오디 알코올 추출물의 항산화 및 미백기능성 분석

Abstract

기존사회와는 다른 현대사회의 향상된 생활수준으로 사람들의 관심이 연령과 성별에 관계없이 노화 방지에 집중되고 있다. 그에 맞게 노화 예방에 효과적인 많은 생리활성 물질들을 이용하는 연구가 많이 진행 중에 있다. 산소는 대기 중의 약 21 %를 차지하는 지구상에 존재하는 가장 많은 원소이다. 모든 호기성 생물은 생명 유지에 절대적으로 필요한 산소를 호흡을 통해 에너지로 획득하게 되는데, 산소와 영양물질이 반응해 에너지를 만드는 대사과정, 체내 효소계, 환원대사의 내적요인, 화학약품, 공해물질, 광화학 반응 (Photochemical reaction) 등 각종 물리적 화학적, 환경적 외적 요인 등에 의해 Superoxide radical, Hydroxyl radical, Hydrogen peroxide, Singlet oxygen의 반응성이 큰 활성산소(Reactive oxygen)로 전환된다. 이런 활성산소는 체내의 미생물에 방어적으로 유용하게 이용되지만 생체에 유해한 산화적 스트레스 (Oxidative stress)를 일으키는 양면성도 지닌다. 사람의 피부색은 인종, 환경, 연령 등에 따라 다른데, 이를 결정하는 가장 큰 요인은 멜라닌 (Melanin)이라는 자연계에 존재하는 생체 고분자 물질이다. 멜라닌은 인간의 피부 색깔을 결정짓는 색소로 알려져 있으며, 자외선이나 활성산소(Free radical)로부터 피부가 손상되는 것을 방지하는 역할을 한다. 그러나 과잉 생성 및 축적이 될 시에는 흑색종, 주근깨, 검버섯 등 색소 침착 증후군과 같은 다양한 피부병 질환을 유발할 수 있다. 멜라닌은 멜라닌 세포 (Melanocyte)의 멜라닌 소체 (Melanosome)라는 소기관에서 생산되어 햇빛, 스트레스 및 멜라닌 자극 인자에 대한 피부의 노출에 대한 반응으로 표피의 기저층에 있는 각질 세포로 전이된다. 멜라닌의 생합성 과정에는 Tyrosine이라는 주요 효소가 작용하는데, Polyphenol oxidase의 일종으로 구리를 함유한 효소이며 Tyrosinase, Tyrosinase related protein-1 (TRP-1), Tyrosinase related protein-2 (TRP-2)과 cyclic Adenosine monophosphate (cAMP) 유도물질인 Adrenocoticotropic hormone (ACTH), Forskoline과 α-Melanocyte stimulating hormone (α-MSH) 등에 의해 조절된다. 멜라닌은 인체표피층의 Melanocyte 내의 Melanosome에서 합성되는데, Tyrosinase가 Tyrosine을 산화시켜 L-3,4-dihydroxyphenylalanine (L-DOPA)으로 변환하고 효소적 화학반응에 의해 DOPA quinone으로 전환된다. 이후 자동산화반응과 효소반응으로 DOPA chrome을 거쳐 흑갈색의 멜라닌을 생성시킨다. 따라서 흑갈색의 공동합체인 멜라닌의 생성을 억제하는 Tyrosinase 활성 억제 실험은 피부 미백제 개발에 유용한 평가법으로 사용되고 있다. 최근 페놀계 합성 항산화제 BHA (Butylated Hydroxyanisole), BHT (Butylated Hydroxytoluene) 등의 부작용들이 밝혀져 천연 자원으로부터 항산화제를 개발하려는 연구가 많이 이루어지고 있다. 천연 자원에서 얻어진 항산화 물질들이 합성 항산화제만큼의 항염증 능력이 있다고 알려진 연구도 나왔으며, 식물로부터 추출한 천연 추출물은 자외선 흡수, 항염증 작용, 항산화 작용, Collagenase와 같은 효소 억제, Tyrosinase 억제와 더불어 경피 (經皮)의 흡수력을 향상 시켜주는 등 다양한 작용을 통해 피부를 보호하는 효과를 나타내기도 한다. 피부의 광노화를 유발하고, 검버섯과 기미, 주근깨 등을 생성시키는 주요 요인으로 산화적 스트레스는 미백과 연관되어 함께 연구되고 있다. 이를 통해 항산화 물질과 활성산소 소거를 통해 산화과정이 진행되면서 형성되는 멜라닌 색소를 막을 수 있다고 보인다. 따라서 식물로부터 추출한 천연 추출물을 이용해 항산화와 미백기능성 등의 기능을 보고 이를 통해 기능성 화장품 연구가 활성화 될 것이다.

오디는 뽕나무의 열매로 달고 차며 독이 없고, 혈기를 통하게 한다. 꽃의 색소는 플라보노이드계에 속하는 안토시아닌으로서 주황색, 분홍색, 보라색 등을 나타내고 열매인 오디는 색소를 다량으로 함유한 과실로서 주로 안토시아닌 계통의 색소를 가지고 있으며 cyanidin-3-glucoside와 cyanidin-3-rutinoside가 주성분이다.

그래서 본 연구에서는 항산화 및 미백기능성을 가지고 있을 것으로 예상되는 오디 (Morus alba)를 추출하여 대식 세포주에서 LPS 자극 후 NO 생성량 감소를 통해 항염증 효능 분석, DPPH radical 소거능, 유사 SOD 활성 측정, 폴리페놀 및 플라보노이드 함량 분석을 통해 추출물의 항산화 효능을 분석하고 Melanoma 세포인 B16F10에서 Tyrosinase 저해 활성 및 Melanin 생성량 억제 효능을 분석하고자 한다. |obic organisms use breathing as energy to obtain oxygen, which is vital to maintaining life. Hydrogen radical, Hydrogen peroxide , Singlet oxygen are various physical and chemical factors such as reactive oxygen photochemical reaction, internal factors that reduce the energy, internal enzymes, metabolism by the reaction of oxygen and nutrients, Chemical and environmental factors. These reactive oxygen species are useful for protecting microorganisms in the body, but they also have both sides that cause oxidative stress that is harmful to the body.

The skin color of a person varies according to race, environment, age, etc. The most important factor in determining this is the biomolecule material in the natural world called melanin. Melanin is known as a pigment that determines the color of human skin, and it prevents skin damage from ultraviolet rays or free radicals. However, overproduction and accumulation may lead to various skin diseases such as melanoma, freckles, black spot, and pigmentation syndrome. Melanin is produced in the organelle called Melanosome of Melanocyte and transformed into keratinocytes in the basal layer of the epidermis in response to exposure of the skin to sunlight, stress and melanin stimulating factors. Tyrosine, tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2) and cyclic adenosine are two enzymes that act as tyrosine enzymes in melanin biosynthesis. Adrenocorticotropic hormone (ACTH), which is a monophosphate (cAMP) inducer, and Forskoline and α-Melanocyte stimulating hormone (α-MSH). Melanin is synthesized in the melanocyte of Melanocyte of the human epidermal layer. Tyrosinase oxidizes Tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and is converted to DOPA quinone by enzymatic chemical reaction. It then undergoes autoxidation and enzymatic reactions to produce dark brown melanin via DOPA chrome. Recently, inhibition of tyrosinase activity, which inhibits the formation of melanin, a dark brown complex, has been used as an effective method for the development of skin whitening agents. Recently, side effects such as BHA (Butylated Hydroxyanisole) and BHT (Butylated Hydroxytoluene) There have been many studies to develop antioxidants from natural resources. Antioxidants obtained from natural resources have been known to have anti-inflammatory properties as much as synthetic antioxidants. Natural extracts from plants have been shown to be effective against UV absorption, antiinflammatory action, antioxidant activity, inhibition of enzymes such as collagenase, inhibition of tyrosinase, (经 皮) to improve the absorption of various effects such as the effect of protecting the skin may also appear. Oxidative stress has been studied together with whitening as a major factor causing skin photoaging, black spot, spots and freckles. As a result, it is possible to prevent the melanin pigment which is formed by the progress of oxidation through antioxidant and reactive oxygen scavenging. Therefore, using natural extracts extracted from plants, antioxidant and whitening function and other functions will be seen through the functional cosmetics research will be activated. In this study, Morus alba, which is expected to have antioxidant and whitening function, was extracted from the macrophage cell line to decrease NO production after LPS stimulation, and to analyze the anti-inflammatory effect, DPPH radical scavenging activity, The antioxidant activity of the extracts was analyzed by the flavonoid content analysis, and the tyrosinase inhibitory activity and the inhibitory effect of melanin formation on melanoma cell line B16F10 were analyzed.

Raw 264.7 macrophage Cell viability was 90% or more and cell viability was 81% at 10 and 50 μg / ml treatment. Thereafter, it was confirmed that the cell survival rate was decreased in a concentration dependent manner.

In this experiment, nitrite scavenging activity was 22.3, 33.1, and 53.4%, respectively, when the concentrations of M. alba pretanol A extracts were 5, 10 and 20 μg / ml, respectively, compared to the LPS (0.5 μg / ml) At a concentration of at least 5 μg / ml, 22.3% of LPS secretion was reduced and compared with previous papers, results were obtained at low concentrations.

The DPPH radical scavenging activities of M. alba pretanol A extracts were 48.8, 55.9 and 66.5% when treated with 5, 10 and 20 μg / ml, respectively.

Total phenol content was 31.4 μg / ml tannic acid equivalent at 20 μg / ml concentration of M. alba pretanol A extract. The total phenol content was also increased proportionally with increasing concentration of extract, and 5, 10 and 20 μg / ml The total phenol contents were 8.3 ± 0.6, 25.0 ± 0.6 and 31.4 ± 0.3 μg / ml tannic acid equivalent, respectively.

The total flavonoid content of 20 μg / ml of M. alba pretanol A extract was 25 μg / ml naringin equivalent, and the content of flavonoid increased proportionally with increasing concentration of extract. As the concentration of M. alba pretanol A extract increased, the naringin equivalents tended to increase.

M. alba extract showed 57.6 ± 0.006, 62.8 ± 0.004 and 72.0 ± 0.003% scavenging activity at concentrations of 5, 10 and 20 μg / ml, respectively. SOD-like activity tended to depend on the concentration, and these results show that the M. alba pretanol A extract is valuable for preventing skin aging by natural materials.

To determine the cytotoxicity of M. alba pretanol A extracts from melanoma cells, B16F10, M. alba pretanol A extracts were treated with 0.1, 1, 10, 50 and 100 μg / ml of extracts and cultured for 24 hours. The cell survival rates of M. alba pretanol A extracts of 0.1, 1, 10 and 50 μg / ml were 81% or more. At 50 μg / ml, there was no toxicity, and thereafter, the cell survival rate was decreased in a concentration-dependent manner.

The tyrosinase activity of M. alba pretanol A extract was decreased in a dose dependent manner. The positive control group, arbutin, showed 10% inhibition activity at 20 μg / ml. The M. alba pretanol A extract showed a tyrosinase inhibitory effect of 32% at a concentration of 20 μg / ml.

Melanin production by M. alba pretanol A extract was 42.8% in the control group Arbutin 20 μg / ml, and the amount of melanin was decreased in a concentration-dependent manner. Melanin biosynthesis at the concentration of 20 ug / ml was 58.3 % Inhibition effect.

As a result of this study, the natural antioxidant and whitening function of the natural extracts extracted from M. alba has been proven to be active. It will also be useful as a material and functional ingredient material that is worthy of natural antioxidants in the future.