KR102668927B1 - Composition for preventing, improving or treating inflammation, wrinkle, allergy and atopic dermatitis containing fermented Centella asiatica extract as effective component - Google Patents
Composition for preventing, improving or treating inflammation, wrinkle, allergy and atopic dermatitis containing fermented Centella asiatica extract as effective component Download PDFInfo
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- KR102668927B1 KR102668927B1 KR1020210049672A KR20210049672A KR102668927B1 KR 102668927 B1 KR102668927 B1 KR 102668927B1 KR 1020210049672 A KR1020210049672 A KR 1020210049672A KR 20210049672 A KR20210049672 A KR 20210049672A KR 102668927 B1 KR102668927 B1 KR 102668927B1
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- KR
- South Korea
- Prior art keywords
- centella asiatica
- extract
- lactic acid
- acid bacteria
- leuconostoc
- Prior art date
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- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
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Abstract
본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방, 개선 또는 치료용 조성물에 관한 것으로, 본 발명의 유효성분인 병풀 발효 추출물은 병풀 추출물 또는 병풀 추출 발효물에 비해 항염, 항주름, 항알러지 및 항아토피 피부염에 대한 효과가 현저하게 증진된 것이다.The present invention relates to a composition for preventing, improving, or treating inflammation, wrinkles, allergies, and atopic dermatitis, comprising a centella asiatica fermented extract as an active ingredient. Compared to this, the anti-inflammatory, anti-wrinkle, anti-allergy and anti-atopic dermatitis effects are significantly improved.
Description
본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating inflammation, wrinkles, allergies and atopic dermatitis, comprising a fermented extract of Centella asiatica as an active ingredient.
병풀(Centella asiatica)은 한반도 남부 섬의 산이나 들에 흔히 나는 여러해살이풀이다. 원줄기는 옆으로 뻗고, 뿌리가 내리는 마디 근처에 2개의 퇴화된 비늘 모양의 잎이 있다. 잎은 마디에서 밀생하고, 신장형, 지름 2~5cm, 표면은 광택이 나고, 가장자리에 둔한 톱니가 있으며, 잎자루의 길이는 4~20cm이다. 꽃은 홍자색, 잎겨드랑이에 3~4송이씩 산형꽃차례로 달리며, 머리 모양이다. 암술대는 2갈래이며, 열매는 분과의 편원형이다. 인도에서는 상처를 입은 호랑이가 병풀이 많이 난 곳에서 뒹굴어 치료하는 것을 보고 호랑이풀이라고 부르며, 오래전부터 약으로 사용하였다. 병풀의 잎과 줄기에 있는 마데카식산이란 성분이 염증을 낫게 하고, 종양과 궤양 등의 상처를 치유하는 힘이 있다는 게 밝혀지면서 연고, 치약 또는 화장품 등의 원료로 쓰인다. Centella asiatica is a perennial plant that commonly grows in the mountains and fields of the southern islands of the Korean Peninsula. The main stem extends to the side, and there are two degenerated scale-shaped leaves near the node where the root comes from. The leaves grow densely at the nodes, are kidney-shaped, 2 to 5 cm in diameter, have a glossy surface, have dull sawtooth edges, and the length of the petiole is 4 to 20 cm. The flowers are red-purple, grow in umbels of 3 to 4 at each leaf axil, and are shaped like a head. The style is two-pronged, and the fruit is an oblong shape. In India, after seeing a wounded tiger rolling around in a place with a lot of centella asiatica to heal, it was called tiger plant and has been used as medicine for a long time. Madecassic acid, a component found in the leaves and stems of Centella asiatica, has been found to have the power to relieve inflammation and heal wounds such as tumors and ulcers, and is now used as a raw material for ointments, toothpaste, and cosmetics.
한편, 염증(inflammation)은 어떤 자극에 대한 생체조직의 방어반응의 하나로, 조직 변질, 순환장애와 삼출(渗出), 조직 증식의 세 가지를 병발하는 복잡한 병변(病變)을 일컫는다. 관절염 또는 아토피 피부염 등과 같이 끝 자가 염(炎)으로 되어 있는 것 외에, 전염성 질환(결핵·매독·이질·디프테리아 등) 등에서 볼 수 있는 병변의 총칭으로, 대부분의 병이 이것에 속한다. 원인은 기계적 상해작용, 온도·방사선 등의 물리적 인자, 독물 등의 화학적 인자, 세균감염 등의 기생체에 의한 것 등이며 이 중 세균에 의한 것이 가장 많다. 이러한 주요 원인 외에도, 여러 부수적 요인과 개체의 소인(素因)이나 면역 등에 의하여 그 발생은 복잡하다.Meanwhile, inflammation is one of the defense responses of living tissue to certain stimuli, and refers to a complex lesion that occurs together with three factors: tissue degeneration, circulatory failure and exudation, and tissue proliferation. It is a general term for lesions that can be seen in infectious diseases (tuberculosis, syphilis, dysentery, diphtheria, etc.), as well as those with terminal inflammation, such as arthritis or atopic dermatitis, and most diseases fall into this category. The causes include mechanical injury, physical factors such as temperature and radiation, chemical factors such as poisons, and parasites such as bacterial infection. Among these, bacteria are the most common. In addition to these main causes, its occurrence is complicated due to various secondary factors and individual predisposition or immunity.
한편, 아토피는 최근 들어, 유병률이 급격히 증가함에 따라 그 위험성이 크게 부각되고 있는 과민증(hypersensitivity)의 일종으로, 면역학적 관점에서는 면역글로불린 E(immunoglobulin E)와 알레르겐(allergen)의 면역반응에 의한 주요증상을 동반하는 유전경향이 강한 알레르기성 질환군으로 정의된다. 특히, 아토피의 대표적 증상인 아토피 피부염(atopic dermatitis)은 전 인구의 0.5~1%, 어린이의 5~10%가 심각한 증상을 호소하고 있지만, 유전적인 요인과 면역계 결핍에 관련 있는 것으로 추정될 뿐 아직 정확한 원인은 밝혀진 바 없고, 환경 및 식생활 개선을 통해 다소 완화되는 것으로 기대될 뿐 근본적인 치료방법이 전무한 실정이다. 그러나 아토피 피부염은 개인차를 보이기는 하지만, 대부분 극심한 가려움증을 비롯한 피부 건조증, 발진, 진물, 부스럼딱지, 비늘 같은 껍질이 있는 피부(인비늘) 등을 수반하며, 이로 인한 정서적 불안, 스트레스, 긴장, 좌절, 분노의 감정과 함께 기타 알레르기 질환인 두드러기, 금속 알레르기, 천식이나 알레르기성 비염 등을 동반하는 경우가 빈번한 바, 오늘날 국민건강을 위협하는 심각한 사회문제로 떠오르고 있다. 한편, 현재까지 아토피 피부염에 효과가 있는 것으로 알려진 대표적 약물로는 스테로이드제(steroid), 항히스타민제(antihistamine), 항생제(antibiotic), 비스테로이드제를 비롯한 기타 진정제와 신경안정제 등을 들 수 있다.Meanwhile, atopy is a type of hypersensitivity whose risk has been greatly highlighted as its prevalence has rapidly increased in recent years. From an immunological perspective, it is a major disease caused by the immune response to immunoglobulin E and allergens. It is defined as a group of allergic diseases with a strong genetic tendency accompanied by symptoms. In particular, atopic dermatitis, a representative symptom of atopy, has serious symptoms in 0.5 to 1% of the general population and 5 to 10% of children, but it is presumed to be related to genetic factors and immune system deficiency. The exact cause has not been revealed, and it is expected to be alleviated somewhat by improving the environment and diet, but there is no fundamental treatment method. However, although there are individual differences in atopic dermatitis, it is mostly accompanied by extreme itching, dry skin, rashes, oozing, scabs, and scaly skin (scaly skin), which results in emotional anxiety, stress, tension, frustration, and other symptoms. Feelings of anger are often accompanied by other allergic diseases such as hives, metal allergy, asthma or allergic rhinitis, which is emerging as a serious social problem that threatens public health. Meanwhile, representative drugs known to be effective for atopic dermatitis to date include steroids, antihistamines, antibiotics, non-steroids, and other sedatives and tranquilizers.
한편, 알러지(allergy)는 면역 시스템의 오작동으로 보통 사람에게는 별 영향이 없는 물질이 어떤 사람에게는 두드러기, 가려움, 콧물 또는 기침 등의 이상 과민 반응을 일으키는 것을 말하며, WHO에서는 이미 알러지 질환을 21세기에 가장 문제가 되는 제7대 질병으로 포함하였다. 주요한 알러지 질환으로는 아토피 피부염, 천식, 비염 등이 있다. 알러지는 현재까지 뚜렷한 치료제가 없을 정도로 현대의학의 난치병에 가까운 질환이다. 현재 알러지 질환 치료에 있어서 근본적인 치료제가 아닌 항히스타민제, 효과는 우수하나 부작용이 많은 스테로이드의 사용으로 질환은 더욱 악화되고 환자의 경제적 부담은 계속 증가하고 있다. Meanwhile, allergy is a malfunction of the immune system that causes abnormal hypersensitivity reactions such as hives, itchiness, runny nose, or cough in some people to substances that have no effect on ordinary people. WHO has already classified allergic diseases in the 21st century. It was included as the 7th most problematic disease. Major allergic diseases include atopic dermatitis, asthma, and rhinitis. Allergy is a disease that is so close to being incurable in modern medicine that there is no clear treatment to date. Currently, in the treatment of allergic diseases, the use of antihistamines, which are not fundamental treatments, and steroids, which are effective but have many side effects, are causing the disease to worsen and the financial burden on patients continues to increase.
대체로, 아토피 증상이나 알러지 질환은 산업의 발달, 식생활의 서구화 및 인스턴트화, 심각한 환경오염 발생, 새집 증후군과 같은 신종 질병 등과 함께 동반되면서, 21세기에 접어들어 더욱 꾸준히 증가하고 있는 추세이다. 알러지 질환의 발병 추세는 국내뿐 아니라 서구 사회나, 심지어 개발이 진행되고 있는 여러 후진국에서조차도 심각하게 발생하고 있으므로, 이들 질환 치료제는 범세계적인 시장수요를 창출할 것으로 예상된다. In general, atopic symptoms and allergic diseases are steadily increasing in the 21st century, accompanied by industrial development, westernization and instantiation of eating habits, serious environmental pollution, and new diseases such as sick building syndrome. Since the incidence of allergic diseases is serious not only in Korea but also in Western societies and even in many underdeveloped countries where development is progressing, treatments for these diseases are expected to create global market demand.
한편, 피부의 노화는 주름 증가와 탄력감소, 색소 침착 등의 여러 형태로 나타난다. 그 중 가장 대표적인 노화의 현상은 바로 주름이다. 이러한 주름을 생성하게 하는 피부의 노화원인은 크게 두 가지로 나누어 볼 수 있다. 나이가 들어감에 따른 자연적인 노화인 내적 요인과 외적 요인으로 인한 노화이다. 외적 요인으로는 자외선, 공해, 병, 음주, 흡연 등이 있고, 내적 요인으로는 프리라디칼과 염증의 발생, 스트레스, 세포 활성의 저하 및 돌연변이화, 세포 대사 불균형 등이다. 피부의 구조에 따라 주름 발생 원인을 좀 더 자세히 설명하면, 피부는 크게 표피와 진피로 나뉜다. 표피에서의 주름 생성 원인으로는 각질 형성 세포의 턴오버(turn-over)의 감소로 각질층의 누적과 이로 인해 각질층이 두꺼워지고, 각질층의 수분 함유 능력이 감소되며, 각질이 경화되어 주름이 발생하게 된다. 표피와 진피의 경계부위는 표피세포 지지, 부착, 영양물질의 이동, 표피 분화의 조절 등에 관여하는데, 각종 노화 요인으로 특히, UV로 인하여 콜라겐 IV, VII이 감소되어 표피, 진피 지지기능이 감소되고, 선별 투과 기능이 약화되어 해로운 성분이 쉽게 진피에 영향을 미치게 되어 주름이 생성된다. Meanwhile, skin aging appears in various forms such as increased wrinkles, decreased elasticity, and pigmentation. Among them, the most representative aging phenomenon is wrinkles. The causes of skin aging that cause these wrinkles can be broadly divided into two. Aging is caused by internal and external factors, which is natural aging as we age. External factors include ultraviolet rays, pollution, illness, drinking, and smoking, and internal factors include the occurrence of free radicals and inflammation, stress, decrease and mutation of cell activity, and cell metabolism imbalance. To explain in more detail the causes of wrinkles depending on the structure of the skin, the skin is largely divided into the epidermis and dermis. The cause of wrinkles in the epidermis is a decrease in the turnover of keratinocytes, leading to accumulation of the stratum corneum, which thickens the stratum corneum, reduces the water-holding capacity of the stratum corneum, and hardens the stratum corneum, causing wrinkles to form. do. The boundary area between the epidermis and dermis is involved in epidermal cell support, adhesion, movement of nutrients, and regulation of epidermal differentiation. Due to various aging factors, especially UV, collagen IV and VII are reduced, leading to a decrease in epidermal and dermal support functions. , the selective penetration function is weakened, allowing harmful ingredients to easily affect the dermis, resulting in wrinkles.
한국공개특허 제2003-0024932호에 병풀 추출물을 함유하는 화장료 조성물에 관해 개시되어 있고, 한국등록특허 제1591872호에 항염, 항알러지 및 항산화 효능이 우수한 Ge-8 혼합물을 함유하는 화장료 조성물이 개시되어 있으나, 아직까지 본 발명의 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방, 개선 또는 치료용 조성물에 대해 개시된 바가 없다.Korean Patent Publication No. 2003-0024932 discloses a cosmetic composition containing centella asiatica extract, and Korean Patent No. 1591872 discloses a cosmetic composition containing a Ge-8 mixture with excellent anti-inflammatory, anti-allergy and antioxidant effects. However, there has been no disclosure yet regarding a composition for preventing, improving, or treating inflammation, wrinkles, allergies, and atopic dermatitis containing the fermented extract of Centella asiatica of the present invention as an active ingredient.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방, 개선 또는 치료용 조성물을 제공하고, 본 발명의 유효성분이 NO 활성을 억제하고, 히스타민 분비를 감소시키며, 케모카인 RANTES의 함량을 감소시키고, 콜라게나아제의 활성을 감소시키며, UV 조사군에 대비하여 MMP-1의 발현량을 감소시키는 효과를 확인함으로써, 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs, and the present invention provides a composition for preventing, improving or treating inflammation, wrinkles, allergies and atopic dermatitis, comprising a fermented extract of Centella asiatica as an active ingredient, and the active ingredient of the present invention By confirming the effect of suppressing NO activity, reducing histamine secretion, reducing the content of the chemokine RANTES, reducing the activity of collagenase, and reducing the expression level of MMP-1 compared to the UV irradiated group, this study The invention was completed.
상기 목적을 달성하기 위하여, 본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In order to achieve the above object, the present invention provides a health functional food composition for preventing or improving inflammation, wrinkles, allergies and atopic dermatitis, containing fermented extract of Centella asiatica as an active ingredient.
또한, 본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방 또는 개선용 화장료 조성물을 제공한다.Additionally, the present invention provides a cosmetic composition for preventing or improving inflammation, wrinkles, allergies, and atopic dermatitis, comprising a fermented extract of Centella asiatica as an active ingredient.
또한, 본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 알러지 및 아토피 피부염의 예방 또는 치료용 약학 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing or treating inflammation, allergies, and atopic dermatitis, comprising a fermented extract of Centella asiatica as an active ingredient.
본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방, 개선 또는 치료용 조성물에 관한 것으로, 본 발명의 유효성분인 병풀 발효 추출물은 병풀 추출물 또는 병풀 추출 발효물에 비해 증진된 NO 활성 억제, 히스타민 분비 감소, RANTES의 함량 감소, 콜라게나아제의 활성 감소 및 MMP-1 발현량 감소 효과가 있으며, 특히, 본 발명의 병풀 발효 추출물 중에서, 병풀 유산균 발효 추출물이 현저한 항염, 항주름, 항알러지 및 항아토피 피부염에 대한 효과가 있다. The present invention relates to a composition for preventing, improving, or treating inflammation, wrinkles, allergies, and atopic dermatitis, comprising a centella asiatica fermented extract as an active ingredient. Compared to this, it has the effect of inhibiting enhanced NO activity, reducing histamine secretion, reducing the content of RANTES, reducing the activity of collagenase, and reducing the expression level of MMP-1. In particular, among the fermented extracts of Centella asiatica of the present invention, the fermented extract of Centella asiatica lactic acid bacteria has a significant anti-inflammatory effect. , has anti-wrinkle, anti-allergy and anti-atopic dermatitis effects.
도 1은 본 발명의 병풀 발효 추출물의 처리에 따른 히스타민 분비량(A) 및 세포 생존률(B)을 확인한 결과이다. *, **은 Compound48/80 처리군 대비 본 발명의 병풀 발효 추출물 또는 병풀 에탄올 추출물 처리군의 히스타민 분비량이 통계적으로 유의미하게 감소하였다는 것으로, *은 p<0.05이고, **은 p<0.01이다.
도 2는 본 발명의 병풀 발효 추출물의 처리에 따른 케모카인(RANTES) 함량(A) 및 세포 생존률(B)을 확인한 결과이다. Vehicle은 아무것도 처리하지 않은 정상군이고, TI는 10ng/㎖의 TNF-α와 10ng/㎖의 IFN-γ 처리군이며, SILY는 양성대조군으로 실리마린(silymarin)을 처리한 군이다. *, **, ***은 TI 처리군 대비 본 발명의 병풀 발효 추출물(CA03~CA05), 병풀 추출물(CA01, CA02) 또는 병풀 추출물의 발효물 처리군(CA06~CA08)의 케모카인(RANTES) 함량이 통계적으로 유의미하게 감소하였다는 것으로, *은 p<0.05이고, **은 p<0.01이며, ***은 p<0.001이다.
도 3은 병풀 추출물 및 본 발명의 병풀 발효 추출물의 콜라게나아제 저해 활성을 확인한 결과이다.
도 4는 병풀 추출물 및 본 발명의 병풀 발효 추출물의 처리에 따른 MMP-1 발현량 변화(A) 및 세포 생존률(B)을 확인한 결과이다. ####은 정상군(NT) 대비 UV 조사군(BLK)의 MMP-1 발현량이 통계적으로 유의미하게 증가하였다는 것으로, p<0.0001이고, ****은 UV 조사군(BLK) 대비 병풀 에탄올 추출물 처리군(CA02) 또는 병풀 유산균 발효 추출물 처리군(CA04)의 MMP-1 발현량이 통계적으로 유의미하게 감소하였다는 것으로, p<0.0001이며, $$$$은 동일 농도의 병풀 에탄올 추출물 처리군에 대비하여 병풀 유산균 발효 추출물 처리군의 MMP-1 발현량이 통계적으로 유의미하게 감소하였다는 것으로, p<0.0001이다.Figure 1 shows the results of confirming the amount of histamine secretion (A) and cell survival rate (B) according to treatment of the fermented extract of Centella asiatica of the present invention. *, ** indicate a statistically significant decrease in histamine secretion in the group treated with the Centella asiatica fermentation extract or the Centella asiatica ethanol extract of the present invention compared to the Compound48/80 treatment group, * indicates p < 0.05, and ** indicates p < 0.01. .
Figure 2 shows the results of confirming the chemokine (RANTES) content (A) and cell survival rate (B) according to the treatment of the fermented extract of Centella asiatica of the present invention. Vehicle is a normal group treated with nothing, TI is a group treated with 10 ng/ml TNF-α and 10 ng/ml IFN-γ, and SILY is a positive control group treated with silymarin. *, **, *** are the chemokines (RANTES) of the Centella asiatica fermented extract (CA03-CA05), Centella asiatica extract (CA01, CA02), or the fermented product of Centella asiatica extract (CA06-CA08) of the present invention compared to the TI treatment group. This means that the content decreased statistically significantly, * means p<0.05, ** means p<0.01, and *** means p<0.001.
Figure 3 shows the results of confirming the collagenase inhibitory activity of the Centella asiatica extract and the Centella asiatica fermented extract of the present invention.
Figure 4 shows the results of confirming the change in MMP-1 expression level (A) and cell survival rate (B) according to treatment of the Centella asiatica extract and the fermented Centella asiatica extract of the present invention. #### indicates a statistically significant increase in MMP-1 expression in the UV irradiated group (BLK) compared to the normal group (NT), p<0.0001, and **** indicates a statistically significant increase in MMP-1 expression compared to the UV irradiated group (BLK). There was a statistically significant decrease in MMP-1 expression in the ethanol extract treatment group (CA02) or the Centella asiatica lactic acid bacteria fermentation extract treatment group (CA04), p<0.0001, and $$$$ refers to the Centella asiatica ethanol extract treatment group at the same concentration. In comparison, the MMP-1 expression level in the group treated with the fermented Centella asiatica lactic acid bacteria extract was statistically significantly reduced, p<0.0001.
본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.The present invention relates to a health functional food composition for preventing or improving inflammation, wrinkles, allergies and atopic dermatitis, containing fermented extract of Centella asiatica as an active ingredient.
상기 병풀 발효 추출물은 하기의 단계를 포함하는 방법에 의해 제조되는 것일 수 있으나, 이에 한정하지 않는다:The fermented extract of Centella asiatica may be prepared by a method including the following steps, but is not limited to this:
1) 병풀 분말에 물을 가하여 멸균하는 단계;1) Sterilizing the Centella asiatica powder by adding water;
2) 상기 단계 1) 이후에, 상온(15~25℃)으로 식혀 발효균을 첨가하고 25~40℃에서 2~4일 동안 발효하는 단계; 2) After step 1), cooling to room temperature (15-25°C), adding fermentation bacteria, and fermenting at 25-40°C for 2-4 days;
3) 상기 단계 2) 이후에, 50~70℃에서 건조하는 단계; 3) After step 2), drying at 50-70°C;
4) 상기 단계 3)에서 건조한 병풀 발효물의 건조 분말에 추출용매를 첨가하여 추출하는 단계;4) extracting by adding an extraction solvent to the dry powder of the fermented product of Centella asiatica dried in step 3);
5) 상기 단계 4)의 병풀 발효 추출물을 여과하는 단계; 및 5) filtering the fermented Centella asiatica extract of step 4); and
6) 상기 단계 5)의 여과한 추출물을 감압 농축하고 건조하여 추출물을 제조하는 단계.6) Preparing an extract by concentrating the filtered extract of step 5) under reduced pressure and drying it.
상기 단계 2)에서 첨가한 발효균은 유산균, 효모 또는 누룩이 바람직하지만, 이에 한정하는 것은 아니며, 상기 유산균은 엔테로코커스 패시움(Enterococcus faecium), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 애시도필러스(Lactobacillus acidophillus), 락토바실러스 플랜타룸(Lactobacillus plantarum), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 메센테로이드스(Leuconostoc mesenteroides) 및 락토바실러스 사케이(Lactobacillus sakei) 중에서 선택된 하나 이상의 유산균인 것이 바람직하며, 더 바람직하게는 락토바실러스 플랜타룸(Lactobacillus plantarum), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 메센테로이드스(Leuconostoc mesenteroides) 및 락토바실러스 사케이(Lactobacillus sakei)로 이루어진 복합 유산균이지만 이에 한정하는 것은 아니다.The fermentation bacteria added in step 2) are preferably lactic acid bacteria, yeast, or yeast, but are not limited thereto, and the lactic acid bacteria include Enterococcus faecium , Lactobacillus casei, and Lactobacillus acidophilus. ( Lactobacillus acidophillus ), Lactobacillus plantarum , Leuconostoc kimchii, Leuconostoc citreum , Leuconostoc mesenteroides and Lactobacillus sakei ( It is preferable that it is one or more lactic acid bacteria selected from Lactobacillus sakei , more preferably Lactobacillus plantarum , Leuconostoc kimchii, Leuconostoc citreum , and Leuconostoc mesen. It is a complex lactic acid bacteria consisting of Leuconostoc mesenteroides and Lactobacillus sakei , but is not limited thereto.
상기 단계 4)에서 추출용매는 물, C1~C4의 저급 알코올 또는 이들의 혼합물인 것이 바람직하며, 더 바람직하게는 에탄올이지만 이에 한정하지 않는다. In step 4), the extraction solvent is preferably water, a C 1 to C 4 lower alcohol, or a mixture thereof, and more preferably ethanol, but is not limited thereto.
상기 제조방법에 있어서, 상기 단계 4)의 병풀 발효물의 추출은 여과, 열수 추출, 침지 추출, 환류 냉각 추출 및 초음파 추출 등의 당 업계에 공지된 모든 통상적인 방법을 이용할 수 있다. 상기 단계 6)의 감압농축은 진공 감압 농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. In the above production method, the extraction of the fermented product of Centella asiatica in step 4) can be performed using all conventional methods known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The vacuum concentration in step 6) is preferably performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto.
또한, 건조는 감압건조, 진공건조, 비등건조, 분무 건조 또는 동결 건조하는 것이 바람직하나 이에 한정하지 않는다.In addition, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.
본 발명에서 용어, "알러지(allergy)"란 이물질(항원, Allergen)에 대한 특이하고 변형된 반응을 나타내는 생화학적 현상을 의미하며, 상기 알러지성 질환은 부종, 과민증(anaphylaxis), 천식(asthma), 두드러기, 소양증, 곤충 알러지, 아토피, 식품 알러지 및 약품 알러지 중에서 선택된 어느 하나인 것이 바람직하지만 이에 한정하지 않는다.In the present invention, the term "allergy" refers to a biochemical phenomenon that represents a specific and modified reaction to a foreign substance (antigen, Allergen), and the allergic disease includes edema, anaphylaxis, and asthma. , urticaria, itching, insect allergy, atopy, food allergy, and drug allergy, but is not limited thereto.
상기 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조되는 것이 바람직하지만, 이에 한정하지 않는다.The composition is preferably manufactured in a dosage form selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물은 병풀 발효 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 혼합하여 제조될 수 있고, 통상적인 방법에 따라 적절하게 제조될 수 있다. 상기 병풀 발효 추출물을 첨가할 수 있는 식품의 예로는 카라멜, 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 중에서 선택된 어느 하나의 형태일 수 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다. 즉, 상기 식품의 종류에는 특별한 제한은 없다. 상기 건강기능식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 및 천연 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 또한, 천연 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 상기의 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 본 발명의 건강기능식품 조성물은 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있으며, 상기 천연 탄수화물은 포도당, 과당과 같은 단당류, 말토스, 슈크로스와 같은 이당류, 덱스트린, 사이클로 덱스트린과 같은 다당류, 자일리톨, 소르비톨, 에리트리톨 등의 당 알코올이다. 상기 천연 탄수화물의 비율은 크게 중요하지 않지만, 본 발명의 조성물 100g에 대하여, 0.01~0.04g인 것이 바람직하고, 더욱 바람직하게는 0.02~0.03g을 포함하는 것이지만 이에 한정하지 않는다. 감미제로는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. The health functional food composition of the present invention can be prepared by adding the fermented extract of Centella asiatica as is or mixing it with other foods or food ingredients, and can be prepared appropriately according to conventional methods. Examples of foods to which the Centella asiatica fermented extract can be added include caramel, meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, It may be in any form selected from tea, drink, alcoholic beverage, and vitamin complex, and includes all health functional foods in the conventional sense. That is, there are no particular restrictions on the type of food. The health functional food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, it may contain pulp for the production of natural fruit juice and vegetable drinks. The above components can be used independently or in combination. In addition, the health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, and the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, dextrins, and cyclosaccharides. These are polysaccharides such as dextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The ratio of the natural carbohydrate is not very important, but is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, per 100 g of the composition of the present invention, but is not limited thereto. Sweeteners include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame.
또한, 본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 주름, 알러지 및 아토피 피부염의 예방 또는 개선용 화장료 조성물에 관한 것이다.Additionally, the present invention relates to a cosmetic composition for preventing or improving inflammation, wrinkles, allergies, and atopic dermatitis, comprising a fermented extract of Centella asiatica as an active ingredient.
상기 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액, 파운데이션, 왁스 파운데이션 및 스프레이 중에서 선택된 어느 하나의 제형으로 제조되는 것이 바람직하지만 이에 한정하는 것은 아니다.The composition is prepared in any one formulation selected from solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion, foundation, wax foundation and spray. It is desirable, but is not limited to this.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1, 3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. These include glycol, 1,3-butylglycol oil, fatty esters of glycerol, fatty acid esters of polyethylene glycol or sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다. 본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판-부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier ingredients. etc. can be used. When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be additionally used. It may contain propellants such as carbon, propane-butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 아세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, and sarcosinate. , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.
본 발명의 화장료 조성물은 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 항염, 항주름, 항알러지 및 항아토피 피부염 효능을 갖는 성분을 1종 이상 함유할 수 있다. In addition to the active ingredient, the cosmetic composition of the present invention may further contain one or more ingredients with anti-inflammatory, anti-wrinkle, anti-allergy, and anti-atopic dermatitis effects that exhibit the same or similar functions.
또한, 본 발명은 병풀 발효 추출물을 유효성분으로 포함하는 염증, 알러지 및 아토피 피부염의 예방 또는 치료용 약학 조성물에 관한 것이다.Additionally, the present invention relates to a pharmaceutical composition for preventing or treating inflammation, allergies, and atopic dermatitis, comprising a fermented extract of Centella asiatica as an active ingredient.
본 발명의 약학 조성물은 상기 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함할 수 있으며, 본 발명의 약학 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여 시 피부 외용 또는 복강 내, 직장, 정맥, 근육 또는 피하주사 방식을 선택하는 것이 바람직하지만 이에 한정하는 것은 아니다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above active ingredients. The pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, it may be used externally on the skin or intraperitoneally. It is preferable to choose, but is not limited to, intravenous, rectal, intravenous, intramuscular or subcutaneous injection.
본 발명의 약학 조성물은 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성 용제 및 현탁 용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, Tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
본 발명에 따른 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도에 따라 그 범위가 다양하게 사용할 수 있다.The dosage of the composition of the present invention can vary depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.
실시예 1. 병풀 발효 추출물, 병풀 추출물 및 병풀 추출 발효물의 제조Example 1. Preparation of Centella asiatica fermented extract, Centella asiatica extract, and Centella asiatica extract fermented product
(1) 병풀 (유산균, 누룩 및 효모) 발효 후, 에탄올 추출물의 제조 (1) Production of ethanol extract after fermentation of Centella asiatica (lactic acid bacteria, yeast and yeast)
발효에 사용된 유산균은 복합 유산균을 이용하였으며, 상기 복합 유산균은 락토바실러스 플랜타룸(Lactobacillus plantarum), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 메센테로이드스(Leuconostoc mesenteroides), 락토바실러스 사케이(Lactobacillus sakei)로 이루어진 것이다. The lactic acid bacteria used for fermentation were complex lactic acid bacteria, and the complex lactic acid bacteria were Lactobacillus plantarum, Leuconostoc kimchii, Leuconostoc citreum , and Leuconostoc mesenteroid. It is composed of Leuconostoc mesenteroides and Lactobacillus sakei .
200g의 병풀을 분쇄한 후, 400㎖의 증류수를 첨가하여 121℃에서 15분 동안 멸균하였다. 상온으로 식힌 상기 병풀에, MRS broth에서 배양한 1×104cfu/㎖의 복합 유산균 20㎖을 첨가한 후, 32℃에서 3일 동안 발효시키고, 60℃에서 건조하여 병풀 유산균 발효물을 제조하였다. After pulverizing 200g of Centella asiatica, 400ml of distilled water was added and sterilized at 121°C for 15 minutes. To the Centella asiatica cooled to room temperature, 20 ml of 1×10 4 cfu/ml complex lactic acid bacteria cultured in MRS broth was added, fermented at 32°C for 3 days, and dried at 60°C to prepare centella asiatica fermentation product. .
또한, 상기 복합 유산균 대신에 10g의 누룩 또는 2g의 효모를 사용한 것을 제외하고는 동일한 방법으로, 병풀 누룩 발효물 및 병풀 효모 발효물을 제조하였다.In addition, centella asiatica yeast fermented product and centella asiatica yeast fermented product were prepared in the same manner, except that 10 g of yeast or 2 g of yeast was used instead of the complex lactic acid bacteria.
상기 병풀 누룩 발효물, 병풀 유산균 발효물 및 병풀 효모 발효물을 70%(v/v) 에탄올로 추출하여 병풀 누룩 발효 추출물(CA03), 병풀 유산균 발효 추출물(CA04) 및 병풀 효모 발효 추출물(CA05)을 제조하고 이를 이용하여 항염, 항알러지, 항주름 및 항아토피 효능평가에 사용하였다.The Centella asiatica yeast fermented product, Centella asiatica lactic acid bacteria fermented product, and Centella asiatica yeast fermented product were extracted with 70% (v/v) ethanol to produce Centella asiatica yeast fermented extract (CA03), Centella asiatica lactic acid bacteria fermented extract (CA04), and Centella asiatica yeast fermented extract (CA05). was prepared and used to evaluate anti-inflammatory, anti-allergy, anti-wrinkle and anti-atopic efficacy.
(2) 병풀 물 또는 에탄올 추출물(2) Centella asiatica water or ethanol extract
병풀 물 추출물은 200g의 병풀을 분쇄한 후, 물 2ℓ를 첨가하여 열수 추출하였고(CA01), 병풀 에탄올 추출물은 200g의 병풀을 분쇄한 후, 70%(v/v) 에탄올 2ℓ를 첨가하여 병풀 에탄올 추출물을 획득하였다(CA02). 이를 항염, 항알러지, 항주름 및 항아토피 효능평가에서 비교예로 사용하였다.Centella asiatica water extract was obtained by grinding 200 g of Centella asiatica and adding 2 liters of water for hot water extraction (CA01), and Centella asiatica ethanol extract was obtained by pulverizing 200 g of Centella asiatica and adding 2 liters of 70% (v/v) ethanol to obtain Centella asiatica ethanol. The extract was obtained (CA02). This was used as a comparative example in evaluating anti-inflammatory, anti-allergy, anti-wrinkle, and anti-atopy efficacy.
(3) 병풀 에탄올 추출물의 제조 및 이의 병풀 유산균, 누룩 및 효모 발효물의 제조(3) Production of Centella asiatica ethanol extract and production of Centella asiatica lactic acid bacteria, nuruk, and yeast fermentation products
상기 획득한 병풀 에탄올 추출물을 감압농축한 후 500㎖의 멸균수를 첨가하고, 여기에 MRS broth에서 배양한 1×104cfu/㎖의 복합 유산균 20㎖을 첨가한 후, 32℃에서 3일 동안 발효하였다. 발효가 완료된 후 60℃에서 감압건조하여 병풀 추출물의 유산균 발효물(CA07)을 제조하였다. 상기 발효에 사용한 복합 유산균은 락토바실러스 플랜타룸(Lactobacillus plantarum), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 메센테로이드스(Leuconostoc mesenteroides), 락토바실러스 사케이(Lactobacillus sakei)로 이루어진 복합균이다.After concentrating the obtained Centella asiatica ethanol extract under reduced pressure, 500 ml of sterilized water was added, and 20 ml of 1 × 10 4 cfu/ml complex lactic acid bacteria cultured in MRS broth was added thereto, followed by 3 days at 32°C. It was fermented. After fermentation was completed, the lactic acid bacteria fermentation product (CA07) of Centella asiatica extract was prepared by drying under reduced pressure at 60°C. The complex lactic acid bacteria used in the fermentation include Lactobacillus plantarum, Leuconostoc kimchii, Leuconostoc citreum , Leuconostoc mesenteroides , and Lactobacillus. It is a complex bacteria consisting of Lactobacillus sakei .
또한, 상기 발효 시, 복합 유산균 대신에 10g의 누룩 또는 2g의 효모를 사용한 것을 제외하고는 동일한 방법으로 병풀 추출물의 누룩 발효물(CA06) 및 병풀 추출물의 효모 발효물(CA08)을 제조하였다. 상기 병풀 추출물과 마찬가지로, 항염, 항알러지, 항주름 및 항아토피 효능평가에서 비교예로 사용하였다.In addition, during the fermentation, yeast fermentation product of Centella asiatica extract (CA06) and yeast fermentation product of Centella asiatica extract (CA08) were prepared in the same manner, except that 10 g of yeast or 2 g of yeast was used instead of complex lactic acid bacteria. Like the above Centella asiatica extract, it was used as a comparative example in evaluating anti-inflammatory, anti-allergy, anti-wrinkle and anti-atopic efficacy.
실시예 2. Raw 274.7 세포에서 NO 생성 억제 효과 확인Example 2. Confirmation of NO production inhibition effect in Raw 274.7 cells
48웰 플레이트에 적정 농도로 분주한 RAW 274.7 세포에 LPS(400ng/㎖)를 처리하고, 상기 실시예 1에서 제조한 다양한 시료를 농도별(100, 200㎍/㎖)로 처리하여 24시간 동안 배양하였다. 배양 상층액을 취한 후 Griess 시약을 사용하여 제조사의 방법에 따라 NO 양을 측정하였다.RAW 274.7 cells dispensed at an appropriate concentration in a 48-well plate were treated with LPS (400 ng/ml), and various samples prepared in Example 1 were treated at different concentrations (100, 200 μg/ml) and cultured for 24 hours. did. After taking the culture supernatant, the amount of NO was measured using Griess reagent according to the manufacturer's method.
그 결과, 표 1에 개시한 바와 같이 LPS에 의해 증가된 NO 저해율이 병풀 추출물, 병풀 추출물의 발효물 및 병풀 유산균 발효 추출물의 순으로 높아졌다. 따라서 본 실시예 2의 결과로부터 병풀 유산균 발효 추출물이 염증에 대한 효과가 가장 우수할 것으로 판단하였다.As a result, as shown in Table 1, the NO inhibition rate increased by LPS increased in the order of Centella asiatica extract, fermented product of Centella asiatica extract, and Centella asiatica lactic acid bacteria fermented extract. Therefore, from the results of Example 2, it was determined that the fermented extract of Centella asiatica lactic acid bacteria would have the best effect on inflammation.
실시예 3. 히스타민 분비량 억제 효능 확인Example 3. Confirmation of efficacy in suppressing histamine secretion
배양된 MC/9 마우스 비만 세포를 96웰 플레이트에 접종하여 25㎍/㎖의 compound 48/80(Sigma, USA)과 정해진 농도(50, 100, 200㎍/㎖)의 시료를 30분 동안 처리하였다. 그 후, 배지에 분비된 알러지성 히스타민(Histamine)의 분비량을 ELISA kit(Oxford Biomedical Research Inc., USA)로 측정하였다. 100% 생성량은 compound 48/80이 처리되지 않은 군에서 처리된 군 사이의 히스타민 분비량의 차이로 계산하였다.Cultured MC/9 mouse mast cells were inoculated into a 96-well plate and treated with 25㎍/㎖ of compound 48/80 (Sigma, USA) and samples at specified concentrations (50, 100, 200㎍/㎖) for 30 minutes. . Afterwards, the amount of allergic histamine secreted in the medium was measured using an ELISA kit (Oxford Biomedical Research Inc., USA). The 100% production amount was calculated as the difference in histamine secretion between the group that was not treated with compound 48/80 and the group that was treated.
그 결과, 도 1(A)에 개시한 바와 같이 50, 100 및 200㎍/㎖의 농도로 병풀 유산균 발효 추출물을 처리한 경우, 히스타민의 분비량이 현저하게 감소한 것을 확인하였다.As a result, it was confirmed that when the fermented Centella asiatica lactic acid bacteria extract was treated at concentrations of 50, 100, and 200 μg/ml as shown in Figure 1(A), the secretion amount of histamine was significantly reduced.
한편, MC/9 마우스 비만 세포에서 병풀 발효 추출물의 세포독성을 확인하기 위하여, CCK-8(Cell Counting Kit-8) 어세이를 실시하였다. 96웰 플레이트(well plate)에서 MC/9 비만세포에 병풀 발효 추출물을 50, 100 및 200㎍/㎖의 농도로 처리하고 배양하였다. 24시간 후에 배지에 CCK-8을 10% 농도로 첨가한 다음 2시간 동안 37℃에서 반응시킨 후, 마이크로플레이트 스펙트로미터(microplate spectrophotometer) (Biorad, Hercules, CA, USA)를 이용하여 450 nm에서 흡광도를 측정하였고, 대조군(vehicle)과의 비교를 통해 상대적인 세포 생존율(%)을 계산하였다. Meanwhile, to confirm the cytotoxicity of the Centella asiatica fermentation extract in MC/9 mouse mast cells, CCK-8 (Cell Counting Kit-8) assay was performed. MC/9 mast cells were treated with Centella asiatica fermentation extract at concentrations of 50, 100, and 200 μg/ml and cultured in a 96-well plate. After 24 hours, CCK-8 was added to the medium at a concentration of 10%, reacted at 37°C for 2 hours, and measured absorbance at 450 nm using a microplate spectrophotometer (Biorad, Hercules, CA, USA). was measured, and the relative cell survival rate (%) was calculated through comparison with the control (vehicle).
그 결과 도 1(B)에 개시한 바와 같이, 본 발명의 병풀 발효 추출물을 50, 100 및 200㎍/㎖의 농도로, MC/9 비만세포에 처리하여도 세포독성이 거의 나타나지 않았다. As a result, as shown in Figure 1 (B), almost no cytotoxicity was observed even when the fermented Centella asiatica extract of the present invention was treated with MC/9 mast cells at concentrations of 50, 100, and 200 μg/ml.
실시예 4. 피부 각질세포(HaCaT cell)에서 케모카인 분비 억제 효과 확인Example 4. Confirmation of the effect of suppressing chemokine secretion in skin keratinocytes (HaCaT cells)
(1) RANTES 감소 효과 확인(1) Confirmation of RANTES reduction effect
피부 각질세포(HaCaT cell)를 6웰 플레이트에 접종하여 50, 100 및 200㎍/㎖의 농도로 시료를 1시간 동안 전 처리한 후, 10ng/㎖의 TNF-α 및 10ng/㎖의 IFN-γ를 처리하고 24시간 동안 배양하였다. Skin keratinocytes (HaCaT cells) were inoculated into a 6-well plate and the samples were pretreated for 1 hour at concentrations of 50, 100, and 200㎍/㎖, followed by 10ng/㎖ of TNF-α and 10ng/㎖ of IFN-γ. were treated and cultured for 24 hours.
양성대조군으로 12 및 25㎍/㎖의 실라마린(silymarin)을 각각 처리하였다. 24시간 후에 배지에 분비된 케모카인(RANTES)의 분비량을 ELISA kit (R&D systems Inc., Minneapolis, MN, USA)로 측정하였다. 100% 활성은 TNF-α 및 IFN-γ이 처리되지 않은 군과 처리된 군의 케모카인 분비량의 차이로 기준하였다. 분비량(%)은 하기 식 (2)에 따라 계산하였다. As a positive control group, 12 and 25 μg/ml of silymarin were treated, respectively. After 24 hours, the amount of secreted chemokine (RANTES) in the medium was measured using an ELISA kit (R&D systems Inc., Minneapolis, MN, USA). 100% activity was based on the difference in the amount of chemokine secretion between the group that was not treated with TNF-α and IFN-γ and the group that was treated. The secretion amount (%) was calculated according to the following formula (2).
식 (2)Equation (2)
케모카인 분비량(%) = [(시료 처리군 사이토카인 분비량 - 무처리군 사이토카인 분비량)/(대조군 사이토카인 분비량 - 무처리군 사이토카인 분비량)]×100Chemokine secretion amount (%) = [(Cytokine secretion amount in sample treatment group - Cytokine secretion amount in untreated group)/(Cytokine secretion amount in control group - Cytokine secretion amount in untreated group)] × 100
그 결과, 도 2(A)에 개시한 바와 같이 TNF-α 및 IFN-γ를 처리한 대조군(TI)의 케모카인(RANTES)의 분비량이 아무것도 처리하지 않은 정상군(vehicle)에 비해 증가하였으나, 본 발명에 따른 병풀 누룩 또는 유산균 발효 추출물을 처리한 군은 TNF-α 및 IFN-γ을 처리한 대조군(TI)에 비해 케모카인의 분비량이 현저하게 감소하였다. As a result, as shown in Figure 2 (A), the secretion amount of chemokine (RANTES) in the control group (TI) treated with TNF-α and IFN-γ increased compared to the normal group (vehicle) not treated with anything, but this The group treated with Centella asiatica yeast or lactic acid bacteria fermented extract according to the invention had a significant decrease in the secretion of chemokines compared to the control group (TI) treated with TNF-α and IFN-γ.
(2) 세포 생존율 분석(2) Cell viability analysis
본 발명의 시료가 피부 각질세포에서 세포독성이 있는지를 확인하고자 CCK-8(Cell Counting Kit-8) 어세이를 실시하였다. 96웰 플레이트(well plate)에서 HaCaT 세포에 시료를 농도별(50, 100 및 200㎍/㎖)로 처리하고, 24시간 동안 배양하였다. 24시간 후 배지에 CCK-8을 10% 농도로 첨가한 다음 2시간 동안 37℃에서 반응시킨 후, 마이크로플레이트 스펙트로미터(Biorad, Hercules, CA, USA)를 이용하여 450nm에서 흡광도를 측정하고, 대조군과의 비교를 통해 상대적인 세포 생존율(%)을 계산하였다. CCK-8 (Cell Counting Kit-8) assay was performed to confirm whether the sample of the present invention was cytotoxic to skin keratinocytes. HaCaT cells were treated with samples at different concentrations (50, 100, and 200 μg/ml) in a 96-well plate and cultured for 24 hours. After 24 hours, CCK-8 was added to the medium at a 10% concentration and reacted at 37°C for 2 hours. Absorbance was measured at 450 nm using a microplate spectrometer (Biorad, Hercules, CA, USA), and the control group Relative cell survival rate (%) was calculated through comparison with .
그 결과 도 2(B)에 개시한 바와 같이, 실시예 1에서 제조한 병풀 추출물(CA01, CA02), 병풀 발효 추출물(CA03~CA05) 및 병풀 추출 발효물(CA06~CA08)을 HaCaT 세포에 200㎍/㎖의 농도까지 처리하여도 세포독성이 거의 나타나지 않았다. As a result, as shown in Figure 2 (B), the Centella asiatica extract (CA01, CA02), Centella asiatica fermented extract (CA03 to CA05), and Centella asiatica extract fermented product (CA06 to CA08) prepared in Example 1 were administered to HaCaT cells for 200 mg. Even when treated at a concentration of ㎍/㎖, almost no cytotoxicity was observed.
실시예 5. 콜라게나아제 억제 효과 확인Example 5. Confirmation of collagenase inhibition effect
상기 실시예 1에서 획득한 시료들의 피부주름 억제 및 개선 효과를 확인하기 위하여, 콜라게나아제(Collageanse) 저해 활성시험을 실시하였다. 콜라게나아제 저해 활성은 2mM의 FALGPA(N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala)를 녹인 기질액 100㎕ 및 시료용액 10㎕를 혼합한 혼합액에, 콜라게나아제(0.08mg/㎖) 30㎕과, 10mM의 CaCl2 및 400mM의 NaCl을 포함하는 50mM의 트리신 완충용액(tricine buffer, pH 7.5) 60㎕를 첨가한 후, 340nm에서 1분 간격으로 총 20분 동안 흡광도를 측정하였다.In order to confirm the effect of the samples obtained in Example 1 on suppressing and improving skin wrinkles, a collagenase inhibition activity test was performed. Collagenase inhibitory activity was tested by mixing 100 ㎕ of substrate solution with 2mM FALGPA (N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala) and 10 ㎕ of sample solution. After adding 30㎕ of enzyme (0.08mg/㎖) and 60㎕ of 50mM tricine buffer (pH 7.5) containing 10mM CaCl 2 and 400mM NaCl, Absorbance was measured for minutes.
콜라게나아제 활성은 시료용액의 첨가구와 무첨가구의 흡광도 감소율(FALGPA 가수분해)로 나타내었고, 양성대조구는 올레아놀산(oleanolic acid)를 사용하여 비교하였다. Collagenase activity was expressed as the rate of decrease in absorbance (FALGPA hydrolysis) of the sample solution added and not added, and the positive control group was compared using oleanolic acid.
그 결과 도 3에 개시한 바와 같이 콜라게나아제 활성에 따른 기질 FALGPA의 흡광도가 감소하는 정도가 다르게 나타났다. 병풀 추출물 또는 본 발명의 병풀 유산균 발효 추출물 첨가한 군 대비 아무것도 첨가하지 않은 대조군의 FALGPA 감소되는 정도가 더 크게 나타나, 본 발명의 병풀 유산균 발효 추출물이 콜라게나아제를 억제하는 효과가 있다는 것을 알 수 있었다. As a result, as shown in Figure 3, the degree to which the absorbance of the substrate FALGPA decreased depending on collagenase activity was different. Compared to the group added with the Centella asiatica extract or the fermented Centella asiatica lactic acid bacteria extract of the present invention, the degree to which FALGPA was reduced in the control group to which nothing was added was greater, indicating that the Centella asiatica lactic acid bacteria fermented extract of the present invention had an effect of inhibiting collagenase. .
실시예 6. 추출 및 발효에 따른 병풀 시료에 함유된 성분 분석Example 6. Analysis of components contained in Centella asiatica samples following extraction and fermentation
본 발명의 실시예 1에서 제조한 병풀 추출물, 병풀 발효 추출물 및 병풀 추출 발효물에 대한 HPLC 분석을 통해, 각 시료 내 마데카소사이드(Madecassocide), 아시아티코사이드(Asiaticoside), 마데카식산(Madecassic acid) 및 아시아틱산(Asiatic acid)의 함량변화를 비교 분석하였다. 각 시료의 HPLC 분석은 동일 조건으로 실시하였고, 마데카소사이드(Madecassocide), 아시아티코사이드(Asiaticoside), 마데카식산(Madecassic acid) 및 아시아틱산(Asiatic acid) 표준품의 머무름시간(RT)에서의 면적(area)으로 함량을 비교하였다.Through HPLC analysis of the Centella asiatica extract, Centella asiatica fermented extract, and Centella asiatica extract fermented product prepared in Example 1 of the present invention, madecassoside, asiaticoside, and madecassic acid were found in each sample. ) and the change in content of asiatic acid were compared and analyzed. HPLC analysis of each sample was performed under the same conditions, and the area at the retention time (RT) of Madecassoside, Asiaticoside, Madecassic acid, and Asiatic acid standards Contents were compared by (area).
그 결과 하기 표 2에 개시한 바와 같이, 병풀 물 추출물에서는 마데카소사이드(Madecassocide), 아시아티코사이드(Asiaticoside), 마데카식산(Madecassic acid) 및 아시아틱산(Asiatic acid)이 전혀 나타나지 않았고, 병풀 발효 추출물은 마데카식산(Madecassic acid) 및 아시아틱산(Asiatic acid)의 함량이 병풀 에탄올 추출물에 비해 높아졌고, 병풀 추출 발효물은 마데카소사이드(Madecassocide) 및 아시아티코사이드(Asiaticoside)의 함량이 병풀 에탄올 추출물에 비해 높아진 것을 확인하였다. As a result, as shown in Table 2 below, Madecassoside, Asiaticoside, Madecassic acid, and Asiatic acid did not appear at all in the Centella asiatica water extract, and Centella asiatica fermentation The content of Madecassic acid and Asiatic acid in the extract was higher compared to the Centella asiatica ethanol extract, and the content of Madecassoside and Asiaticoside in the fermented product from Centella asiatica was higher than that in the Centella asiatica ethanol extract. It was confirmed that it was higher than the extract.
(MS)Madecassoside
(MS)
(AS)Asiaticoside
(AS)
(MA)Madecassic acid
(MA)
(AA)Asiatic acid
(AA)
실시예 7. 병풀 에탄올 추출물(CA02) 및 병풀 유산균 발효 추출물(CA04)의 처리에 따른 MMP-1 함량 변화 분석Example 7. Analysis of changes in MMP-1 content according to treatment of Centella asiatica ethanol extract (CA02) and Centella asiatica lactic acid bacteria fermentation extract (CA04)
인간 각질세포인 HaCaT 세포를 10% FBS와 페니실린/스트렙토마이신 100 unit/㎖이 함유된 DMEM(Dulbecco’s modified Eagle’s medium)를 사용하여 37℃, 5% CO2 인큐베이터에서 배양하였다. HaCaT cells, which are human keratinocytes, were cultured in an incubator at 37°C and 5% CO 2 using DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS and 100 units/ml of penicillin/streptomycin.
이후, HaCaT 세포를 웰당 1×105개의 세포가 되도록 6웰 플레이트에 분주하고 24시간 동안 배양하고, PBS(Phosphate buffered salin)으로 2회 세척 후 UVB 25 mJ/cm2을 조사하고 UV 조사된 buffer를 제거한 다음, 병풀 추출물 및 병풀 발효 추출물을 각각 농도별(50, 100㎍/㎖)로 처리하여 24시간 동안 배양하였다. 이후 배양액을 이용하여 Human MMP-1 ELISA kit를 이용하여 MMP-1의 농도를 마이크로플레이트 리더로 흡광도를 측정하였다. Afterwards, HaCaT cells were dispensed into a 6-well plate at 1×10 5 cells per well and cultured for 24 hours, washed twice with PBS (Phosphate buffered salin), irradiated with UVB 25 mJ/cm 2 , and washed with UV-irradiated buffer. After removal, Centella asiatica extract and Centella asiatica fermented extract were treated at different concentrations (50 and 100 μg/ml) and cultured for 24 hours. Afterwards, the absorbance of the culture medium was measured using a Human MMP-1 ELISA kit to determine the concentration of MMP-1 using a microplate reader.
그 결과 도 4A에 개시한 바와 같이, UV 조사에 의해 MMP-1 발현량이 통계적으로 유의미하게 증가하였으며, 본 발명의 병풀 유산균 발효 추출물을 처리한 경우 MMP-1 발현량이 현저하게 감소하였다는 것을 확인하였다. As a result, as shown in Figure 4A, it was confirmed that the amount of MMP-1 expression was statistically significantly increased by UV irradiation, and that the amount of MMP-1 expression was significantly decreased when treated with the fermented extract of Centella asiatica lactic acid bacteria of the present invention. .
한편, 상기 HaCaT 세포에 대하여, 병풀 에탄올 추출물 및 병풀 유산균 발효 추출물의 세포생존률를 확인하기 위하여, HaCaT 세포를 웰당 5×103개의 세포가 되도록 96웰 플레이트에 분주하여 37℃, 5% CO2 인큐베이터에서 배양하였다. 이후, 병풀 추출물 및 병풀 발효 추출물을 각각 농도별(50, 100㎍/㎖)로 처리하여 24시간 배양한 후 MTT(3-(4,5-dimethylthiazole-2-yl)-2,5-di-phenyl-tetrazolium bromide) 어세이를 이용하여 세포 생존률을 확인하였다.Meanwhile, in order to check the cell survival rate of the Centella asiatica ethanol extract and the Centella asiatica lactic acid bacteria fermentation extract for the HaCaT cells, HaCaT cells were distributed in a 96-well plate to have 5 × 10 cells per well and incubated in an incubator at 37°C and 5% CO 2. Cultured. Afterwards, Centella asiatica extract and Centella asiatica fermented extract were treated at different concentrations (50 and 100 μg/ml), cultured for 24 hours, and MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-di- Cell viability was confirmed using a phenyl-tetrazolium bromide) assay.
그 결과 도 4B에 개시한 바와 같이, UV 조사 및 본 발명의 시료를 처리한 경우에도 세포 생존율이 거의 정상군 수준으로 유지되는 것을 확인하였다.As a result, as shown in Figure 4B, it was confirmed that the cell survival rate was maintained at almost the normal level even when treated with UV irradiation and the sample of the present invention.
Claims (7)
1) 병풀 분말에 물을 가하여 멸균하는 단계;
2) 상기 단계 1) 이후에, 상온으로 식혀 락토바실러스 플랜타룸(Lactobacillus plantarum), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 메센테로이드스(Leuconostoc mesenteroides) 및 락토바실러스 사케이(Lactobacillus sakei)로 이루어진 복합유산균을 첨가하고 25~40℃에서 2~4일 동안 발효하는 단계;
3) 상기 단계 2) 이후에, 50~70℃에서 건조하는 단계;
4) 상기 단계 3)에서 건조한 병풀 유산균 발효물의 건조 분말에 에탄올을 첨가하여 추출하는 단계;
5) 상기 단계 4)의 병풀 유산균 발효 에탄올 추출물을 여과하는 단계; 및
6) 상기 단계 5)의 여과한 추출물을 감압 농축하고 건조하는 단계;를 포함하는 제조방법으로 제조된 것을 특징으로 하는 염증, 주름, 알러지 및 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물.The method of claim 1, wherein the ethanol extract of Centella asiatica lactic acid bacteria fermentation
1) Sterilizing the Centella asiatica powder by adding water;
2) After step 1), cool to room temperature and mix with Lactobacillus plantarum , Leuconostoc kimchii, Leuconostoc citreum , and Leuconostoc mesenteroides. ) and Lactobacillus sakei ( Lactobacillus sakei ) and fermenting at 25-40°C for 2-4 days;
3) After step 2), drying at 50-70°C;
4) extracting by adding ethanol to the dried powder of the fermented Centella asiatica lactic acid bacteria dried in step 3);
5) filtering the ethanol extract from the fermentation of Centella asiatica lactic acid bacteria in step 4); and
6) A health functional food composition for preventing or improving inflammation, wrinkles, allergies and atopic dermatitis, which is prepared by a manufacturing method comprising the step of concentrating and drying the filtered extract of step 5) under reduced pressure.
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