KR20240041582A - Manufcaturing method of cell activator with excellent skin penetration rate improvement effect using antioxidant matter delivery system - Google Patents
Manufcaturing method of cell activator with excellent skin penetration rate improvement effect using antioxidant matter delivery system Download PDFInfo
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- KR20240041582A KR20240041582A KR1020220120758A KR20220120758A KR20240041582A KR 20240041582 A KR20240041582 A KR 20240041582A KR 1020220120758 A KR1020220120758 A KR 1020220120758A KR 20220120758 A KR20220120758 A KR 20220120758A KR 20240041582 A KR20240041582 A KR 20240041582A
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- antioxidant
- glutathione
- penetration rate
- delivery system
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Abstract
본 발명은 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법에 관한 것으로, 더욱 상세하게는 글루타치온을 제조하는 글루타치온제조단계, 상기 글루타치온제조단계를 통해 제조된 글루타치온에 피크노제놀을 혼합하는 항산화물질혼합단계, 상기 항산화물질혼합단계를 통해 제조된 항산화물질의 표면에 인지질 막을 형성하는 인지질막형성단계 및 상기 인지질막형성단계를 통해 항상화 물질의 표면에 형성된 인지질 막 표면에 엑소좀-12을 부착하는 엑소좀부착단계로 이루어진다.
상기의 과정을 통해 제조되는 세포활성화제는 항산화물질의 피부침투율이 93% 이상을 나타내며, 세포를 활성화하여 면역력을 향상시키고 피부노화를 억제하는 효과를 나타낸다.The present invention relates to a method for producing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system. More specifically, it relates to a glutathione production step of producing glutathione, and mixing pycnogenol with the glutathione produced through the glutathione production step. An antioxidant mixing step, a phospholipid film forming step of forming a phospholipid film on the surface of the antioxidant prepared through the antioxidant mixing step, and an exosome on the surface of the phospholipid film formed on the surface of the antioxidant material through the phospholipid film forming step. It consists of the exosome attachment step of attaching 12.
The cell activator prepared through the above process exhibits a skin penetration rate of over 93% of antioxidants, and has the effect of activating cells to improve immunity and inhibit skin aging.
Description
본 발명은 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법에 관한 것으로, 더욱 상세하게는 항산화물질의 피부침투율이 93% 이상을 나타내며, 세포를 활성화하여 면역력을 향상시키고 피부노화를 억제하는 효과를 나타내는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법에 관한 것이다.The present invention relates to a method for manufacturing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system. More specifically, the skin penetration rate of antioxidants is over 93%, activating cells to improve immunity and skin This relates to a method for manufacturing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system that has an anti-aging effect.
최근 피부장벽의 기능과 중요성에 대한 관심이 높아지면서 기존의 항산화 기전을 중심으로 수행되어 온 연구가 점차 피부장벽 손상의 회복 및 조절에 관한 연구로 다양화되고 있다. 피부는 표피(epidermis), 진피(dermis), 피하조직(hypodermis)으로 구성된 기관으로 수분 유지 및 병원균을 포함한 외부 감염원의 침입을 방어하는 장벽기능(barrier function)을 수행한다. 피부장벽은 낮은 투과성으로 외부환경으로부터 인체를 보호하는 물리적 장벽뿐만 아니라, 천연보습인자(NMF, Natural Moisturizing Factor)를 통해 체내 수분을 유지하는 보습장벽, 항균 펩타이드(antimicrobial peptide)를 포함한 다양한 항균면역물질로 피부면역기능을 담당하는 화학적 장벽 기능을 동시에 수행하고 있다.Recently, as interest in the function and importance of the skin barrier has increased, research that has been conducted focusing on existing antioxidant mechanisms is gradually diversifying into research on the recovery and control of skin barrier damage. The skin is an organ composed of the epidermis, dermis, and hypodermis and performs a barrier function to retain moisture and protect against invasion by external infectious agents, including pathogens. The skin barrier is not only a physical barrier that protects the human body from the external environment with low permeability, but also a moisturizing barrier that retains moisture in the body through natural moisturizing factor (NMF), and various antibacterial immune substances including antimicrobial peptides. It simultaneously performs a chemical barrier function responsible for skin immune function.
한편, 피부노화에는 다양한 원인이 있는데, 특히 자외선과 호흡을 통해 생성되는 활성산소는 피부노화에 가장 중요한 원인으로 꼽힌다. 활성산소는 정상적인 대사과정에서 생성이 되며, 질병상태나 신체 스트레스가 높은 경우 과잉으로 발생하는데, 자외선에 노출되어 있는 경우에도 생성된다.Meanwhile, there are various causes of skin aging. In particular, ultraviolet rays and active oxygen generated through respiration are considered the most important causes of skin aging. Active oxygen is generated during normal metabolic processes, and is generated in excess during disease or high physical stress. It is also generated when exposed to ultraviolet rays.
활성산소는 피부 세포 및 조직 손상을 주도한다. 정상의 경우 비정상적인, 노화된 세포를 사멸하여 균형을 만들지만 과하게 발생될 경우 피부 항산화 방어망을 파괴하고 지질 과산화, 단백질 산화, 세포간질 성분을 파괴, 콜라겐과 엘라스틴 등 피부 단백질을 파괴하며, 그 결과 탄력감소, 주름, 색소 침착 등 피부 노화로 이어진다.Free radicals lead to skin cell and tissue damage. Normally, it creates balance by killing abnormal and aged cells, but if it occurs excessively, it destroys the skin's antioxidant defense network, causes lipid peroxidation, protein oxidation, destroys interstitial components, and destroys skin proteins such as collagen and elastin, resulting in elasticity. This leads to skin aging, including reduction, wrinkles, and pigmentation.
진피층의 결합조직을 포함하는 피부 단백질은 피부의 탄력과 보습 유지에 중요한 역할을 하며, 이는 세포 구성과 생성에 필수적인 단백질에서 기인한다고 볼 수 있다. 최근 단백질 섭취와 피부 수분의 증가에 양의 상관관계를 입증하는 연구 결과 있으며, 영양학적인 측면에서 양질의 단백질 섭취가 피부건강에 밀접한 관련이 있다고 유추할 수 있다. 그러나, 식품을 통한 단백질 섭취가 피부에 미치는 영향은 미미한 수준에 불과하다.Skin proteins, including the connective tissue of the dermal layer, play an important role in maintaining skin elasticity and moisture, and this can be attributed to proteins essential for cell composition and production. There has been recent research demonstrating a positive correlation between protein intake and an increase in skin moisture, and from a nutritional perspective, it can be inferred that high-quality protein intake is closely related to skin health. However, the effect of protein intake through food on the skin is only minimal.
항산화, 항노화 등의 특정 약리 작용을 가진 식물 추출물은 국내에 약 500여종 이상이 알려져 있지만, 대부분 피부에 미치는 효과가 검증되지 않은 상태이며, 식물추출물 안에는 수많은 성분들이 다양한 비율로 존재하고 있기 때문에 어떤 효과를 가진 유효물질을 찾는 것은 어려운 과정이며, 식물 성분 중 효과를 나타내는 유효물질이 하나의 성분으로써 단독으로 작용할 수도 있지만 여러 성분들의 상호작용을 통해서만 그 효과가 나타날 수도 있다. 더욱이 기존 다양한 식물 추출물은 피부 장벽 통과가 어려워 단지 이를 피부에 바르는 것만으로는 효과가 제한적인 문제점이 있었다.More than 500 types of plant extracts with specific pharmacological effects such as antioxidant and anti-aging are known in Korea, but most of them have not been verified for their effects on the skin, and since numerous components exist in various ratios in plant extracts, there is no doubt that some Finding an effective substance is a difficult process. Among plant ingredients, an effective substance may act alone as a single ingredient, but its effect may only appear through the interaction of several ingredients. Moreover, various existing plant extracts have difficulty passing through the skin barrier, so simply applying them to the skin has limited effectiveness.
본 발명의 목적은 항산화물질의 피부침투율이 93% 이상을 나타내며, 세포를 활성화하여 면역력을 향상시키고 피부노화를 억제하는 효과를 나타내는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법을 제공하는 것이다.The purpose of the present invention is to manufacture a cell activator with an excellent skin penetration rate improvement effect using an antioxidant delivery system that exhibits a skin penetration rate of 93% or more and has the effect of activating cells to improve immunity and inhibit skin aging. It provides a method.
본 발명의 목적은 글루타치온을 제조하는 글루타치온제조단계, 상기 글루타치온제조단계를 통해 제조된 글루타치온에 피크노제놀을 혼합하는 항산화물질혼합단계, 상기 항산화물질혼합단계를 통해 제조된 항산화물질의 표면에 인지질 막을 형성하는 인지질막형성단계 및 상기 인지질막형성단계를 통해 항상화 물질의 표면에 형성된 인지질 막 표면에 엑소좀-12을 부착하는 엑소좀부착단계로 이루어지는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법을 제공함에 의해 달성된다.The purpose of the present invention is to produce glutathione, a glutathione production step, an antioxidant mixing step of mixing pycnogenol with the glutathione produced through the glutathione production step, and forming a phospholipid film on the surface of the antioxidant produced through the antioxidant mixing step. Skin penetration rate improvement effect using an antioxidant delivery system, characterized in that it consists of a phospholipid film formation step and an exosome attachment step of attaching exosome-12 to the surface of the phospholipid film formed on the surface of the antioxidant substance through the phospholipid film formation step. This is achieved by providing a method for producing an excellent cell activator.
본 발명의 바람직한 특징에 따르면, 상기 글루타치온제조단계는 글루타치온을 고발현하는 균주를 탄소원, 질소원 및 아미노산이 함유된 배지에서 배양하여 이루어지는 것으로 한다.According to a preferred feature of the present invention, the glutathione production step is performed by culturing a strain that highly expresses glutathione in a medium containing a carbon source, a nitrogen source, and amino acids.
본 발명의 더 바람직한 특징에 따르면, 상기 글루타치온을 고발현하는 균주는 사카로마이세스 세레비지애 BB-01(S. cerevisiae BB-01) 균주(KCTC 18262P)인 것으로 한다.According to a more preferred feature of the present invention, the strain expressing high levels of glutathione is Saccharomyces cerevisiae BB-01 ( S. cerevisiae BB-01) strain (KCTC 18262P).
본 발명의 더욱 바람직한 특징에 따르면, 상기 항산화물질혼합단계는 글루타치온 100 중량부에 피크노제놀 50 내지 150 중량부를 혼합하여 이루어지는 것으로 한다.According to a more preferred feature of the present invention, the antioxidant mixing step is performed by mixing 50 to 150 parts by weight of pycnogenol with 100 parts by weight of glutathione.
본 발명의 더욱 더 바람직한 특징에 따르면, 상기 인지질막형성단계는 항산화물질이 구형 인지질의 이중막으로 감싸지도록 이루어지는 것으로 한다.According to an even more preferred feature of the present invention, the phospholipid film forming step is performed so that the antioxidant substance is surrounded by a double film of spherical phospholipids.
본 발명의 더욱 더 바람직한 특징에 따르면, 상기 구형 인지질 막은 180 내지 530 나노미터의 두께로 형성되는 것으로 한다.According to an even more preferred feature of the present invention, the spherical phospholipid film is formed to have a thickness of 180 to 530 nanometers.
본 발명의 더욱 더 바람직한 특징에 따르면, 상기 엑소좀-12은 항산화인자의 발현이 증가되어 있는 것으로 한다.According to an even more preferred feature of the present invention, the exosome-12 has increased expression of antioxidant factors.
본 발명에 따른 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법은 항산화물질의 피부침투율이 93% 이상을 나타내며, 세포를 활성화하여 면역력을 향상시키고 피부노화를 억제하는 효과를 나타내는 세포활성화제를 제공하는 탁월한 효과를 나타낸다.The method for producing a cell activator with excellent skin penetration rate improvement effect using the antioxidant delivery system according to the present invention shows a skin penetration rate of antioxidants of more than 93%, and has the effect of activating cells to improve immunity and inhibit skin aging. It has an excellent effect in providing a cell activator.
도 1은 본 발명에 따른 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법을 나타낸 순서도이다.
도 2는 본 발명에서 항산화 물질로 사용되는 피크노제놀의 항산화 효과를 비교하여 나타낸 그래프이다.
도 3은 본 발명을 통해 제조되는 세포활성화제가 피부에 침투하는 과정을 나타낸 개략도이다.Figure 1 is a flowchart showing a method for manufacturing a cell activator with excellent skin penetration rate improvement effect using the antioxidant delivery system according to the present invention.
Figure 2 is a graph comparing the antioxidant effect of pycnogenol, which is used as an antioxidant in the present invention.
Figure 3 is a schematic diagram showing the process by which the cell activator produced through the present invention penetrates the skin.
이하에는, 본 발명의 바람직한 실시예와 각 성분의 물성을 상세하게 설명하되, 이는 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 발명을 용이하게 실시할 수 있을 정도로 상세하게 설명하기 위한 것이지, 이로 인해 본 발명의 기술적인 사상 및 범주가 한정되는 것을 의미하지는 않는다.Below, preferred embodiments of the present invention and the physical properties of each component are described in detail, but are intended to be described in detail so that a person skilled in the art can easily carry out the invention. This does not mean that the technical idea and scope of the present invention are limited.
본 발명에 따른 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법은 글루타치온을 제조하는 글루타치온제조단계(S101), 상기 글루타치온제조단계(S101)를 통해 제조된 글루타치온에 피크노제놀을 혼합하는 항산화물질혼합단계(S103), 상기 항산화물질혼합단계(S103)를 통해 제조된 항산화물질의 표면에 인지질 막을 형성하는 인지질막형성단계(S105) 및 상기 인지질막형성단계(S105)를 통해 항상화 물질의 표면에 형성된 인지질 막 표면에 엑소좀-12을 부착하는 엑소좀부착단계(S107)로 이루어진다.The method for producing a cell activator with excellent skin penetration rate improvement effect using the antioxidant delivery system according to the present invention includes a glutathione production step (S101) of producing glutathione, and mixing pycnogenol with the glutathione produced through the glutathione production step (S101). antioxidant substance mixing step (S103), a phospholipid film forming step (S105) of forming a phospholipid film on the surface of the antioxidant prepared through the antioxidant mixing step (S103), and the phospholipid film forming step (S105). It consists of an exosome attachment step (S107) in which exosome-12 is attached to the surface of the phospholipid membrane formed on the surface of the material.
상기 글루타치온제조단계(S101)는 글루타치온을 항상화 물질로 사용할 수 있도록 배양하여 제조하는 단계로, 글루타치온을 고발현하는 균주를 탄소원, 질소원 및 아미노산이 함유된 배지에서 배양하여 이루어지는데, 더욱 상세하게는 생막걸리(전남 영암군 삼호읍 생막걸리)로부터 글루타치온을 생산하는 균주를 다음과 같이 분리하였다.The glutathione production step (S101) is a step of culturing and producing glutathione so that it can be used as an antioxidant, and is achieved by culturing a strain that highly expresses glutathione in a medium containing a carbon source, a nitrogen source, and amino acids. In more detail, A strain producing glutathione was isolated from Saengmakgeolli (Saengmakgeolli, Samho-eup, Yeongam-gun, Jeollanam-do) as follows.
먼저 채취한 생막걸리 시료 1 ㎖을 10 ㎖의 멸균 증류수와 잘 혼합한 후, 상기 시료를 멸균 증류수로 103, 104 및 105배 희석하고 각 희석 시료 0.1 ㎖을 취하여 YM 아가 평판 배지에 균일하게 자라도록 도말하고 35℃에서 하루 동안 배양하여 군락(colony)을 형성하였다. 군락의 크기가 2 ㎜이상 되는 군락을 선택한 후, 선택된 균주들을 3 ㎖의 YM 액체 배지를 포함하는 캡튜브에 접종하고 35℃에서 200 rpm으로 24시간 동안 진탕 배양한 후, 원심분리(12,000 rpm, 5분)하여 세포를 회수하였다. 회수한 세포에 1 ㎖의 식염수를 가하여 현탁한 후 원심분리하여 세포를 잘 세척하였다. 다시, 회수한 세포에 1 ㎖의 1% SDS 용액을 가하여 잘 현탁한 후 각각 10개의 유리구슬(직경 2-3 mm)을 넣어주고 미니비드비터(Biospec Products사, 미국)로 분당 2,000 ocillations의 속도로 1시간 동안 처리하여 세포를 파쇄하였다. 파쇄한 세포를 원심분리(13,000 rpm, 10분)하여 상층액을 회수하고 글루타치온 양을 측정하였다. 그 결과, 글루타치온 함량이 가장 뛰어난 균주를 선별하였고, 선별한 균주를 동정하였다. 상기 균주의 18S 리보소말 RNA의 염기서열을 분석한 결과, 서열목록 제1서열에 기재된 염기서열의 18S 리보소말 RNA를 갖는 것으로 밝혀졌고, 상기 염기서열을 분석한 결과 본 발명의 균주는 사카로마이세스 속 균주이며, 더욱 상세하게는 사카로마이세스 세레비지애 BB-01(S. cerevisiae BB-01) 균주(기탁번호: KCTC18262P)로 확인되었다.First, 1 ml of the collected fresh makgeolli sample was mixed well with 10 ml of sterilized distilled water, then the sample was diluted 10 3 , 10 4 and 10 5 times with sterilized distilled water, and 0.1 ml of each diluted sample was taken and distributed uniformly on YM agar plate medium. It was plated to grow well and cultured at 35°C for one day to form colonies. After selecting a colony with a colony size of 2 mm or more, the selected strains were inoculated into a cap tube containing 3 ml of YM liquid medium, cultured with shaking at 35°C at 200 rpm for 24 hours, and then centrifuged (12,000 rpm, 5 minutes) and the cells were recovered. 1 ml of saline was added to the recovered cells to suspend them, and the cells were washed well by centrifugation. Again, 1 ml of 1% SDS solution was added to the recovered cells and suspended well, then 10 glass beads (diameter 2-3 mm) were added to each, and the cells were beaten at a speed of 2,000 ocillations per minute using a mini bead beater (Biospec Products, USA). The cells were disrupted by treatment for 1 hour. The disrupted cells were centrifuged (13,000 rpm, 10 minutes) to recover the supernatant, and the amount of glutathione was measured. As a result, the strain with the highest glutathione content was selected, and the selected strain was identified. As a result of analyzing the nucleotide sequence of the 18S ribosomal RNA of the strain, it was found to have 18S ribosomal RNA of the nucleotide sequence described in the first sequence of the sequence list, and as a result of analyzing the nucleotide sequence, the strain of the present invention was Saccharomyces. It is a strain of the genus Seth, and more specifically, it was identified as Saccharomyces cerevisiae BB-01 ( S. cerevisiae BB-01) strain (accession number: KCTC18262P).
이때, 상기 글루타치온의 정량은 96웰 플레이트에 50 ㎕의 시료 용액 또는 글루타치온 표준 용액을 넣고 100 ㎕의 반응용액(575 ㎖의 반응 완충용액(100 mM 인산타트륨 완충용액,pH 75/1 mM EDTA), 01 ㎖의 GSH 환원효소(200 U/ml), 5 ㎖의 1 mM DTNB((5,5-dithio-bis 2-nitrobenzoic acid) 및 5 ㎖의 1 mM NADPH 혼합액, 사용하기 직전에 혼합하여 사용)을 가한 후 간단하게 혼합하여 준 후, 상온에서 20분간 방치한 후 반응액의 흡광도는 410 nm(Spectramax PLUS 384, Molecular Device사. 미국)에서 측정하였다. 이 때, 0-1 ㎍의 글루타치온을 이용하여 표준곡선을 구하였고 시료의 흡광도를 글루타치온 표준 곡선에 대입하여 시료의 글루타치온 양을 계산하는 방법을 이용하였다.At this time, the quantification of glutathione was performed by adding 50 ㎕ of sample solution or glutathione standard solution to a 96-well plate and adding 100 ㎕ of reaction solution (575 ㎖ of reaction buffer (100 mM potassium phosphate buffer, pH 75/1 mM EDTA)). , 01 ml of GSH reductase (200 U/ml), 5 ml of 1 mM DTNB ((5,5-dithio-bis 2-nitrobenzoic acid) and 5 ml of 1 mM NADPH mixture, mixed immediately before use. ) was added, mixed briefly, left at room temperature for 20 minutes, and the absorbance of the reaction solution was measured at 410 nm (Spectramax PLUS 384, Molecular Device, USA). At this time, 0-1 μg of glutathione was added. A standard curve was obtained using a method to calculate the amount of glutathione in the sample by substituting the absorbance of the sample into the glutathione standard curve.
글루타치온(glutathione, GSH)은 글루탐산(glutamate), 시스테인(cystein), 글라이신(glycine) 세 가지 아미노산으로 구성된 트리펩타이드(L-γ-glutamyl-L-cysteinylglycine)이며, 티올기(-SH)를 가지고 있는 세포 내 수용성 항산화제 역할을 할 뿐만 아니라 여러 가지 항산화 효소들(예; 글루타치온 퍼록시다아제 등)의 보조인자 역할을 하는 중요한 물질이다(Kidd, PM, Alt Met Rev 1997, 2, 155-176).Glutathione (GSH) is a tripeptide (L-γ-glutamyl-L-cysteinylglycine) composed of three amino acids: glutamate, cysteine, and glycine, and has a thiol group (-SH). It is an important substance that not only acts as an intracellular water-soluble antioxidant but also acts as a cofactor for various antioxidant enzymes (e.g. glutathione peroxidase, etc.) (Kidd, PM, Alt Met Rev 1997, 2, 155-176) .
글루타치온은 대부분의 생물에 존재하며, 사람의 경우 조직에 따라 차이가 있지만 세포 내에서 01-10 mM의 농도로 존재하며, 간에서 가장 많은 농도로 존재하며(10 mM 까지), 그 외, 비장, 신장 등에서도 많은 농도로 존재한다. 글루타치온의 항산화활성은 이 물질이 가지고 있는 티올기의 강력한 전자 제공 능력 때문이며, 환원형(GSH) 및 산화형(GS-SG)으로 존재할 수 있으며, 환원형의 시스테인의 티올기가 전자를 제공하여 자신은 다른 환원형 글루타치온과 반응하여 산화형인 이황화 글루타치온(GS-SG)을 형성하면서 항산화 기능을 나타낸다(GSH + GSH GS-SG) 세포내에서 산화형 글루타치온은 GSH 환원효소에 의해 다시 환원형으로 재생되어 항산화 활성을 얻게 된다.Glutathione exists in most living things. In humans, it exists at a concentration of 01-10mM within cells, although there are differences depending on the tissue. It is present at the highest concentration in the liver (up to 10mM), and in other places, the spleen, It is also present in large concentrations in the kidneys. The antioxidant activity of glutathione is due to the strong electron-donating ability of the thiol group of this substance. It can exist in a reduced form (GSH) and an oxidized form (GS-SG), and the thiol group of cysteine in the reduced form donates electrons to protect itself. It reacts with other reduced forms of glutathione to form the oxidized form of glutathione disulfide (GS-SG) and exhibits antioxidant properties (GSH + GSH GS-SG). Within cells, the oxidized glutathione is regenerated back to its reduced form by GSH reductase and acts as an antioxidant. becomes active.
또한, 글루타치온은 일반 폴리페놀에 비해 100만배나 강한 항산화 효과를 나타내는 것으로 알려져 있으며, 알츠하이머 치매, 파킨슨, 백내장, 천식 및 관절염등과 같은 다양한 질병의 개선 및 보조제로 사용되고 있으며, 주로 셀레늄, 엉겅퀴(밀크시슬), 비타민 C, 비타민 E등에 다량함유되어 있는데, 항산화 작용을 돕는 결정적인 역할을 한다.In addition, glutathione is known to have an antioxidant effect that is 1 million times stronger than that of general polyphenols, and is used as an improvement and supplement for various diseases such as Alzheimer's dementia, Parkinson's, cataracts, asthma, and arthritis. It is mainly used as a supplement for selenium and thistle (milk). Thistle), vitamin C, vitamin E, etc. are contained in large quantities, and play a critical role in supporting antioxidant action.
상기 항산화물질혼합단계(S103)는 상기 글루타치온제조단계(S101)를 통해 제조된 글루타치온에 피크노제놀을 혼합하는 단계로, 상기 글루타치온제조단계(S101)를 통해 제조된 글루타치온 100 중량부에 피크노제놀 50 내지 150 중량부를 혼합하여 이루어지는 것이 바람직하다.The antioxidant mixing step (S103) is a step of mixing pycnogenol with the glutathione produced through the glutathione production step (S101), and 50 to 150 parts by weight of pycnogenol per 100 parts by weight of glutathione produced through the glutathione production step (S101). It is preferable to mix parts.
상기 피크노제놀(Pycrngenol)은 프랑스 남부 해안송의 껍질에서 추출한 성분으로 4,000가지 파이토케미컬이 포함되어 있는 천연물질로서 세찬 바닷바람, 습기, 자외선 등과 같은 열악한 자연환경에서 해안송이 자라다보면 5 내지 10 센티미터의 두꺼운 껍질이 형성되는데, 이러한 해안송의 껍질에 항균, 항암, 항바이러스 및 항알레르기 작용을 하는 플라보노이드가 다량 함유되어 있다.Pycrngenol is a natural substance extracted from the bark of coastal pine in southern France and contains 4,000 phytochemicals. When coastal pine grows in harsh natural environments such as strong sea breezes, moisture, and ultraviolet rays, it grows to a thickness of 5 to 10 centimeters. The bark is formed, and the bark of coastal pine contains a large amount of flavonoids that have antibacterial, anticancer, antiviral, and antiallergic effects.
또한, 피크노제놀은 코엔자임 Q10과 비타민 C가 다량함유되어 매우 우수한 항산화 기능을 나타내는데, 종래에 항산화 물질인 코엔자임 Q10, 비타민 C, 리포산, 비타민 E 및 포도씨와의 항산화 효능을 비교하여 아래 표 2에 나타내었다.In addition, Pycnogenol contains a large amount of coenzyme Q10 and vitamin C, showing excellent antioxidant function. The antioxidant efficacy of pycnogenol with conventional antioxidants such as coenzyme Q10, vitamin C, lipoic acid, vitamin E, and grape seed is compared and shown in Table 2 below. .
아래 표 2에 나타낸 것처럼, 피크노제놀은 종래에 항산화 물질인 코엔자임 Q10, 비타민 C, 리포산, 비타민 E 및 포도씨에 비해 항산화효능이 매우 우수한 것을 알 수 있다.As shown in Table 2 below, it can be seen that pycnogenol has very excellent antioxidant efficacy compared to conventional antioxidant substances such as coenzyme Q10, vitamin C, lipoic acid, vitamin E, and grape seed.
또한, 상기 글루타치온(Glutathion)은 아래 구조식 1에 나타낸 것처럼, In addition, glutathione is as shown in structural formula 1 below,
<구조식 1><Structural Formula 1>
시스테인(Cysteine), Glutamic acid 및 Glycine이라는 아미노산 3개로 이루어진 작은 단백질로, DNA 회복에 필수 효소로 매우 강력한 항산화 효과를 발휘하며, 알코올 중독이나 급성간염 및 만성간염 치료 보조제로 사용되고 있다.It is a small protein made up of three amino acids, Cysteine, Glutamic acid, and Glycine. It is an essential enzyme for DNA repair and has a very strong antioxidant effect. It is used as an adjunct in the treatment of alcoholism, acute hepatitis, and chronic hepatitis.
또한, 피부의 흑색멜라닌을 만드는 타이로시나제의 활성을 억제함으로서 대사를 활성화시키고 피부재생 내지는 피부색을 맑게 만들어주는 역할을 한다.In addition, it plays a role in activating metabolism and regenerating skin or making skin color clear by suppressing the activity of tyrosinase, which produces black melanin in the skin.
인체는 노화가 진행됨에 따라 근육, 뼈, 인지 기능 및 혈관 건강이 자연스럽게 저하될 뿐만아니라 치매나 알츠하이머와 같은 노인성 질환의 위험성이 빠르게 증가하는데, 글루타치온은 퇴행성 신경 질환의 진행을 억제하며 노화와 관련하여 발생하는 건강 문제를 효과적으로 예방하는 역할을 한다.As the human body ages, not only does muscle, bone, cognitive function, and vascular health naturally decline, but the risk of geriatric diseases such as dementia and Alzheimer's rapidly increases. Glutathione inhibits the progression of degenerative neurological diseases and helps with aging. It effectively prevents health problems from occurring.
상기 인지질막형성단계(S105)는 상기 항산화물질혼합단계(S103)를 통해 제조된 항산화물질의 표면에 인지질 막을 형성하는 단계로, 상기 항산화물질혼합단계(S103)를 통해 제조된 항산화물질의 표면을 구형 인지질의 이중막으로 감싸지도록 이루어는데, 상기 구형 인지질 막은 180 내지 530 나노미터의 두께로 형성되는 것이 바람직하다.The phospholipid film forming step (S105) is a step of forming a phospholipid film on the surface of the antioxidant material prepared through the antioxidant mixing step (S103), and the surface of the antioxidant material prepared through the antioxidant mixing step (S103) is formed. It is made to be surrounded by a double membrane of spherical phospholipids, and the spherical phospholipid membrane is preferably formed to have a thickness of 180 to 530 nanometers.
상기와 같이 인지질막으로 감싸진 항산화 물질은 피부의 표피층이나 진피층으로 진입하기 전이나 진입하는 과정에서 손상이 억제되어 우수한 항산화 효과를 피부 세포에 전달할 수 있다.As described above, antioxidant substances wrapped in a phospholipid membrane can deliver excellent antioxidant effects to skin cells by suppressing damage before or during entry into the epidermal or dermal layer of the skin.
이때, 상기 구형 인지질은 180 내지 530 나노미터의 두께를 나타내는 것이 바람직한데. 상기 구형 인지질의 두께가 180 나노미터 미만이면 상기의 효과가 미미하며, 상기 구형 인지질의 두께가 530 나노미터를 초과하게 되면 구형 인지질의 두께가 지나치게 두꺼워져 세포활성화제의 크기를 증가시키기 때문에, 피부의 표피층이나 진피층의 침투효과가 저하될 수 있다.At this time, the spherical phospholipid preferably has a thickness of 180 to 530 nanometers. If the thickness of the spherical phospholipid is less than 180 nanometers, the above effect is minimal, and if the thickness of the spherical phospholipid exceeds 530 nanometers, the thickness of the spherical phospholipid becomes too thick, increasing the size of the cell activator, so that the skin The penetration effect of the epidermis or dermis layer may be reduced.
상기 엑소좀부착단계(S107)는 상기 인지질막형성단계(S105)를 통해 항상화 물질의 표면에 형성된 인지질 막 표면에 엑소좀-12을 부착하는 단계로, 상기 인지질막형성단계(S105)를 통해 인지질 막의 표면에 엑소좀-12을 부착하여 본 발명을 통해 제조된 세포활성화제가 진피층에 흡수된 후 활성산소의 자유 라디칼을 흡입할 수 있도록 하는 단계다.The exosome attachment step (S107) is a step of attaching exosome-12 to the surface of the phospholipid membrane formed on the surface of the antioxidant through the phospholipid membrane formation step (S105). This is the step of attaching exosome-12 to the surface of the phospholipid membrane so that the cell activator prepared through the present invention can be absorbed into the dermal layer and then absorb free radicals of active oxygen.
이때, 상기 엑소좀-12의 부착은 특별히 한정되지 않고 인지질 막에 통상적으로 엑소좀-12을 부착하는 과정으로 이루어질 수 있으며, 상기 엑소좀-12은 항산화인자의 발현이 증가되어 있는 것을 사용하는 것이 바람직하다.At this time, the attachment of the exosome-12 is not particularly limited and can be accomplished through a process of attaching the exosome-12 to a phospholipid membrane, and the exosome-12 that has increased expression of antioxidant factors is used. desirable.
본 발명에서 '엑소좀-12(exosome-12)'이란 다양한 세포들로부터 분비되는 막 구조의 작은(대략 30-100 nm의 직경) 소낭을 의미하며, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미하는데, 상기 엑소좀-12은 자연적으로 분비된 것이거나, 혹은 인공적으로 분비된 것을 포함한다.In the present invention, 'exosome-12' refers to a small (diameter of approximately 30-100 nm) membrane-structured vesicle secreted from various cells, and the fusion of the multivesicular body and the plasma membrane occurs and enters the extracellular environment. It refers to a vesicle that is released, and the exosome-12 includes those secreted naturally or artificially.
또한, 본 발명에서 엑소좀-12을 분리하는 방법에는 제한이 없으며, 예를 들면 배양액에서, 원심분리, 초고속 원심분리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 모세관 전기영동, 폴리머를 이용한 분리 등의 방법 및 이들의 조합을 이용하여 분리할 수 있으며, 바람직하게는 원심분리/초원심분리이다. 이때, 원심분리/초원심분리는, 4℃, 3,000-100,000g에서 10분 내지 5시간 동안 행하는 것이 바람직하다.In addition, there is no limitation to the method of isolating exosome-12 in the present invention, for example, from culture medium, centrifugation, ultra-high-speed centrifugation, filtration by filter, gel filtration chromatography, free-flow electrophoresis, capillary electrophoresis. , separation using polymers, etc., and combinations thereof, preferably centrifugation/ultracentrifugation. At this time, centrifugation/ultracentrifugation is preferably performed at 4°C and 3,000-100,000g for 10 minutes to 5 hours.
본 발명에서 세포 배양에 이용되는 배지는, 당, 아미노산, 각종 영양물질, 혈청, 성장인자, 무기질 등의 세포의 성장 및 증식 등에 필수적인 요소를 포함하는 생체 외(in vitro)에서 줄기세포 등의 세포의 성장 및 증식을 위한 혼합물을 의미한다. 본 발명에서 이용될 수 있는 배지는 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM(α-Minimal essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMDM(Isocove's Modified Dulbecco's Medium) 및 KnockOut DMEM 등의 상업적으로 제조된 배지 또는 인위적으로 합성한 배지를 포함하나, 이에 한정되지 않는다.The medium used for cell culture in the present invention contains essential elements for cell growth and proliferation, such as sugars, amino acids, various nutrients, serum, growth factors, and minerals, and can be used to cultivate cells such as stem cells in vitro. refers to a mixture for growth and proliferation. The media that can be used in the present invention are DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10) , DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM. This includes, but is not limited to, commercially produced media or artificially synthesized media such as these.
상기의 엑소좀부착단계를 거치면, 본 발명에 따른 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조가 완료되는데, 상기의 과정을 통해 제조된 세포활성화제는 항산화물질의 피부침투율이 93% 이상을 나타내며, 세포를 활성화하여 면역력을 향상시키고 피부노화를 억제하는 효과를 나타내며, 진피층까지 침투하여 진피층 내에 활성산소를 흡수하여 항산화효과를 부여할 수 있다.After going through the above exosome attachment step, the production of a cell activator with excellent skin penetration rate improvement effect using the antioxidant delivery system according to the present invention is completed. The cell activator manufactured through the above process has a skin penetration rate of antioxidants. It represents more than 93%, and has the effect of activating cells to improve immunity and suppress skin aging. It can also penetrate into the dermis layer and absorb active oxygen within the dermis layer to provide an antioxidant effect.
뿐만 아니라, 엑소좀부착단계를 통해 인지질 막의 표면에 엑소좀-12이 부착되면 인지질 막으로 감싸진 항산화원료가 피부의 표면층 뿐만 아니라 두피의 진피층에 대한 침투율이 월등하게 향상되어 피부에 항산화효과를 부여할 수 있다.In addition, when exosome-12 is attached to the surface of the phospholipid membrane through the exosome attachment step, the penetration rate of the antioxidant raw material wrapped in the phospholipid membrane into not only the surface layer of the skin but also the dermal layer of the scalp is significantly improved, giving an antioxidant effect to the skin. can do.
상기의 효과를 나타내는 세포활성화제는 본 발명을 통해 제조되는 항산화물질 추출을 통한 피부 침투력 증강과 면역성 제고와 노화예방을 위한 엑소좀-12 조성물 1ml당 1억개 이상 함유되어야 상기의 효과를 나타낼 수 있다.The cell activator that exhibits the above effects must contain at least 100 million per ml of the exosome-12 composition for enhancing skin penetration, improving immunity, and preventing aging through the extraction of antioxidants prepared through the present invention to exhibit the above effects. .
따라서, 본 발명에 따른 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법은 항산화물질의 피부침투율이 93% 이상을 나타내며, 세포를 활성화하여 면역력을 향상시키고 피부노화를 억제하는 효과를 나타내는 세포활성화제를 제공한다.Therefore, the method for producing a cell activator with excellent skin penetration rate improvement effect using the antioxidant delivery system according to the present invention has a skin penetration rate of antioxidants of more than 93%, and activates cells to improve immunity and inhibit skin aging. Provides an effective cell activator.
이상에서 본 발명은 실시예를 중심으로 상세히 설명되었지만 본 발명의 기술사상 범위 내에서 다양한 변형 및 수정이 가능함은 당업자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속함은 당연한 것이다.In the above, the present invention has been described in detail focusing on the embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the technical scope of the present invention, and it is natural that such changes and modifications fall within the scope of the appended patent claims.
S101 ; 글루타치온제조단계
S103 ; 항산화물질혼합단계
S105 ; 인지질막형성단계
S107 ; 엑소좀부착단계S101 ; Glutathione manufacturing steps
S103 ; Antioxidant mixing step
S105 ; Phospholipid membrane formation stage
S107 ; Exosome attachment step
Claims (7)
상기 글루타치온제조단계를 통해 제조된 글루타치온에 피크노제놀을 혼합하는 항산화물질혼합단계;
상기 항산화물질혼합단계를 통해 제조된 항산화물질의 표면에 인지질 막을 형성하는 인지질막형성단계; 및
상기 인지질막형성단계를 통해 항상화 물질의 표면에 형성된 인지질 막 표면에 엑소좀-12을 부착하는 엑소좀부착단계;로 이루어지는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.
Glutathione production step of producing glutathione;
An antioxidant mixing step of mixing pycnogenol with the glutathione produced through the glutathione production step;
A phospholipid film forming step of forming a phospholipid film on the surface of the antioxidant material prepared through the antioxidant material mixing step; and
Cell activation with excellent skin penetration rate improvement effect using an antioxidant delivery system, characterized in that it consists of an exosome attachment step of attaching exosome-12 to the surface of the phospholipid membrane formed on the surface of the antioxidant material through the phospholipid membrane formation step. Manufacturing method.
상기 글루타치온제조단계는 글루타치온을 고발현하는 균주를 탄소원, 질소원 및 아미노산이 함유된 배지에서 배양하여 이루어지는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.
In claim 1,
The glutathione production step is a method for producing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system, characterized in that the strain that highly expresses glutathione is cultured in a medium containing a carbon source, a nitrogen source, and an amino acid.
상기 글루타치온을 고발현하는 균주는 사카로마이세스 세레비지애 BB-01(S. cerevisiae BB-01) 균주(KCTC 18262P)인 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.
In claim 2,
The strain that highly expresses glutathione is Saccharomyces cerevisiae BB-01 ( S. cerevisiae BB-01) strain (KCTC 18262P). Cell activation with excellent skin penetration rate improvement effect using an antioxidant delivery system. Manufacturing method.
상기 항산화물질혼합단계는 글루타치온 100 중량부에 피크노제놀 50 내지 150 중량부를 혼합하여 이루어지는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.
In claim 1,
The antioxidant mixing step is a method of producing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system, characterized in that it is achieved by mixing 50 to 150 parts by weight of pycnogenol with 100 parts by weight of glutathione.
상기 인지질막형성단계는 항산화물질이 구형 인지질의 이중막으로 감싸지도록 이루어지는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.
In claim 1,
The phospholipid film forming step is a method of producing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system, characterized in that the antioxidant is wrapped in a double membrane of spherical phospholipids.
상기 구형 인지질 막은 180 내지 530 나노미터의 두께로 형성되는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.
In claim 5,
A method for producing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system, wherein the spherical phospholipid film is formed with a thickness of 180 to 530 nanometers.
상기 엑소좀-12은 항산화인자의 발현이 증가되어 있는 것을 특징으로 하는 항산화물질 전달 시스템을 이용한 피부침투율 향상효과가 우수한 세포활성화제의 제조방법.In claim 1,
The exosome-12 is a method of producing a cell activator with excellent skin penetration rate improvement effect using an antioxidant delivery system, characterized in that the expression of antioxidant factors is increased.
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