg / ml) substance of Amara-Hibiscus showed high efficacy and showed high cell proliferating ability at a ratio of 7: 3 and 8: 2 at 9: 1 to 5: 5.
실험예 2: Dermal papilla cell을 이용한 유효성 검증 (탈모관련 인자 발현 변화 검증)Experimental Example 2: Validation using Dermal papilla cells (Verification of expression of hair follicle-related factors)
1) Human 5-alpha reductase 활성 측정1) Measurement of human 5-alpha reductase activity
탈모 유발 인자인 5 alpha reductase의 활성을 Elisa kit의 매뉴얼에 따라 측정하였다. 조건에 맞는 물질이 처리된 세포 상등액 100 μl의 샘플과 표준시약을 분주한 플레이트를 37℃에서 120분 동안 반응시켰다. 이 후 용액을 제거한 플레이트에 100 μl의 detection reagent A를 첨가하여 37℃에서 60분간 반응시켰다. 반응이 끝나면 용액을 제거한 뒤 3차례 세척한 후 100 μl의 detection reagent B를 첨가하여 37℃에서 60분간 반응시켰다. 반응이 끝난 후 용액 제거 후 5차례 세척하였다. 이후 90 μl의 TMB 기질용액을 넣고 37℃에서 30분간 반응시키며, 반응이 끝나면 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.The activity of 5 alpha reductase, an alopecia inducer, was measured according to Elisa kit manual. A 100 μl sample of the cell supernatant treated with the condition-matched material and a plate in which the standard reagent was dispensed was reacted at 37 ° C for 120 minutes. Then, 100 μl of detection reagent A was added to the plate from which the solution had been removed, and the reaction was carried out at 37 ° C for 60 minutes. After the reaction was completed, the solution was removed and washed three times. Then, 100 μl of detection reagent B was added and reacted at 37 ° C. for 60 minutes. After the reaction was completed, the solution was washed five times. After that, 90 μl of TMB substrate solution was added and reacted at 37 ° C for 30 minutes. When the reaction was completed, 50 μl of stop solution was added and the value was measured at 450 nm.
도 3은 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 5a-reductase 활성을 측정한 그래프이다. 5alpha reductase는 Testosterone을 Dehydro-testosterone으로 치환하여 탈모를 유발 시키는 주요 인자로 인식된다. 따라서 5alpha reductase의 활성 억제 효과 분석은 탈모 예방 효과를 분석할 수 있는 기초 data로 활용할 수 있다.FIG. 3 is a graph showing the activity of 5a-reductase according to the ratio and concentration of Amara-Hibiscus blend extract using Dermal Papilla cell. 5alpha reductase is recognized as a major factor causing hair loss by replacing Testosterone with Dehydro-testosterone. Therefore, the inhibitory effect of 5alpha reductase can be used as the basic data to analyze the effect of preventing hair loss.
Testosterone을 처리하여 탈모 환경을 유발한 모유두 세포에 암라:히비스커스 복합 물질을 배합비율 및 농도별로 처리한 결과 암라:히비스커스=9:1 내지 5:5의 비율에서 5 alpha reductase의 활성이 많이 감소되었고, 7:3 의 비율에서 5 alpha reductase의 활성이 가장 많이 감소하는 것을 확인하였으며, 농도별 에서는 50 mg/ml와 100 mg/ml 두 농도가 농도간 유의하지 않은 차이로 가장 효과가 좋은 것으로 분석되었다. 특히, 히비스커스 단독(0:10)의 경우는 거의 5 alpha reductase 활성 감소 효과가 나타나지 않았으나, 암라와 조합한 경우 암라 또는 히비스커스 단독에 비해 시너지 효과가 나타남을 확인하였다. Testosterone was applied to the hair dermal papilla cells to induce hair loss, and the amount of 5-alpha reductase was significantly reduced in the ratio of Amara: Hibiscus = 9: 1 to 5: 5, The highest concentration of 5 alpha reductase activity was observed at the ratio of 7: 3. The concentrations of both 50 mg / ml and 100 mg / ml were the most effective at the concentration. Especially, Hibiscus alone (0:10) showed almost no effect of reducing 5 alpha reductase activity, but it was confirmed that when combined with Amaran, synergistic effect was shown compared to Amara or Hibiscus alone.
2)TGF-beta 1 Human Enzyme-Linked Immunosorbent Assay2) TGF-
탈모 유발 인자인 TGF-beta 1의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated TGF-beta1 detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응함. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척함. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응함. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.Expression of TGF-beta1, an alopecia inducer, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Subsequently, 100 μl of 1X biotinylated TGF-beta1 detection antibody was applied to each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, wash 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well, and reacted at room temperature for 30 minutes under dark conditions. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 4는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 TGF-β-1 활성을 측정한 그래프이다. TGF-β1은 outer root sheath(외모근초)에서 생성되어 정상적인 모낭의 성장기를 조기에 퇴행기로 촉진하는 역할을 하는 것으로 알려진 인자이다.FIG. 4 is a graph showing TGF-.beta.-1 activity according to the ratio and concentration of Amara-hibiscus blend extract using Dermal Papilla cell. TGF-β1 is a factor known to play a role in promoting early growth of the hair follicle early in the outer root sheath.
Testosterone을 처리하여 탈모 환경을 유발한 모유두 세포에 암라:히비스커스 복합 물질을 배합비율 및 농도별로 처리한 결과 암라:히비스커스=9:1 내지 5:5의 비율에서 TGF-β1의 활성이 많이 감소되었고, 6:4 비율에서 TGF-β1의 활성이 가장 많이 감소하는 것을 확인하였으며, 농도별 에서는 100 mg/ml 농도가 가장 효과가 좋은 것으로 분석되었다. 특히, 히비스커스 단독(0:10)의 경우는 거의 TGF-β1 활성 감소 효과가 나타나지 않았으나, 암라와 조합한 경우 암라 또는 히비스커스 단독에 비해 시너지 효과가 나타남을 확인하였다.Testosterone was applied to the dermal papilla cells to induce hair loss environment, and the amount of TGF-β1 activity was significantly decreased in the ratio of Amara: Hibiscus = 9: 1 to 5: 5, At the 6: 4 ratio, the highest activity of TGF-β1 was observed, and the concentration of 100 mg / ml was the most effective. In particular, Hibiscus alone (0:10) showed almost no effect of reducing TGF-β1 activity, but when combined with amaranthine, synergistic effect was observed compared to either Amara or Hibiscus alone.
3) DKK1 Human Enzyme-Linked Immunosorbent Assay3) DKK1 Human Enzyme-Linked Immunosorbent Assay
탈모 유발 인자인 DKK1의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated DKK1 detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응함. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.The expression of DKK1, an alopecia inducer, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated DKK1 detection antibody was treated on each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 5는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 DKK-1 (Dickkopf-1) 활성을 측정한 그래프이다. DKK-1은 세포괴사를 촉진하여 탈모를 유발하는 대표적인 negative cytokines 중의 하나로, 남성호르몬(DHT) 유도물질인 DKK-1 단백질이 Wnt/β-catenin 경로의 작용을 억제하여 탈모를 일으키는 것으로 알려져 있다.FIG. 5 is a graph showing DKK-1 (Dickkopf-1) activity according to the ratio and concentration of a mixture of Amara-Hibiscus extracts using Dermal Papilla cells. DKK-1 is one of the typical negative cytokines that promote apoptosis by promoting cell necrosis. It is known that DKK-1 protein, a male hormone (DHT) inducer, inhibits the action of the Wnt / β-catenin pathway and causes hair loss.
Testosterone을 처리하여 탈모 환경을 유발한 모유두 세포에 암라:히비스커스 복합 물질을 배합비율 및 농도별로 처리한 결과, 암라:히비스커스=9:1 내지 5:5의 비율에서 DKK-1의 활성이 많이 감소되었고, 6:4 비율에서 DKK-1의 활성이 가장 많이 감소하는 것을 확인하였으며, 농도별 에서는 50 mg/ml 농도가 가장 효과가 좋은 것으로 분석되었다. 특히, 암라 단독(10:0) 또는 히비스커스 단독(0:10)의 경우는 DKK-1 활성 감소 효과가 매우 미약하였으나, 이들을 조합한 경우 암라 또는 히비스커스 단독에 비해 시너지 효과가 나타남을 확인하였다.Testosterone was applied to the dermal papilla cells to induce hair loss, and the amount of DKK-1 was decreased at a ratio of Amara: Hibiscus = 9: 1 to 5: 5 , And 6: 4 ratio, respectively. The concentration of DKK-1 was most effective at 50 mg / ml concentration. In particular, in the case of Ala (10: 0) or Hibiscus alone (0:10), the effect of decreasing DKK-1 activity was very weak, but when they were combined, synergistic effect was observed compared with Ala or Hibiscus alone.
4) IGF1 Human Enzyme-Linked Immunosorbent Assay4) IGF1 Human Enzyme-Linked Immunosorbent Assay
발모 성장 인자인 IGF1의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated IGF1 detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응하였다. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.Expression of IGF1, a growth promoting factor, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated IGF1 detection antibody was treated on each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 6는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 IGF-1 활성을 측정한 그래프이다. IGF-1은 모낭 및 상피세포의 성장을 촉진하는 대표적인 성장인자로 알려져 있다. 또한, 모유두세포의 세포사멸을 방지하며, 모유두세포에서 IGF-1 생성에 남성호르몬이 직접적으로 영향을 미치는 것으로 알려져 있다.FIG. 6 is a graph showing IGF-1 activity according to the ratio and concentration of Amara-Hibiscus blend extracts using Dermal Papilla cells. IGF-1 is known to be a typical growth factor promoting the growth of hair follicles and epithelial cells. In addition, it is known that apoptosis of apopto cell is prevented and male hormone directly affects IGF-1 production in dermal papilla cells.
Testosterone을 처리하여 탈모 환경을 유발한 모유두 세포에 암라:히비스커스 복합 물질을 배합비율 및 농도별로 처리한 결과, 암라:히비스커스=9:1 내지 5:5의 비율에서 IGF-1의 활성이 많이 증가하였고, 7:3 비율에서 IGF-1의 활성이 가장 많이 증가하는 것을 확인하였으며, 농도별 에서는 100 mg/ml 농도가 가장 효과가 좋은 것으로 분석되었다. 특히, 암라 단독(10:0) 또는 히비스커스 단독(0:10)의 경우는 IGF-1 활성 증가 효과가 매우 미약하였으나, 이들을 조합한 경우 암라 또는 히비스커스 단독에 비해 시너지 효과가 나타남을 확인하였다.Testosterone was applied to the hair dermal papilla cells to induce hair loss environment. Amla: hibiscus complex was treated by blending ratio and concentration to increase the activity of IGF-1 at a ratio of hibiscus = 9: 1 to 5: 5 , And IGF-1 at the 7: 3 ratio. The concentration of IGF-1 was the most effective at 100 mg / ml concentration. Particularly, in the case of Ala (10: 0) or Hibiscus alone (0:10), the effect of increasing IGF-1 activity was very weak, but when they were combined, it was confirmed that synergy was more effective than that of Amara or Hibiscus alone.
5) VEGF Human Enzyme-Linked Immunosorbent Assay5) VEGF Human Enzyme-Linked Immunosorbent Assay
발모 성장 인자인 VEGF의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated VEGF detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응하였다. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.Expression of VEGF, a hair growth factor, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated VEGF detection antibody was added to each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 7는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 VEGF 활성을 측정한 그래프이다. VEGF는 혈관 내피를 성장시켜 혈액 순환을 개선하여 모발의 성장과 모근세포의 분화를 유도시키는 인자로 알려져 있으며 모발 관련 연구에 적용되는 중요지표인자 중의 하나이다.FIG. 7 is a graph showing VEGF activity according to the ratio and concentration of Amara-Hibiscus blend extract using Dermal Papilla cell. VEGF is known as a factor that induces hair growth and differentiation of hair follicle cells by improving blood circulation by growing vascular endothelium and is one of the important index factors applied to hair related research.
Testosterone을 처리하여 탈모 환경을 유발한 모유두 세포에 암라:히비스커스 복합 물질을 배합비율 및 농도별로 처리한 결과, 암라:히비스커스=9:1 내지 5:5의 비율에서 VEGF의 활성이 많이 증가하였고, 6:4 비율에서 VEGF의 활성이 가장 많이 증가하는 것을 확인하였으며, 농도별 에서는 50 mg/ml 농도가 가장 효과가 좋은 것으로 분석되었다. 특히, 암라 단독(10:0) 또는 히비스커스 단독(0:10)의 경우는 VEGF 활성 증가 효과가 매우 미약하였으나, 이들을 조합한 경우 암라 또는 히비스커스 단독에 비해 시너지 효과가 나타남을 확인하였다.Testosterone was applied to the hair dermal papilla cells to induce hair loss, and the amount of VEGF was increased in the ratio of Amara: Hibiscus = 9: 1 to 5: 5. : 4 ratio, the highest activity of VEGF was found to be the most effective at concentrations of 50 mg / ml. Especially, the effect of increasing the VEGF activity was very weak in the case of Ala (10: 0) or Hibiscus alone (0:10), but it was confirmed that synergistic effect was obtained when the combination of Ala or Hibiscus alone was used.
6) FGF Human Enzyme-Linked Immunosorbent Assay6) FGF Human Enzyme-Linked Immunosorbent Assay
발모 성장 인자인 FGF의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated FGF detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응하였다. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.The expression of FGF, a growth promoting factor, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated FGF detection antibody was treated on each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 8는 Dermal Papilla cell을 이용한 암라-히비스커스 혼합 추추물의 비율 및 농도에 따른 FGF 활성을 측정한 그래프이다. FGF는 모근을 보호하며 모발의 인장력을 강화시켜주는 성장인자이다. 콜라겐 합성유도 및 두피 환경을 개선하며 케라틴 재생 세포를 증식시켜 모발의 성장을 돕는 것으로 알려져 있다.FIG. 8 is a graph showing FGF activity according to the ratio and concentration of Amara-Hibiscus blend extract using Dermal Papilla cell. FGF is a growth factor that protects hair follicles and strengthens hair tension. It is known to improve collagen synthetic oil and scalp environment and to support hair growth by growing keratinocyte regenerating cells.
Testosterone을 처리하여 탈모 환경을 유발한 모유두 세포에 암라:히비스커스 복합 물질을 배합비율 및 농도별로 처리한 결과, 암라-히비스커스 배합물의 처리에 따른 FGF 활성이 증가하지 않는 것으로 확인되었으며, 대조군인 미녹시딜 처리군에서는 FGF 활성이 증가되는 것을 확인하였다. 따라서, 본원발명의 암라:히비스커스 복합 물질은 FGF와는 다른 성장인자를 통해 발모 성장을 촉진하는 것으로 보여진다.Testosterone was applied to the dermal papilla cells to induce hair loss environment. The results showed that the treatment of Amara: Hibiscus complex with the blend ratio and concentration resulted in no increase in FGF activity following treatment with Amara-Hibiscus blend, , It was confirmed that the FGF activity was increased. Thus, the inventive Amaras: Hibiscus complex is believed to promote hair growth through growth factors other than FGF.
실험예 3: NIH3T3 cell을 이용한 모세혈관 확장 효과 확인Experimental Example 3: Examination of capillary vasodilation effect using NIH3T3 cells
1) 암라:히비스커스 최적 배합비 및 최적 농도에서의 세포증식 촉진효과 확인1) Amla: Confirmation of cell proliferation promoting effect at optimal blending ratio and optimal concentration of hibiscus
암라:히비스커스 최적배합비 및 최적농도에 따른 세포증식효과를 확인하기 위하여 NIH3T3 세포를 이용하여 세포증식시험을 실시하였다. 세포는 12well 플레이트 기준 1x105/well으로 분주하며, 각각의 조건에 따른 물질을 처리하여 시험을 진행하였다. 물질처리 24시간 후 각 well에 MTT solution(5 mg/ml)을 20 μl씩 처리하고 1시간 동안 5% CO2, 37℃ 인큐베이터에서 반응을 진행하였다. 이후 용액 제거 후 1X PBS를 이용하여 세척한 후 DMSO 100 μl를 분주하여 540 nm에서 OD값을 측정하였다.Amla: Cell proliferation test was performed using NIH3T3 cells in order to confirm the cell proliferation effect according to the optimum blending ratio and optimum concentration of hibiscus. Cells were plated at 1 × 10 5 / well on a 12-well plate and processed according to the respective conditions. Twenty-four hours after the material treatment, 20 μl of MTT solution (5 mg / ml) was added to each well, and reaction was carried out in a 5% CO2 incubator at 37 ° C for 1 hour. After washing with 1X PBS, 100 μl of DMSO was added and the OD value was measured at 540 nm.
도 9는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 세포증식촉진 효과를 측정한 그래프이다. 전세계적으로 탈모치료제로 널리 알려진 미녹시딜이 모세 혈관 확장 효과를 나타내는 것으로 나타남에 따라 최적배합비 및 최적농도군에서의 암라:히비스커스 복합 물질의 NIH3T3 cell의 세포증식 촉진 효과를 확인한 결과, 음성대조군 대비 물질처리군(50 mg/ml, 7:3)에서 최대 130%까지 증가하는 것이 확인되었다.FIG. 9 is a graph showing the optimal mixing ratio of Amara-hibiscus blend extracts using the NIH3T3 cell line and promoting cell proliferation in the optimum concentration group. Minoxidil, which is widely known as a hair loss treatment drug worldwide, showed a capillary vasodilating effect. As a result of confirming the effect of the optimal blending ratio and the optimal concentration of Amaras: Hibiscus complex on cell proliferation of NIH3T3 cells, (50 mg / ml, 7: 3) up to 130%.
2) NIH3T3 cell에서의 발모 성장인자 분석 2) Analysis of hair growth factors in NIH3T3 cells
① IGF1 Human Enzyme-Linked Immunosorbent Assay① IGF1 Human Enzyme-Linked Immunosorbent Assay
발모 성장 인자인 IGF1의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated IGF1 detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응하였다. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.Expression of IGF1, a growth promoting factor, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated IGF1 detection antibody was treated on each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 10는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 IGF-1 활성을 측정한 그래프이다. NIH3T3 세포에서 발모성장인자인 IGF-1의 활성을 확인한 결과, 음성대조군 및 미녹시딜처리군 대비 물질처리군에서는 성장인자인 IGF-1의 값이 크게 증가하지 않는 것으로 확인되었다. 따라서, NIH3T3 cell에서는 다른 성장인자를 통해 모세혈관 확장 효과를 나타내는 것으로 보여진다.FIG. 10 is a graph showing the optimum blending ratio of amla-hibiscus blended extracts using the NIH3T3 cell line and the IGF-1 activity in the optimum concentration group. As a result of confirming the activity of IGF-1, a growth promoting factor in NIH3T3 cells, the value of IGF-1, which is a growth factor, was not significantly increased in the group treated with negative control and minoxidil. Therefore, NIH3T3 cells have been shown to exhibit capillary vasodilation effects through other growth factors.
② VEGF Human Enzyme-Linked Immunosorbent Assay② VEGF Human Enzyme-Linked Immunosorbent Assay
발모 성장 인자인 VEGF의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated VEGF detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응하였다. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응함. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다.Expression of VEGF, a hair growth factor, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated VEGF detection antibody was added to each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. Add 100 μl of 1X HRP-Streptavidin solution and react at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 11는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 VEGF 활성을 측정한 그래프이다. NIH3T3 세포에서 발모성장인자인 VEFG의 활성을 확인한 결과, 50 mg/ml, 7:3비율의 물질처리군에서 미녹시딜처리군과 유사한 정도의 VEGF값의 증가가 확인되었다.FIG. 11 is a graph showing the optimum mixing ratio of VHF and the optimum concentration of VHF activity in the mixture of Amara-Hibiscus using the NIH3T3 cell line. The activity of VEFG, a growth promoting factor in NIH3T3 cells, was confirmed to be similar to that of minoxidil treated group at 50 mg / ml and 7: 3 ratio.
③ FGF Human Enzyme-Linked Immunosorbent Assay ③ FGF Human Enzyme-Linked Immunosorbent Assay
발모 성장 인자인 FGF의 발현을 Enzyme-Linked Immunosorbent Assay 방법으로 측정하였다. 조건에 맞는 물질이 처리된 세포상등액과 표준시약 100 μl을 첨가한 플레이트를 상온에서 150분 동안 반응시켰다. 반응 후 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 이후 1X biotinylated FGF detection antibody 100 μl를 각 플레이트에 처리하여 1시간 동안 상온에서 반응하였다. 용액을 제거하고 1X 세척용액을 이용하여 4차례 세척하였다. 1X HRP-Streptavidin solution을 100 μl씩 첨가하고 45분 동안 상온에서 반응하였다. 용액을 제거한 후 1X 세척용액을 이용하여 4차례 세척하였다. 이후 TMB One-Step substrate reagent를 각 웰에 100 μl씩 첨가하여 암조건 하에서 상온에 30분간 반응하였다. 마지막으로 stop 용액을 50 μl 첨가하고 450 nm에서 값을 측정하였다. The expression of FGF, a growth promoting factor, was measured by Enzyme-Linked Immunosorbent Assay. The cell supernatants treated with the appropriate substances and the plate containing 100 μl of the standard reagent were reacted at room temperature for 150 minutes. After the reaction, the solution was removed and washed 4 times with 1X wash solution. Then, 100 μl of 1X biotinylated FGF detection antibody was treated on each plate and reacted at room temperature for 1 hour. The solution was removed and washed four times with 1X wash solution. 100 μl of 1 × HRP-Streptavidin solution was added and reacted at room temperature for 45 minutes. After removing the solution, it was washed 4 times with 1X washing solution. Then, 100 μl of TMB One-Step substrate reagent was added to each well and reacted at room temperature for 30 minutes under dark condition. Finally, 50 μl of stop solution was added and the value was measured at 450 nm.
도 12는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 FGF 활성을 측정한 그래프이다. NIH3T3 세포에서 발모성장인자인 FGF의 활성을 확인한 결과, 음성대조군 및 미녹시딜처리군 대비 물질처리군에서는 성장인자인 FGF 값이 크게 증가하지 않는 것으로 확인되었다. 따라서, NIH3T3 cell에서는 다른 성장인자를 통해 모세혈관 확장 효과를 나타내는 것으로 보여진다.FIG. 12 is a graph showing the optimum compounding ratio and the FGF activity in the optimum concentration group of the mixture of Amara-hibiscus using the NIH3T3 cell line. As a result of confirming the activity of FGF, a growth promoting factor in NIH3T3 cells, it was confirmed that the FGF value as a growth factor was not significantly increased in the group treated with negative control and minoxidil. Therefore, NIH3T3 cells have been shown to exhibit capillary vasodilation effects through other growth factors.
④ K+Channel opening effect확인 시험 ④ K + Channel opening effect confirmation test
혈관 확장과 관련된 K+Channel opening effect를 확인하였다. NIH3T3 세포를 12well 플레이트 기준 1x/well으로 분주하며, K+Channelinhibitor(Tolbutamide)1시간 선처리 후 각각의 조건에 따른 물질을 처리하여 시험을 진행하였다. 물질처리 24시간 후 각 well에 MTT solution(5 mg/ml)을 20 μl씩 처리하고 1시간 동안 5% CO2,37℃인큐베이터에서 반응을 진행하였다. 이후 용액 제거 후 1X PBS를 이용하여 세척한 후 DMSO 100 μl를 분주하여 540 nm에서 OD값을 측정하였다.The K + channel opening effect associated with vasodilation was confirmed. NIH3T3 cells were plated at 1 × / well on a 12-well plate and pretreated for 1 hour with K + channel inhibitor (Tolbutamide). Twenty-four hours after the material treatment, 20 μl of MTT solution (5 mg / ml) was added to each well and incubated for 1 hour in a 5% CO2 incubator at 37 ° C. After washing with 1X PBS, 100 μl of DMSO was added and the OD value was measured at 540 nm.
도 13는 NIH3T3 cell line을 이용한 암라-히비스커스 혼합 추추물의 최적배합비 및 최적농도군에서의 K channel opening effect를 확인한 그래프이다. 탈모치료제로 널리 알려진 미녹시딜이 NIH 3T3 세포에서 K+channel에 작용하여 혈관 팽창 및 모발 성장을 촉진시키는 것으로 알려져 있으며, 물질처리에 따라 K+channel이 열리면 NIH3T3세포가 증식이 촉진된다는 것을 추정할 수 있다.FIG. 13 is a graph showing the optimum compounding ratio of Amla-Hibiscus mixture using NIH3T3 cell line and the K channel opening effect in the optimum concentration group. It is known that minoxidil, which is widely known as a hair loss treatment, acts on K + channel in NIH 3T3 cells and promotes vascular expansion and hair growth. It can be estimated that NIH3T3 cells proliferate when K + .
이를 확실하게 확인하기 위하여 K+channel을 차단하는 것으로 알려진 K+channelinhibitor인 Tolbutamide를 선처리하여 K+channel차단 환경을 조성한 후에 시험물질 및 미녹시딜을 처리하여 NIH3T3세포의 증식능을 확인하였다. 그 결과 K+channel을 억제한 환경에서도 50 mg/ml 8:2, 7:3 비율의 물질에서 115%까지 세포증식이 일어나는 것을 확인할 수 있었다.To confirm this clearly, the K + channel blocking environment was prepared by pretreating Tolbutamide, a K + channel inhibitor known to block K + channel, and then the test substance and minoxidil were treated to confirm the proliferative activity of NIH3T3 cells. As a result, it was confirmed that the cell proliferation was up to 115% in the 50 mg / ml 8: 2 and 7: 3 ratio materials even in the K + channel-restricted environment.
Claims (6)
A composition for preventing hair loss or promoting hair growth, comprising an extract of Amara and a mixture of Hibiscus as an active ingredient.
The composition for promoting hair loss prevention or hair growth according to claim 1, wherein the mixed extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
The composition for promoting hair loss prevention or hair growth according to claim 1, wherein the mixed extract is obtained by mixing each plant extract or extracting a plant mixture.
The composition for promoting hair loss prevention or hair growth according to claim 1, wherein the mixed extract is mixed at a weight ratio of 9: 1 to 1: 9.
[2] The composition for promoting hair loss prevention or hair growth according to claim 1, wherein the extract is concentrated and powdered to be used as a material for a functional tea bag.
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