KR20210096840A - Manufacturing method of Hwangryunhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects - Google Patents

Manufacturing method of Hwangryunhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects Download PDF

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KR20210096840A
KR20210096840A KR1020200010367A KR20200010367A KR20210096840A KR 20210096840 A KR20210096840 A KR 20210096840A KR 1020200010367 A KR1020200010367 A KR 1020200010367A KR 20200010367 A KR20200010367 A KR 20200010367A KR 20210096840 A KR20210096840 A KR 20210096840A
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coptis chinensis
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서상완
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호남대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A23L31/10—Yeasts or derivatives thereof
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age

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Abstract

The present invention relates to a method for manufacturing a Coptis chinensis detoxification decoction with enhanced antibacterial, anti-inflammatory, and antioxidant effects. According to the present invention, the method comprises the following steps: preparing a Coptis chinensis detoxification decoction consisting of a Coptis chinensis extract, a Scutellaria baicalensis extract, and a Gardenia jasminoides Ellis seed extract; inoculating the Coptis chinensis detoxification decoction with a Lactobacillus plantarum strain; and performing fermentation after inoculation of the strain. Accordingly, a fermented Coptis chinensis detoxification decoction has antibacterial activity and, in particular, has excellent growth inhibitory activity against Cutibacterium acnes, thereby reducing dermatitis such as acne. In addition, through 1,1-diphenyl-2-picryhydrazyl (DPPH) radical scavenging activity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, and superoxide dismutase (SOD)-like activity, antioxidant activity has increased by 12.3% more than that of a non-fermented Coptis chinensis detoxification decoction. In addition, at a concentration of 400 μg /ml, the fermented Coptis chinensis detoxification decoction has a 43.1% nitric oxide expression inhibitory effect than that of a non-fermented hot-water extraction Coptis chinensis detoxification decoction, and anti-inflammatory is provided by inhibiting production of intrinsic inflammatory substances (tumor necrosis factor (TNF)-α, Interleukin (IL)-1β and IL-6, etc.).

Description

항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법 {Manufacturing method of Hwangryunhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects}{Manufacturing method of Hwangryunhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects}

본 발명은 황련해독탕의 제조방법에 관한 것으로, 더욱 상세하게는 황련해독탕을 Lactobacillus plantarum 균주를 이용하여 발효시킴으로써 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법에 관한 것이다.The present invention relates to a method for preparing Hwangryeonhaedok-tang, and more particularly, to a method for preparing Hwangryeonhaedok-tang with improved antibacterial, anti-inflammatory and antioxidant effects by fermenting Hwangryeonhaedok-tang using a Lactobacillus plantarum strain.

약식동원(藥食同源)이란 약(藥)과 먹는 것(食)의 근원은 하나를 뜻한다. 음식으로 고치지 못하는 병은 약으로도 고치지 못한다는 말인데 우리가 먹는 음식을 체질에 맞게 섭취하면 바로 약이 된다는 말이다. 예로부터 우리 조상들은 좋은 식재료로 함유되어 있는 몸에 필요한 영양소를 섭취함으로서 건강을 지켜왔다. Yakshik mobilization (藥食同源) means that the source of medicine and eating is one. A disease that cannot be cured with food cannot be cured even with medicine, which means that if we consume the food we eat according to our constitution, it becomes medicine. From time immemorial, our ancestors have maintained their health by consuming the nutrients necessary for the body, which is contained in good ingredients.

동양의학에서는 약을 처방하기 전에 먼저 음식 조절을 통해 병을 예방 또는 치료하고, 그 후에 효과가 없을 경우에 약을 사용하였다. 한방 천연물도 함부로 쓰지 않고, 철저하게 인체의 기운을 상하지 않게 보존하는 것이 올바른 치료 방법임을 후대에 전하여 왔다. 조선시대 명의 허준이 집필한 동의보감(1610년)에서도 질병을 치료하기 위해 약처방과 침뜸 방법보다는 음식으로 예방·치료하는 식이문화에 대하여 매우 깊이 있게 다루고 있다(Ryu, 2015, 비특허문헌 1). 조선후기 명의 이제마에 의해 창안된 조선시대 의학이론인 사상체질 또한 태양인, 태음인, 소양인, 소음인 등 각각 체질에 따라 건강에 좋은 음식과 피해야 할 음식에 대해 상세히 소개하고 있다(Park, 1999, 비특허문헌 2). In oriental medicine, before prescribing a drug, the disease was prevented or treated through food control, and then the drug was used when it was not effective. It has been handed down to the next generation that it is the right treatment method to thoroughly preserve the energy of the human body without using natural herbal medicines carelessly. Even in Donguibogam (1610), written by Heo Jun, a master of the Joseon Dynasty, the dietary culture of preventing and treating diseases with food rather than prescription drugs and acupuncture to treat diseases is dealt with very deeply (Ryu, 2015, Non-Patent Document 1). Sasang constitution, a medical theory of the Joseon Dynasty, created by Je-ma, a master of the late Joseon Dynasty, also introduces in detail healthy foods and foods to avoid according to each constitution, such as Taeyangin, Taeeumin, Soyangin, and Soeumin (Park, 1999, Non-Patent Literature). 2).

최근에는 국민소득의 상승과 급격한 산업화로 생활수준이 향상되었고, 의학기술의 발달로 인해 평균수명이 크게 늘어남에 따라 육체적·정신적 건강의 조화를 통한 아름답고 건강한 외모에 대한 관심 또한 높아졌는데, 서구화된 식습관으로 인하여 비만과 각종 피부질환도 발생하게 되었다. 외모관리에 있어서 피부 트러블은 심각한 스트레스로 작용할 수 있다.Recently, the standard of living has improved due to the rise in national income and rapid industrialization, and as the average life expectancy has increased significantly due to the development of medical technology, interest in beautiful and healthy appearance through the harmony of physical and mental health has also increased. As a result, obesity and various skin diseases have occurred. Skin problems in appearance management can act as a serious stress.

피부 미용분야에서 여드름의 원인은 아직까지도 정확하게 밝혀져 있지 않으며 다양한 인자의 상호작용에 의해 유발되기 때문에 현재까지 효과적인 치료방법은 미비한 실정이다. 여드름은 특히 사춘기에 유발되며 결절, 구진, 농포, 면포 등 병변을 특징으로 하는 모피지성 질환(pilosebaceous disease)으로 최근 성장기가 지난 성인에게도 여드름 발생이 증가하고 있다. 여드름의 병인은 남성호르몬에 의한 피지 분비의 증가, 과각화된 모낭벽의 증가, 여드름 원인 미생물인 Cutibacterium acnes 세균 증식에 의한 염증유발, 환경적 요인, 유전적 요인, 피부장벽 이상, 모낭의 반응성 등 복합적인 요인으로 발생하는 것으로 보고하고 있다(Farrar & Ingham, 2004, 비특허문헌 3). 이 중에서 C. acnes 여드름 유발균이 여드름 발생의 중요한 원인으로 알려져 있다. C. acnes는 혐기성 상재균으로서 모낭 내에서 성장하는 지방 친화성 미생물이다. 황색포도상구균으로 알려져 있는 Staphylococus aureus와 S. epidermidis는 세포집괴를 형성하는 그람양성 통성혐기성 세균이다. 피부 내에 정상적으로 분포하는 호기성 피부 상재균으로 염증반응의 일차적인 원인은 아니지만, 염증의 발생 부위를 더욱 확장시켜 결국 여드름을 악화시키며 화농성 감염증을 유발하는 것으로 알려져 있다.. In the field of skin care, the cause of acne is still not precisely known, and since it is caused by the interaction of various factors, an effective treatment method is insufficient so far. Acne is a pilosebaceous disease that is especially induced during puberty and is characterized by lesions such as nodules, papules, pustules, and comedones. The etiology of acne is complex such as increase in sebum secretion by male hormone, increase in hyperkeratinized hair follicle wall, inflammation caused by bacterial growth of Cutibacterium acnes , a microorganism that causes acne, environmental factors, genetic factors, skin barrier abnormality, hair follicle reactivity, etc. It has been reported that it occurs due to a negative factor (Farrar & Ingham, 2004, Non-Patent Document 3). Among them, C. acnes acne-causing bacteria is known to be an important cause of acne. C. acnes is an anaerobic flora, a lipophilic microorganism that grows in hair follicles. Staphylococcus aureus and S. epidermidis , also known as Staphylococcus aureus, are gram-positive facultative anaerobes that form agglomerates. It is an aerobic dermatophyte that is normally distributed in the skin and is not the primary cause of the inflammatory reaction, but it is known that it further expands the site of inflammation, exacerbating acne and causing purulent infections.

C. acnes는 면역 반응과 염증 유발을 유도하고 각질형성 세포를 자극하여 다량의 superoxide anion(O2-)을 생성한다. 이렇게 생성된 O2-는 nitric oxide (NO)와 함께 peroxynitrites 형태로 각질형성 세포의 억제에 관여한다(Bowe et al., 2008, 비특허문헌 4). 면포가 형성된 피부에서는 정상인 피부보다 linoleic acid가 현저하게 감소하고, palmitic acid는 증가하는 것으로 보고되어 있다. Linoleic acid는 활성 산소종(O2-, OH-, H2O2 -) 생성 자체를 억제하는 작용이 있으며, palmitic acid는 단지 H2O2 - 생성만을 감소시킨다(Akamatsu et al., 2003, 비특허문헌 5). 면포 부위에서 활성 산소종(reactive oxygen species, ROS)의 과도한 생성은 C. acnes 증식 저해물질의 결핍을 초래하고, palmitic acid는 여드름 염증반응에 관여한다. 또한 피지성분 중의 squalen은 triterpenoid 계열로 지질과산화 과정에서 생성되는 superoxide 소거능이 있어 피부를 보호하지만, squalen peroxide가 생성되면 면포형성과 inflammatory mediator를 생성하며, 세균의 집락화에 관여한다(Akamatsu et al., 1998, 비특허문헌 6; Ottavianiv et al., 2006, 비특허문헌 7). 활성 산소종 중에서 1O2 (singlet oxygen) 및 OH-은 단백질 및 DNA 산화, 지질 과산화반응의 개시, 멜라닌 생성과정, 결합조직의 사슬절단 및 비정상적인 교차결합에 의한 주름생성과 피부 항산화제 파괴 등에 관여하여 피부노화를 가속화시키는 것으로 알려져 있다. 활성 산소종은 이러한 생체 구성성분의 산화적 손상뿐만 아니라 UVA 의존성 세포사멸이나 유전자 활성화에 중요한 역할을 한다(Scharffetter, 1997, 비특허문헌 8; Vile, 1995, 비특허문헌 9). 이와 같이 항산화제는 피부노화 방지와 피부 여드름의 염증 제거 또는 상처 재생과정에서 중요한 역할을 한다. C. acnes induces immune response and inflammation, and stimulates keratinocytes to produce large amounts of superoxide anion (O 2 - ). O 2 generated in this way is involved in the inhibition of keratinocytes in the form of peroxynitrites together with nitric oxide (NO) (Bowe et al., 2008, Non-Patent Document 4). It has been reported that linoleic acid is significantly decreased and palmitic acid is increased in skin with comedones than in normal skin. Linoleic acid inhibits the generation of reactive oxygen species (O 2 - , OH - , H 2 O 2 - ), and palmitic acid only reduces H 2 O 2 - production (Akamatsu et al., 2003, Non-patent document 5). Excessive production of reactive oxygen species (ROS) in the comedonal area leads to a deficiency of C. acnes proliferation inhibitors, and palmitic acid is involved in the acne inflammatory response. In addition, squalen in the sebum component is a triterpenoid type and has the ability to remove superoxide generated in the lipid peroxidation process to protect the skin. 1998, Non-Patent Document 6; Ottavianiv et al., 2006, Non-Patent Document 7). Among reactive oxygen species, 1O 2 (singlet oxygen) and OH - are involved in protein and DNA oxidation, initiation of lipid peroxidation, melanin production, connective tissue chain cutting and abnormal crosslinking, wrinkle formation and destruction of skin antioxidants. It is known to accelerate skin aging. Reactive oxygen species play an important role in UVA-dependent cell death or gene activation as well as oxidative damage of these biological components (Scharffetter, 1997, Non-Patent Document 8; Vile, 1995, Non-Patent Document 9). As such, antioxidants play an important role in preventing skin aging, removing inflammation of skin acne, or regenerating wounds.

김치는 대표적인 전통 발효식품으로 모든 채소류가 김치의 주원료로 사용할 수 있으며, 김치 원·부재료 유래 미생물에 의해서 발효가 진행된다(Cheigh et al., 1994, 특허문헌 10; Koh, 2004, 비특허문헌 11). 특히 김치 발효과정에 관여하는 유산균에 대한 관심이 점차 증가하고 있으며, 다양한 기능성 연구도 진행되고 있다. 김치유산균이 생산하는 대사산물은 김치의 향미뿐 아니라, 이를 섭취하였을 때 장내 군총 증가와 같은 기능성도 보고되어 있으며, 김치유산균은 유산균 제제 뿐만 아니라, 건강 기능성 식품이나 건강식품 등에 함유하여 인체의 영양 강화 또는 생리활성 기능을 촉진하고 있다(Atrih et al., 1993, 비특허문헌 12; Klaver, 1993, 비특허문헌 13). 최근에는 프로바이오틱스(probiotics)가 주목을 받고 있으며 프로바이오틱스는 살아있는 균주를 섭취하는 것이라 항생제 섭취 문제인 잔류 및 내성문제를 해결할 수 있다(Femandez et al., 2003, 비특허문헌 14; O’Sullivan, 2001, 비특허문헌 15). Kimchi is a representative traditional fermented food, and all vegetables can be used as the main raw material for kimchi, and fermentation is carried out by microorganisms derived from raw and sub-materials of kimchi (Cheigh et al., 1994, Patent Document 10; Koh, 2004, Non-Patent Document 11) ). In particular, interest in lactic acid bacteria involved in the fermentation process of kimchi is increasing, and various functional studies are being conducted. The metabolites produced by Kimchi Lactobacillus are reported not only for the flavor of Kimchi, but also for functions such as an increase in intestinal colonization when ingested. Or it promotes the physiologically active function (Atrih et al., 1993, Non-Patent Document 12; Klaver, 1993, Non-Patent Document 13). Recently, probiotics have been attracting attention, and probiotics can solve the problems of antibiotic intake, such as retention and resistance, by ingesting live strains (Femandez et al., 2003, Non-Patent Document 14; O'Sullivan, 2001, B. Patent Document 15).

김치에는 다양한 항산화 물질들이 함유되어 있어 활성산소에 의한 DNA 손상 억제, superoxide anion 및 과산화수소 소거능 및 지질과산화 억제 등과 같은 효능이 보고되어 있다(Kang, 1988, 비특허문헌 16). 또한 섭취시 혈중 중성지방과 총콜레스테롤 농도를 저하시키고 면역기능을 조절하는 효과가 있다. 노화촉진 쥐(SAM)에게 김치 섭취한 결과, 간과 뇌 안에서 유리기 생성이 유의적으로 억제되었고, 항산화 효소활성과 항노화 활성이 증대되었음을 확인하였다(Kim et al., 2002, 비특허문헌 17). 특히 피부세포에 산화적 스트레스를 유발시키고 김치 추출물을 처리한 결과, 활성산소에 의한 독성이 완화되는 효과가 있었다고 보고한 바 있다(Ryu et al., 1997, 비특허문헌 18). 또한 마우스에 다양한 김치(배추김치, 갓김치, 부추김치)를 섭취시켰을 때, 지질 및 단백질 산화가 억제되었다는 연구보고가 있다(Ryu et al., 2004, 비특허문헌 19).Since kimchi contains various antioxidants, effects such as inhibition of DNA damage caused by free radicals, superoxide anion and hydrogen peroxide scavenging ability, and inhibition of lipid peroxidation have been reported (Kang, 1988, Non-Patent Document 16). In addition, when ingested, it has the effect of lowering blood triglyceride and total cholesterol levels and regulating the immune function. As a result of ingestion of kimchi to accelerated aging mice (SAM), it was confirmed that free radical production was significantly inhibited in the liver and brain, and antioxidant enzyme activity and anti-aging activity were increased (Kim et al., 2002, Non-Patent Document 17). In particular, it has been reported that oxidative stress was induced in skin cells and that the kimchi extract had the effect of alleviating the toxicity caused by free radicals (Ryu et al., 1997, Non-Patent Document 18). In addition, there is a research report that lipid and protein oxidation was inhibited when mice ingested various types of kimchi (baechu kimchi, gat kimchi, leek kimchi) (Ryu et al., 2004, Non-Patent Document 19).

종양괴사 인자(tumor necrosis factor, TNF-α)는 병원성 미생물에 의한 급성 염증반응의 매개자이며, 중증 감염 형태인 전신적인 합병증의 원인이 된다. TNF-α는 종양에 대하여 출혈성 괴사를 일으키는 혈청인자와 염증을 통한 생체방어 체계에 관여하는 사이토카인으로도 알려져 있다. 주로 대식세포에서 생성되어지는 사이토카인이지만 염증반응에서 주 역할을 담당하는 호중구, 림프구, 섬유모세포, NK cell, 각질세포, 비만세포에서도 생성이 된다. TNF-α 기능으로는 항균활성, 항종양활성, 분화 증식 조절작용, 염증반응에 따른 면역작용 등이 보고되어 있으나, 과도한 분비시 류마티스성 질환의 원인이 되기도 한다(Felson et al., 1995, 비특허문헌 20).Tumor necrosis factor (TNF-α) is a mediator of an acute inflammatory response caused by pathogenic microorganisms and causes systemic complications in the form of severe infections. TNF-α is also known as a serum factor that causes hemorrhagic necrosis against tumors and a cytokine involved in the biological defense system through inflammation. Although it is a cytokine produced mainly by macrophages, it is also produced by neutrophils, lymphocytes, fibroblasts, NK cells, keratinocytes, and mast cells that play a major role in the inflammatory response. As the function of TNF-α, antibacterial activity, antitumor activity, regulation of differentiation and proliferation, and immune function according to inflammatory response have been reported, but excessive secretion can also cause rheumatic diseases (Felson et al., 1995, B. Patent Document 20).

Interleukin-1β(IL-1β)의 주요 기능은 TNF-α와 유사하나 감염이나 외부의 자극에 대한 숙주의 염증반응 매개자로 알려져 있다. IL-1β는 미생물을 비롯한 여러 가지 생성 유도물질의 자극을 통해 대식세포, NK cell, 각질세포, 비만세포, 섬유모세포, T-cell, B-cell 등 다양한 세포들로부터 생성되며, TNF-α와 동일하게 과다 발현시 류머티스성 질환이 발생하는 것으로 알려져 있다(Chung et al., 2009, 비특허문헌 21)The main function of Interleukin-1β (IL-1β) is similar to that of TNF-α, but it is known as a mediator of the host's inflammatory response to infection or external stimuli. IL-1β is produced from various cells such as macrophages, NK cells, keratinocytes, mast cells, fibroblasts, T-cells, and B-cells through stimulation of various production inducers including microorganisms, and TNF-α and Similarly, overexpression is known to cause rheumatic diseases (Chung et al., 2009, Non-Patent Document 21)

Interleukin-6(IL-6)는 선천면역과 적응면역 모두에서 기능을 나타내는 사이토카인이다. IL-6는 다기능성 사이토카인으로 대식세포, T-림프구와 B-림프구에서 주로 생성되며 대표적인 기능으로는 조혈세포 및 신경세포의 증식과 분화 및 면역반응 등에 관여하고 있으며, IL-6가 과다하게 분비될 경우 림프계 종양과 다양한 면역이상 증상이 발생하는 것으로 알려져 있다(Toshio, 2014, 비특허문헌 22). 이와 같이 인체에 유익한 사이토카인이지만 과도한 발현시 오히려 인체에 해로운 작용을 하기 때문에 분비량 조절이 반드시 필수적이다. Interleukin-6 (IL-6) is a cytokine that functions in both innate immunity and adaptive immunity. IL-6 is a multifunctional cytokine and is mainly produced in macrophages, T-lymphocytes and B-lymphocytes, and its representative functions are involved in the proliferation and differentiation of hematopoietic cells and nerve cells, and immune response. When secreted, it is known that lymphoid tumors and various immune abnormalities occur (Toshio, 2014, Non-Patent Document 22). Although this cytokine is beneficial to the human body, it is essential to control the amount of secretion because excessive expression of the cytokine has a detrimental effect on the human body.

황금, 황련, 황백 및 치자를 주원료로 함유한 황련 해독탕은 한방에서 예로부터 피부질환 치료에 널리 사용했다고 보고되어 있으며, 아토피성 피부염, 습진, 지루성 피부염, 접촉성 피부염 등에 효과적인 것으로 알려져 있다. 황련에는 berberine과 worenine 같은 알칼로이드성 물질이 함유되어 항균작용이 뛰어나며, 해열, 진통, 진정, 국소마비, 혈압저하 및 담(痰)을 원활하게 해주는 작용을 한다(Yang et al., 1995, 비특허문헌 23). 황금은 습사(濕邪)와 습열사(濕熱邪)를 제거하고 지혈 및 해독하는 효능을 가지고 있으며 체외에서 각종 병원성 피부사상균을 억제시키며 baicalin과 baicalein을 다량 함유하고 있어 항염증 및 항알러지 효능과 알러지성 천식을 완화하는 것으로 알려져 있다. 황백은 phellodendrine과 berberine 같은 알칼로이드와 obaculactone 및 obacunone 물질이 함유되어 있다. 황련과 같이 피부 진균 및 각종 병원성 세균을 억제하는 항균활성이 있으며 담(痰)을 흩어주고 해열, 진통하는 작용도 보고되어 있다. 이와 같이 황련, 황금, 황백은 공통적으로 청열 해독의 기능을 가지므로 이들을 혼합하면 효과가 더 증가될 뿐 아니라 천연 방부효과도 있는 것으로 알려져 있다. 이와 같은 기능성으로 각종 목욕용품과 화장품 배합제로 사용되어 알러지성 피부염 및 다양한 피부병의 개선 또는 예방하는 것으로 알려져 있다(주영승, 2004, 비특허문헌 24). 치자는 꼭두서니과에 속하는 상록활엽 관목으로 한방에서 소염, 해열, 이뇨, 지혈, 진정, 이담, 혈압강하제로 사용되어 왔다. 치자의 황금색 색소는 식용 및 염료로 오랫동안 사용된 천연색소로 carotenoid 계열 중 crocetin의 카복실기(-COOH)에 여러 가지 당이 결합된 배당체로 구성되어 있으며, 그 중 crocetin digentiobioside인 crocin이 주성분으로 알려져 있다(Jeong et al., 1999, 비특허문헌 25). 치자 추출물은 피부 미백, monoamine oxidase(MAO) 저해 및 anti-inflamatory 효과가 있는 것으로 보고되어 있다(Hwang et al, 2007, 비특허문헌 26). Hwangryeon detoxification soup, which contains gold, yellow lily, yellow lily and gardenia as the main ingredients, has been reported to have been widely used in oriental medicine for the treatment of skin diseases since ancient times, and is known to be effective in atopic dermatitis, eczema, seborrheic dermatitis, and contact dermatitis. Yellow lotus contains alkaloids such as berberine and worenine, which have excellent antibacterial action, and have antipyretic, analgesic, sedative, local paralysis, blood pressure lowering and biliary effects (Yang et al., 1995, non-patented). literature 23). Gold has the effect of removing wet and moist heat, hemostasis and detoxification, and inhibiting various pathogenic dermatophytes outside the body. It is known to relieve sexual asthma. Yellow white contains alkaloids such as phellodendrine and berberine, as well as obaculactone and obacunone substances. It has antibacterial activity that suppresses skin fungus and various pathogenic bacteria like yellow lotus, and it has also been reported to dissipate phlegm and relieve fever and pain. As described above, yellow lotus, gold, and yellow white have a function of detoxifying heat and heat in common, so mixing them increases the effect and is known to have a natural antiseptic effect. It is known to improve or prevent allergic dermatitis and various skin diseases by being used as a formulation for various bath products and cosmetics due to such functionality (Joo Young-seung, 2004, Non-Patent Document 24). Gardenia is an evergreen broad-leaved shrub belonging to the Apiaceae family and has been used as an anti-inflammatory, antipyretic, diuretic, hemostasis, sedative, edema, and blood pressure lowering agent in oriental medicine. Gardenia's golden pigment is a natural pigment that has been used for a long time as food and dye. It is composed of glycosides in which various sugars are bonded to the carboxyl group (-COOH) of crocetin among carotenoids, and among them, crocin, a crocetin digentiobioside, is known as the main component. (Jeong et al., 1999, Non-Patent Document 25). Gardenia extract has been reported to have skin whitening, monoamine oxidase (MAO) inhibition and anti-inflamatory effects (Hwang et al, 2007, Non-Patent Document 26).

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본 발명에서 해결하고자 하는 과제는 우수한 기능성 김치유산균을 이용하여 황련해독탕을 발효함으로써 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법을 제공하는 것이다.An object to be solved in the present invention is to provide a method for preparing Hwangryeonhaedoktang with improved antibacterial, anti-inflammatory and antioxidant effects by fermenting Hwangryeonhaedoktang using excellent functional kimchi lactic acid bacteria.

위와 같은 과제를 해결하기 위한 본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 황련, 황금, 황백 및 치자 추출물로 이루어지는 황련해독탕을 제조하는 단계; 상기 황련해독탕에 Lactobacillus plantarum 균주를 접종하는 단계; 및 상기 균주 접종 후 발효시키는 단계를 포함하여 구성되는 것을 기술적 특징으로 한다.According to the present invention for solving the above problems, there is provided a method for preparing hwangryeonhaedok-tang with enhanced antibacterial, anti-inflammatory and antioxidant effects, comprising: preparing hwangryeonhaedok-tang consisting of extracts of hwangryeon, gold, hwangbaek and gardenia; inoculating the Lactobacillus plantarum strain into the Hwangnyeonhaedoktang; And it is technically characterized in that it is configured to include the step of fermenting after inoculation of the strain.

또한, 위와 같은 과제를 해결하기 위한 본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 상기 균주는 Lactobacillus plantarum CK9인 것을 기술적 특징으로 한다.In addition, the method for producing Hwangryeonhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects according to the present invention for solving the above problems is characterized in that the strain is Lactobacillus plantarum CK9.

또한, 위와 같은 과제를 해결하기 위한 본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 상기 발효시키는 단계는 상기 균주 접종 후 36시간 동안 발효하는 것을 기술적 특징으로 한다.In addition, in the method for producing Hwangryeonhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects according to the present invention for solving the above problems, the fermenting step is fermented for 36 hours after inoculation of the strain.

또한, 위와 같은 과제를 해결하기 위한 본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 상기 황련해독탕을 제조하는 단계는, 황련, 황금, 황백, 치자 및 yeast extract를 물에 넣는 단계; 상기 혼합물을 전탕하는 단계; 및 상기 전탕된 혼합물을 여과한 다음, 원심분리하여 상층액만을 분리하는 단계를 포함하여 구성되는 것을 기술적 특징으로 한다.In addition, the manufacturing method of Hwangryeonhaedok-tang with enhanced antibacterial, anti-inflammatory and antioxidant effect according to the present invention for solving the above problems is the step of preparing the hwangryeonhaedok-tang, hwangryeon, gold, hwangbaek, gardenia and yeast extract putting it in water; boiling the mixture; and filtering the scalded mixture and then centrifuging to separate only the supernatant.

본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 항균활성이 있고, 특히 Cutibacterium acnes에 대해 우수한 생육저해능을 가짐으로써 여드름과 같은 피부염을 감소시킬 수 있다. The method for preparing Hwangryeonhaedok-tang with enhanced antibacterial, anti-inflammatory and antioxidant effects according to the present invention has antibacterial activity and, in particular, has excellent growth inhibitory activity against Cutibacterium acnes, thereby reducing dermatitis such as acne.

또한, 본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 DPPH radical 소거능, ABTS radical 소거능, SOD 유사활성을 통해 비발효 황련해독탕보다 항산화 활성이 12.3% 더 증가된 효과를 갖는다.In addition, the method for preparing Hwangryeonhaedok-tang with improved antibacterial, anti-inflammatory and antioxidant effects according to the present invention has the effect of increasing antioxidant activity by 12.3% more than non-fermented Hwangryeonhaedok-tang through DPPH radical scavenging activity, ABTS radical scavenging activity, and SOD-like activity. has

또한, 본 발명에 따른 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법은 400 μg/ml 농도에서 비발효 열수추출 황련해독탕보다 43.1% nitric oxide 발현 억제 효과가 있고,내재적 염증물질(TNF-α, IL-1β과 IL-6 등)의 생산을 억제함으로써 항염증 효과를 갖는다.In addition, the method for producing Hwangryeonhaedok-tang with enhanced antibacterial, anti-inflammatory and antioxidant effects according to the present invention has an effect of inhibiting the expression of 43.1% nitric oxide than non-fermented hot water extract Hwangryeonhaedok-tang at a concentration of 400 μg/ml, and intrinsic inflammatory substances ( It has an anti-inflammatory effect by inhibiting the production of TNF-α, IL-1β and IL-6).

도 1은 김치로부터 분리한 균주 Leuconostoc, Weissella, Lactobacillus 속에 대한 배양상등액의 항산화 활성(SOD)을 측정한 결과를 나타내는 그래프
도 2는 1차 선발된 15균주를 황련해독탕에 접종한 뒤 생육 결과를 나타내는 그래프
도 3은 Lactobacillus plantarum CK9 균주의 Cutibacterium acnes 균주 배지에 대한 agar well diffusion 실험결과 사진
도 4a는 Lactobacillus plantarum CK9 균주의 염기서열
도 4b는 Lactobacillus plantarum CK9 균주의 계통수
도 5는 비발효 및 발효된 황련해독탕의 Cutibacterium acnes 균주 배지에 대한 agar well diffusion 실험결과 사진
도 6은 황백, 황금, 황련 및 치자 발효물에 대한 생균수 측정 실험 사진
도 7은 황련해독탕 발효물에 대한 생균수 측정 실험 사진
도 8은 ABTS radical 소거능 실험 결과를 나타내는 그래프
도 9는 DPPH radical 소거능 실험 결과를 나타내는 그래프
도 10은 Superoxide dismutase 유사활성 측정 결과를 나타내는 그래프
도 11은 Raw 264.7 세포에 대한 세포 생존율 측정 결과를 나타내는 그래프
도 12는 Nitric oxide 생성량 측정 결과를 나타내는 그래프
도 13은 TNF-α 발현량 측정 결과를 나타내는 그래프
도 14는 IL-1β 발현량 측정 결과를 나타내는 그래프
도 15는 IL-6 발현량 측정 결과를 나타내는 그래프1 is a graph showing the results of measuring the antioxidant activity (SOD) of the culture supernatant for the strains Leuconostoc, Weissella, Lactobacillus isolated from kimchi;
Figure 2 is a graph showing the growth results after inoculating the first 15 strains in Hwangryeonhaedoktang
Figure 3 is a photograph of agar well diffusion test results for the Cutibacterium acnes strain medium of Lactobacillus plantarum CK9 strain;
Figure 4a is the base sequence of the Lactobacillus plantarum CK9 strain
Figure 4b is a phylogenetic tree of Lactobacillus plantarum CK9 strain
5 is a photograph of agar well diffusion test results for Cutibacterium acnes strain medium of non-fermented and fermented Hwangryeonhaedoktang;
Figure 6 is a photograph of a live cell count measurement experiment for Hwangbaek, gold, hwangryeon and gardenia fermented products;
7 is a photograph of a live cell count measurement experiment for Hwangryeonhaedoktang fermented product
8 is a graph showing the results of the ABTS radical scavenging activity
9 is a graph showing the results of DPPH radical scavenging activity
10 is a graph showing the measurement result of superoxide dismutase-like activity
11 is a graph showing the cell viability measurement results for Raw 264.7 cells
12 is a graph showing the measurement result of nitric oxide production
13 is a graph showing the measurement result of TNF-α expression
14 is a graph showing the measurement result of IL-1β expression level;
15 is a graph showing the measurement result of IL-6 expression level;

본 명세서 및 청구범위에 사용된 용어나 단어는 "발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙"에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야지, 통상적이거나 사전적인 의미로 한정해서 해석되서는 안 된다.The terms or words used in the present specification and claims conform to the technical idea of the present invention based on the "principle that the inventor can appropriately define the concept of a term in order to best describe his invention" It should be interpreted as the meaning and concept that

따라서 본 명세서에 기재된 실시예와 도면에 도시된 구성은 본 발명의 가장 바람직한 실시예에 불과할 뿐이고, 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형 예들이 있을 수 있음을 이해해야 한다.Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical ideas of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be water and variations.

실시예 1. 황련해독탕의 제조Example 1. Preparation of Hwangnyeon Haedoktang

본 실험에서 사용한 황련해독탕의 주성분인 황련, 황금, 황백, 치자는 명신당(광주)에서 구입하여 사용하였다. 황련, 황금, 황백, 치자는 각각 물 1L당 20g씩 넣고 yeast extract를 5g 첨가하여 121℃에서 2시간 동안 전탕한 후, 부직포로 1차 여과하고 원심분리(8,500×g, 15분)하고 얻어진 상층액을 취하여 동결건조를 수행하였다. 동결건조한 시료는 6.25, 12.5, 25, 50, 100, 200, 400 μg/ml 농도로 희석하여 아래의 실험예에서 실험하였다.Hwangryeon, gold, hwangbaek, and gardenia, which are the main ingredients of Hwangryeonhaedoktang used in this experiment, were purchased from Myeongsindang (Gwangju) and used. Yellow lily, gold, yellow white, and gardenia, each 20 g per 1 liter of water, 5 g of yeast extract added, simmered at 121° C. for 2 hours, first filtered with a nonwoven fabric, and centrifuged (8,500 × g, 15 minutes), and the resulting upper layer The solution was collected and freeze-dried. The freeze-dried samples were diluted to 6.25, 12.5, 25, 50, 100, 200, and 400 μg/ml concentrations and tested in the following experimental examples.

이하의 실험예에서 사용되는 황련해독탕은 동결건조하여 시료로서 사용하였으며, HHT(비발효 황련해독탕)는 34.4%의 수율을 보였으며, FHHT(발효 황련해독탕은) 32.6%의 수율을 확인하였다. 시료의 농도에 따라 멸균증류수로 희석하여 사용하였다.Hwangryeonhaedoktang used in the following experimental examples was freeze-dried and used as a sample. did. It was used after dilution with sterile distilled water according to the concentration of the sample.

실시예 2. 유산균 분리Example 2. Isolation of lactic acid bacteria

다양한 김치(배추김치, 깍두기, 양배추김치, 총각김치, 동치미 등)를 각각 파쇄 및 여과하고 김치여과액을 0.85% 멸균 생리식염수로 십진 희석하였다. MRS agar 고체배지에 100 μl씩 도말하고 30℃ 배양기에서 혐기조건으로 48시간동안 배양 후, 미생물 집락 형태에 따라 서로 다른 미생물을 분리하였다. 피검균으로 사용한 Cutibacterium acnes 배양은 cooked meet medium(Difco Lab., USA) 액체배지에 접종하여 30℃에서 48시간 혐기적으로 배양하였고, Staphylococcus aureus 배양은 tryptic soy broth를 사용하였고, Candida albicans 배양은 yeast pepton dextrose broth에서 배양하였다. Various kimchi (baechu kimchi, kkakdugi, cabbage kimchi, bachelor kimchi, dongchimi, etc.) were crushed and filtered, respectively, and the kimchi filtrate was diluted decimal with 0.85% sterile physiological saline. 100 μl of MRS agar was plated on solid medium and cultured for 48 hours under anaerobic conditions in an incubator at 30° C., and then different microorganisms were separated according to the type of microbial colony. Cutibacterium acnes culture used as the test bacteria was inoculated in cooked meet medium (Difco Lab., USA) liquid medium and cultured anaerobically at 30° C. for 48 hours. Tryptic soy broth was used for Staphylococcus aureus culture, and Candida albicans culture was yeast. Cultured in pepton dextrose broth.

그 결과, 다양한 김치로부터 분리한 Leuconostoc, Weissella, Lactobacillus 속 300종에 대한 배양상등액의 항산화 활성(SOD)을 측정한 결과, 항산화 활성이 우수한 15균주를 1차 선발하였다(도 1 참고).As a result, as a result of measuring the antioxidant activity (SOD) of the culture supernatant for 300 species of Leuconostoc , Weissella , and Lactobacillus genus isolated from various kimchi, 15 strains with excellent antioxidant activity were first selected (see FIG. 1 ).

그리고 천연물 발효능이 우수한 김치유산균을 선발하기 위해 1차 선발한 15균주를 대상으로 황련해독탕의 주원료인 황련, 치자, 황백 및 황금 열수추출물에 각각 선발미생물을 접종한 후, 생육을 비교한 결과(도 2 참고), 김치유산균 9종(C2, CK9, DK4, E1, F1, JC7, KP1, L01, M2)을 2차 선발하였다. In addition, in order to select kimchi lactic acid bacteria with excellent fermenting ability of natural products, the selected microorganisms were inoculated with the main ingredients of Hwangryeonhaedoktang, hwangryeon, gardenia, hwangbaek, and golden hot water extracts, respectively, and the growth was compared. (See FIG. 2), 9 types of Kimchi lactic acid bacteria (C2, CK9, DK4, E1, F1, JC7, KP1, L01, M2) were secondarily selected.

그리고 여드름 원인 미생물인 C. acnes 균주에 대한 항균활성을 확인한 결과, 선발미생물 모두 항균활성이 존재하였으나 그 중에서 분리균주 isolate CK9가 가장 우수한 생육 억제환을 나타내어 최종 선발하였다(도 3 참고).And as a result of confirming the antibacterial activity against the C. acnes strain, which is the microorganism causing acne, all of the selected microorganisms had antibacterial activity, but among them, the isolate CK9 wasolate showed the best growth inhibitory ring and was finally selected (refer to FIG. 3).

실시예 3. 유산균 동정Example 3. Identification of lactic acid bacteria

최종 선발한 미생물의 동정은 Gram 염색과 현미경 관찰을 통해 형태학적 특성을 조사하였다. API 50 CHB kit (bioMerieux Co., France)으로 당이용성 실험을 하고 API 50 CHB database V3.0 (http://apiweb.biomerieux. com)을 이용하여 생화학적 동정을 수행하였다. For the identification of the finally selected microorganisms, the morphological characteristics were investigated through Gram staining and microscopic observation. Sugar availability was tested with API 50 CHB kit (bioMerieux Co., France), and biochemical identification was performed using API 50 CHB database V3.0 (http://apiweb.biomerieux. com).

또한 선발되어진 균주를 30℃에서 48시간 배양한 후 원심분리(10,000 xg)하여 멸균 생리식염수(0.85% NaCl)에 3회 세척한 후, DNeasy tissue kit(Qiagen, ValeCia, CA, USA)를 사용하여 Genomic DNA를 추출하고, 2% agarose gel에 전기영동 하였다. 추출된 DNA의 16S ribosomal DNA gene 증폭 Primer는 1492R (5'-GGATACCTTGTTACGACTT-3')와 27F (5'-AGAGTTTGATCATGGCTCAG-3')를 사용하였다. PCR 증폭에서 reaction mixture의 구성은 DNA template (20 μg/ml) 1 μL, 0.4 mM dNTP, 0.5 units Taq polymerase, 4 mM Mg2+이 함유된 Takara Perfect Premix (Takara, Japan) 10 ㎕에 1.0 μM forward primer와 1.0 μM reverse primer를 각각 1 μL씩 첨가하고, 총 부피가 20 μL가 되도록 나머지는 증류수를 첨가하여 제조하였다. PCR 반응은 initial denaturation (95℃에서 5분), denaturation (94℃에서 45초), annealing (52℃에서 45초), extension (72℃에서 1분) 반응을 총 30회 반복 실시하였고, 72℃에서 5분간 최종 extension을 실시하였다. 증폭된 약 1,400 bp의 fragment를 T vector (Invitrogen, Carlsbad, CA, USA)에 결합시킨 후 형질전환 하였다. T vector sequencing primer를 이용하여 염기서열 결정을 수행하였고, 염기서열간의 상동성을 확인하기 위하여 BLAST(Basic Local Alignment Search Tool) search program (http://www.ncbi.nlm.nih.gov)을 이용하여 GenBank database (NCBI, Bethesda, MD, USA)의 ribosomal RNA gene sequencing과 비교 분석하였다. 16S rRNA gene의 결정된 염기서열은 Clustal_W 및 BioEdit 프로그램으로 정렬하였다. 정렬 결과, 발생한 gap은 이후 분석과정에서 결여형질로 처리하였다. 염기서열 간의 유전적 거리 계산과 neighbor-joining, maximum-likelihood 및 parsimony 계통수의 제작은 Mega 프로그램을 사용하여 수행하였다(1,000 replicates). In addition, the selected strain was incubated at 30℃ for 48 hours, centrifuged (10,000 x g), washed 3 times in sterile physiological saline (0.85% NaCl), and then using a DNeasy tissue kit (Qiagen, ValeCia, CA, USA). Genomic DNA was extracted and electrophoresed on 2% agarose gel. For the 16S ribosomal DNA gene amplification primers of the extracted DNA, 1492R (5'-GGATACCTTGTTACGACTT-3') and 27F (5'-AGAGTTTGATCATGGCTCAG-3') were used. The composition of the reaction mixture in PCR amplification is 1.0 μM forward in 10 μl of Takara Perfect Premix (Takara, Japan) containing 1 μL of DNA template (20 μg/ml), 0.4 mM dNTP, 0.5 units Taq polymerase, and 4 mM Mg 2+ 1 μL of primer and 1.0 μM reverse primer were added each, and the remainder was prepared by adding distilled water so that the total volume was 20 μL. For the PCR reaction, the initial denaturation (5 minutes at 95°C), denaturation (45 seconds at 94°C), annealing (45 seconds at 52°C), and extension (1 minute at 72°C) reactions were repeated 30 times in total, 72°C A final extension was performed for 5 minutes. After binding the amplified fragment of about 1,400 bp to T vector (Invitrogen, Carlsbad, CA, USA), it was transformed. Base sequence determination was performed using T vector sequencing primer, and BLAST (Basic Local Alignment Search Tool) search program (http://www.ncbi.nlm.nih.gov) was used to confirm homology between base sequences. and ribosomal RNA gene sequencing in the GenBank database (NCBI, Bethesda, MD, USA). The determined nucleotide sequence of the 16S rRNA gene was aligned with Clustal_W and BioEdit programs. As a result of the alignment, gaps that occurred were treated as deficient traits in the subsequent analysis process. Genetic distance calculation between nucleotide sequences and generation of neighbor-joining, maximum-likelihood, and parsimony phylogenetic trees were performed using the Mega program (1,000 replicates).

Figure pat00001
Figure pat00001

그 결과, 선발한 isolate CK9 균주를 API 50 CHL kit(Biomerieux, France)로 생화학적 동정한 결과는 표 1에 나타내었으며, 보다 정확한 동정을 위하여 16S rRNA의 염기서열을 결정하고 GenBenk에 등록된 다른 균주의 염기서열과 비교한 결과, Lactobacillus plantarum과 높은 상동성을 나타내었다. 따라서 선발균주는 Lactobacillus plantarum로 동정하였고 Lactobacillus plantarum CK9로 명명하였다. CK9의 염기서열은 도 4(a)에 기재된 바와 같고, CK9의 계통수는 도 4(b)에 도시된 바와 같다.As a result, the results of biochemical identification of the selected isolate CK9 strain with the API 50 CHL kit (Biomerieux, France) are shown in Table 1, and for more accurate identification, the base sequence of 16S rRNA was determined and other strains registered in GenBenk. As a result of comparison with the nucleotide sequence of Lactobacillus plantarum , it showed high homology. Therefore starter strains were identified as Lactobacillus plantarum was named Lactobacillus plantarum CK9. The nucleotide sequence of CK9 is as shown in Fig. 4(a), and the phylogenetic tree of CK9 is as shown in Fig. 4(b).

실험예 1. 항균활성Experimental Example 1. Antibacterial activity

배양상등액의 항균활성을 검증하기 위해 agar well diffusion method를 사용하였다. Tryptic soy agar(TSA, Difco, USA)에 피검균주를 접종한 중층배양 bioassay plate를 제조하였다. 선발균주의 배양액을 원심분리(8,000 rpm, 10 min)하고, membrane filter(0.45 μm, Advantec Co., Japan)를 이용하여 제균하였다. 피검균이 접종된 TSA 고체배지 상에 well을 만들고 200 μL 배양상등액을 분주하여 30℃에서 배양한 후, 생육억제환 형성여부를 관찰하였다.To verify the antimicrobial activity of the culture supernatant, an agar well diffusion method was used. A multilayer culture bioassay plate inoculated with the test strain on tryptic soy agar (TSA, Difco, USA) was prepared. The culture medium of the selection strain was centrifuged (8,000 rpm, 10 min) and sterilized using a membrane filter (0.45 μm, Advantec Co., Japan). After making a well on the TSA solid medium inoculated with the test bacteria, aliquoting 200 μL of the culture supernatant and culturing at 30° C., the formation of growth inhibitory rings was observed.

김치유산균 Lactobacillus plantarum CK9로 발효한 황련해독탕(FHHT)의 항균활성을 확인한 결과, 발효 황련해독탕은 Cutibacterium acnes에 대해 항균활성이 우수한 것으로 확인되었으나, 비발효 황련해독탕(HHT)은 항균활성을 확인할 수 없었다. 이와 같이 Lactobacillus plantarum CK9 발효를 통해 천연물이 가수분해 또는 생물전환능에 의해 항균물질이 생성되어 나타난 결과로 사료되었다(도 5 참고). As a result of confirming the antibacterial activity of Hwangryeon Haedok-tang (FHHT) fermented with Kimchi Lactobacillus Lactobacillus plantarum CK9, it was confirmed that the fermented Hwangryeon Haedok-tang had excellent antibacterial activity against Cutibacterium acnes. could not confirm As such, it was considered as a result of the production of antibacterial substances by hydrolysis or bioconversion of natural products through Lactobacillus plantarum CK9 fermentation (refer to FIG. 5).

여드름 유발 미생물인 Cutibacterium acnes에 대한 발효 황련해독탕의 우수한 항균활성을 통해 화장품 혹은 피부질환 치료소재로서 응용 가능성이 매우 높을 것으로 판단되었다. The excellent antibacterial activity of fermented Hwangryeonhaedok-tang against Cutibacterium acnes , an acne-causing microorganism, was judged to have a very high application potential as a cosmetic or skin disease treatment material.

실험예 2. 생균수 조사Experimental Example 2. Investigation of the number of viable cells

황련, 치자, 황백 및 황금 열수추출물에 선발미생물인 Lactobacillus plantarum CK9를 각각 접종(105 CFU/ml)한 후, 생균수를 측정하여 황련, 치자, 황백 및 황금 천연물의 발효능을 조사하였다. After each inoculation (10 5 CFU/ml) of Lactobacillus plantarum CK9, a selection microorganism, into hot water extracts of yellow lotus, gardenia, yellow-white, and golden hot water extracts, the number of viable cells was measured to investigate the fermentability of yellow lotus, gardenia, yellow-white and gold natural products.

그 결과, 배양 12시간 간격으로 생육정도를 확인하였으며 36시간 배양시 최대 생육에 도달하였다. Lactobacillus plantarum CK9 생균수는 MRS agar 배지에 도말하여 확인하였는데, 황백 발효물(1.7×109 CFU/mL), 황금 발효물 (3.5×1010 CFU/mL), 황련 발효물 (1.3×109 CFU/mL), 치자 발효물 (2.4×1010 CFU/mL)로 각각 우수한 발효능과 생균수를 확인하였다(도 6 참고). As a result, the growth rate was confirmed at 12-hour intervals of culture, and the maximum growth was reached at 36-hour incubation. Lactobacillus plantarum CK9 live cell number were confirmed by spread on MRS agar medium, yellowish fermentation water (1.7 × 10 9 CFU / mL ), gold fermentation (3.5 × 10 10 CFU / mL ), goldthread fermentation (1.3 × 10 9 CFU /mL) and gardenia fermented product (2.4×10 10 CFU/mL), respectively, and excellent fermentation ability and viable cell count were confirmed (see FIG. 6 ).

황련, 치자, 황백 및 황금 천연물을 혼합하여 추출한 황련해독탕(HHT)에 Lactobacillus plantarum CK9을 접종(105 CFU/ml)하여 36시간 후, 생균수를 측정한 결과, 3.4×1011 CFU/mL로 매우 우수한 발효능을 확인하였다(도 7 참고).Goldthread, gardenia, yellowish and inoculated with Lactobacillus plantarum CK9 the goldthread to doktang (HHT) is extracted by mixing gold natural products (10 5 CFU / ml) and after 36 hours, results of the measurement of the viable cell count, 3.4 × 10 11 CFU / mL very excellent fermentation ability was confirmed (see FIG. 7).

따라서 각각 천연물 추출물에 개별적으로 유산균을 배양한 것보다 HHT에 배양한 유산균의 생육이 우수한 것을 확인하였다.Therefore, it was confirmed that the growth of lactic acid bacteria cultured in HHT was superior to that of lactic acid bacteria individually cultured in each natural product extract.

실험예 3. ABTS radical 소거능 측정Experimental Example 3. ABTS radical scavenging activity measurement

총 항산화력 측정은 ABTS-cation decolorization assay 방법으로 수행하였다. 70 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 과 24.5 mM potassium persulfate을 12시간동안 암소 반응하여 ABTS 양이온을 형성시킨 후, 이 용액을 734nm에서 흡광도 값이 0.7∼0.8±0.02값이 나오도록 희석하였다. 희석된 ABTS용액 100μl에 시료를 50μl씩 농도별로 처리하여 30분 후 734nm에서 흡광도를 측정하였다. 양성대조군으로 L-ascorbic acid를 시료와 동일량 처리하였다. Total antioxidant capacity was measured by ABTS-cation decolorization assay method. 70 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 24.5 mM potassium persulfate were reacted in the dark for 12 hours to form ABTS cations. It was diluted to give a value of 0.7-0.8±0.02. Samples were treated at each concentration by 50 μl in 100 μl of the diluted ABTS solution, and absorbance was measured at 734 nm after 30 minutes. As a positive control, L-ascorbic acid was treated with the same amount as the sample.

ABTS radical 소거능을 측정한 결과, 유산균 발효추출물(FHHT) 농도 25 μg/ml 농도부터 100% 소거능으로 확인하였으나 비발효 열수추출물(HHT) 은 50 μg/ml에서 100% 소거능으로 확인하였다. 그리고, 50 μg/ml 농도 이상은 발효추출물이나 비발효추출물이나 동일한 ABTS radical 100% 소거능으로 항산화 활성에 있어서 차이가 없음을 확인하였다(도 8 참고). As a result of measuring the ABTS radical scavenging ability, it was confirmed that the lactic acid bacteria fermented extract (FHHT) had 100% scavenging activity from the concentration of 25 μg/ml, but the non-fermented hot water extract (HHT) was confirmed with the scavenging ability of 100% at 50 μg/ml. In addition, it was confirmed that there was no difference in antioxidant activity at a concentration of 50 μg/ml or more with the same 100% scavenging ability of ABTS radicals as fermented or non-fermented extracts (refer to FIG. 8).

실험예 4. DPPH radical 소거능 측정Experimental Example 4. DPPH radical scavenging activity measurement

1,1-Diphenyl-2-picryhydrazyl(DPPH)용액 100μl에 시료 100μl를 잘 혼합하여 30분간 암소반응 시킨 후 microplate reader로 517nm에서 흡광도 값을 측정하여 농도에 따른 DPPH radical 소거능을 확인하였다. Blank는 시료의 희석용매를 사용하였고, control로는 시료 대신 시료의 희석 용매에 DPPH시약을 넣어 사용하였다. 양성대조군으로 L-ascorbic acid를 시료와 동일량 처리하였다. 100 μl of the sample was mixed well with 100 μl of 1,1-Diphenyl-2-picryhydrazyl (DPPH) solution and reacted in the dark for 30 minutes. The absorbance value was measured at 517 nm with a microplate reader to confirm the DPPH radical scavenging ability according to the concentration. Blank used the dilution solvent of the sample, and as a control, DPPH reagent was added to the dilution solvent of the sample instead of the sample. As a positive control, L-ascorbic acid was treated with the same amount as the sample.

DPPH radical 소거능을 측정한 결과, FHHT 농도 25 μg/ml 농도부터 95% 소거능으로 확인하였으나 HHT는 100 μg/ml에서 100% 소거능으로 확인하였다. 그리고, 400 μg/ml 농도 이상은 FHHT나 HHT나 동일한 DPPH radical 100% 소거능으로 항산화 활성에 있어서 차이가 없음을 확인하였다(도 9 참고). 이와 같이 천연물을 발효함으로써 항산화 효과가 더 증가된 것을 확인할 수 있었다. 항산화 효과의 증대로 미루어 보아 황련해독탕을 L. plantarum CK9으로 발효시 적은 함량으로 동일한 항산화 효과를 기대할 수 있는 기능성 화장품의 소재로서 사용할 수 있을 것으로 판단되었다.As a result of measuring the DPPH radical scavenging ability, it was confirmed that the FHHT concentration was 95% scavenging at a concentration of 25 μg/ml, but HHT was confirmed as a 100% scavenging ability at 100 μg/ml. In addition, it was confirmed that there was no difference in antioxidant activity at a concentration of 400 μg/ml or more with 100% scavenging ability of DPPH radicals, which is the same as FHHT or HHT (see FIG. 9 ). As such, it was confirmed that the antioxidant effect was further increased by fermenting the natural product. Judging by the increase in antioxidant effect, it was judged that Hwangryeonhaedoktang could be used as a material for functional cosmetics that can expect the same antioxidant effect with a small content when fermented with L. plantarum CK9.

실험예 5. Superoxide dismutase 유사활성 측정 Experimental Example 5. Superoxide dismutase-like activity measurement

Superoxide dismutase(SOD)의 유사활성 측정을 위해 OxiTec™ SOD assay kit(Biomax, Seoul, Korea)를 사용하였으며 실험법은 해당 protocol에 따라 수행하였다. 과산화수소(H2O2)로 전환시키는 반응을 촉매하는 pyrogallol의 생성량을 측정하여 SOD 유사활성으로 나타내었다. 시료 0.2 ml에 pH8.5 Tris·HCl buffer[50mM tris(hydroxymethyl) aminomethane, 10mM EDTA, pH 8.5] 2.6ml과 7.2mM pylogallol 0.2ml를 첨가한다. 실온에서 10분동안 반응시킨 후, 1N HCl을 가하여 반응을 정지한다. ELISA microplate reader(Infinite 200 pro, TECAN, Austria)를 이용하여 450 nm에서 흡광도를 측정하였고 SOD 유사활성은 추출물 첨가구와 무첨가구의 흡광도 차이를 백분율로 나타내었다.To measure the similar activity of superoxide dismutase (SOD), OxiTec™ SOD assay kit (Biomax, Seoul, Korea) was used, and the experimental method was performed according to the corresponding protocol. The amount of pyrrogallol, which catalyzes the conversion to hydrogen peroxide (H 2 O 2 ), was measured and expressed as SOD-like activity. To 0.2 ml of the sample, 2.6 ml of pH8.5 Tris-HCl buffer [50 mM tris(hydroxymethyl) aminomethane, 10 mM EDTA, pH 8.5] and 0.2 ml of 7.2 mM pylogallol are added. After reacting at room temperature for 10 minutes, 1N HCl was added to stop the reaction. Absorbance was measured at 450 nm using an ELISA microplate reader (Infinite 200 pro, TECAN, Austria), and SOD-like activity was expressed as a percentage of the difference in absorbance between the group with and without the extract.

Superoxide anion radical 소거에 관련된 SOD 효소활성은 NBT 환원법에 의해 검정하였다. 유산균 발효추출물 25 μg/ml에서 86.64% 환원 저해율을 나타낸 반면, 비발효 열수추출물은 74.39% 환원 저해율로 확인되어, Lactobacillus plantarum CK9으로 발효시 항산화 활성이 12.25% 더 높아진다는 것을 확인하였다(도 10 참고). SOD enzyme activity related to superoxide anion radical scavenging was assayed by NBT reduction method. On the other hand shows the lactic acid bacteria fermentation extract, 25 μg / ml 86.64% reduction inhibition in, non-fermented hot-water extract has been confirmed is identified as 74.39% reduction inhibition, antioxidant activity during fermentation increases further 12.25% by Lactobacillus plantarum CK9 (Figure 10 reference ).

황련해독탕의 우수한 항산화 활성 및 phenol 화합물의 높은 함량에 대하여서는 이미 연구보고된 바 있으나, 다양한 항산화 활성 측정 결과를 통해 김치유산균 Lactobacillus plantarum CK9의 발효로 황련해독탕의 항산화 활성이 더 증가하는 것을 확인하였다. 이와 같이 항산화 기능성 향상을 통해 기능성 식품, 기능성 화장품, 천연 보존제 및 의약품으로 개발할 수 있을 것으로 판단되었다. 앞으로의 연구에서는 유산균을 발효시킴으로서 증대되는 생리활성에 대해 구체적으로 규명하고 그 이용성을 증대시킬 수 있는 연구가 수행되어야 할 것으로 사료되었다. Although research has already been reported on the excellent antioxidant activity and high content of phenolic compounds in Hwangryeonhaedoktang, it was confirmed that the antioxidant activity of Hwangryeonhaedoktang was further increased by fermentation of Kimchi Lactobacillus Lactobacillus plantarum CK9 through various antioxidant activity measurement results. did. As such, it was determined that it could be developed into functional foods, functional cosmetics, natural preservatives and pharmaceuticals through the improvement of antioxidant functionality. In future research, it was considered that a study that could specifically identify the physiological activity increased by fermenting lactic acid bacteria and increase its usefulness should be carried out.

실험예 6. 세포 생존율 측정Experimental Example 6. Measurement of cell viability

대식세포주 Raw 264.7 cell은 한국세포주은행(Seoul, Korea)에서 분양받아 사용하였으며, 10% fetal bovine serum(FBS)와 1% penicillin-streptomycin(100 U/ml)가 함유된 dulbecco’s modified Eagle’s medium(Gibco Co., USA)(DMEM) 배지에서 37℃, 5% CO2 incubator 조건으로 배양하였다.Macrophage Raw 264.7 cells were purchased from Korea Cell Line Bank (Seoul, Korea) and used, and dulbecco's modified Eagle's medium (Gibco Co) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/ml). ., USA) (DMEM) medium, 37 ℃, 5% CO 2 Incubator conditions.

Raw 264.7 cell은 96-well plate에 1×105 cell/well로 분주한 다음, 각 농도별로 시료를 처리하고 1시간 후, lipopolysaccharide(LPS)를 처리한 군과 처리하지 않은 군으로 나누어 24시간동안 배양하였다. 배양 후 supernatant는 새로운 96-well plate에 옮겨 동일한 양의 Griess reagent를 첨가하여 15분 후에 흡광도(540nm)를 측정하였고 NO 농도는 sodium nitrate를 이용한 표준 곡선으로 계산하였다. Raw 264.7 cell 배양 96-well plate에 10% MTS solution이 함유된 DMEM medium을 well당 100μl씩 넣어주고 3시간동안 배양한 후, 흡광도(490nm)를 측정하여 cell viability 값을 계산하였다. 흡광도 측정은 ELISA microplate reader(Infinite 200 pro, TECAN, Austria)을 사용하였다.Raw 264.7 cells were aliquoted in a 96-well plate at 1×10 5 cells/well, and after each concentration sample was treated, 1 hour later, it was divided into a group treated with lipopolysaccharide (LPS) and a group without treatment for 24 hours. cultured. After incubation, the supernatants were transferred to a new 96-well plate, the same amount of Griess reagent was added, and absorbance (540 nm) was measured after 15 minutes, and the NO concentration was calculated using a standard curve using sodium nitrate. 100 μl of DMEM medium containing 10% MTS solution was put into a 96-well plate of raw 264.7 cell culture and cultured for 3 hours, and then the absorbance (490 nm) was measured to calculate the cell viability value. Absorbance was measured using an ELISA microplate reader (Infinite 200 pro, TECAN, Austria).

생존율(viability, % control)viability (% control)

= 100×/(absorbance of treated sample)/(absorbance of control)= 100×/(absorbance of treated sample)/(absorbance of control)

MTS assay를 통해 raw 264.7 cell의 생존율을 확인한 결과, 도 11에 도시된 바와 같이 유산균 발효 황련해독탕(FHHT) 농도 25 μg/ml에서 400 μg/ml까지 89.2-99.9% 세포 생존율을 나타내었다. 비발효 황련해독탕(HHT)은 25 μg/ml에서 400 μg/ml 농도까지 88.7-101.9% 세포 생존율을 나타내었다. 유산균 발효와 비발효 황련해독탕 모두 raw 264.7 cell에 대하여 유의적인 독성반응을 보이지 않는 것으로 확인하여 해당 농도로 이하의 실험예 7에서 NO assay를 실시하였다.As a result of confirming the viability of raw 264.7 cells through MTS assay, 89.2-99.9% cell viability was exhibited from 25 μg/ml to 400 μg/ml of lactic acid fermented Hwangryeonhaedoktang (FHHT) concentration as shown in FIG. 11 . Unfermented Hwangnyeonhaedoktang (HHT) exhibited 88.7-101.9% cell viability from 25 μg/ml to 400 μg/ml concentration. It was confirmed that both lactic acid bacteria fermentation and non-fermented Hwangryeonhaedoktang did not show a significant toxic reaction to raw 264.7 cells, and NO assay was performed at the corresponding concentration in Experimental Example 7 below.

실험예 7. Nitric oxide 생성량 조사Experimental Example 7. Nitric oxide production amount investigation

농도별로 희석한 sodium nitrate 표준곡선으로 NO assay를 수행하였다. LPS로 자극한 raw 264.7 cell에 유산균 발효와 비발효 황련해독탕 시료를 각각 처리한 후, nitric oxide 소거능을 확인하였다.NO assay was performed with a sodium nitrate standard curve diluted by concentration. After the LPS-stimulated raw 264.7 cells were treated with lactic acid bacteria fermented and non-fermented Hwangryeonhaedoktang samples, the scavenging ability of nitric oxide was confirmed.

그 결과, 도 12에 도시된 바와 같이 LPS(1㎍/ml) 처리시 NO 농도는 43.1 μM 이었으나, 유산균 발효와 비발효 황련해독탕 모두 농도 비례적으로 점차 감소하는 것으로 확인하였다. 유산균 발효와 비발효 황련해독탕 400 ㎍/ml 농도에서 유산균 발효 황련해독탕이 비발효 열수 추출 황련해독탕보다 5.7 μM 함량이 더 감소된 차이를 나타내었다. 모든 농도 처리군에서도 Lactobacillus plantarum CK9으로 발효시킬 경우 nitric oxide 생성 억제력이 더 증가된 것을 확인하였다. As a result, as shown in FIG. 12 , the NO concentration during LPS (1㎍/ml) treatment was 43.1 μM, but it was confirmed that both the lactic acid bacteria fermentation and the non-fermented Hwangryeonhaedoktang decreased in proportion to the concentration. At 400 μg/ml concentration of lactic acid bacteria fermented and non-fermented Hwangryeonhaedoktang, the difference between lactic acid bacteria fermented Hwangryeonhaedoktang and nonfermented hot water extracted Hwangryeonhaedoktang was further reduced by 5.7 μM. When fermented with Lactobacillus plantarum CK9 in all concentration treatment groups, it was confirmed that the inhibition of nitric oxide production was further increased.

NO는 원래 염증성 또는 항염증성의 기능을 동시에 하는 것으로 알려져 있으나, 생체 내 과도한 분비는 오히려 세포독성을 통해 세포를 파괴하고 shock에 의한 혈관 확장 및 염증반응을 촉진하여 조직 손상을 유발하는 것으로 알려져 있다(Nathan, 1992, Lowenstein, 1992). 이러한 NO의 발현량이 HHT와 FHHT를 처리하였을 때 통계적 유의성이 있게 감소하였으며 발효를 통해 효과가 증대 되는 것을 확인하였고 염증성 피부질환의 치료에 응용 가능할 것으로 생각된다.NO is originally known to have inflammatory or anti-inflammatory functions at the same time, but excessive secretion in vivo is known to cause tissue damage by rather destroying cells through cytotoxicity and promoting vasodilation and inflammatory response by shock ( Nathan, 1992; Lowenstein, 1992). The expression level of NO was decreased with statistical significance when HHT and FHHT were treated, and it was confirmed that the effect was increased through fermentation, and it is thought to be applicable to the treatment of inflammatory skin diseases.

실험예 8. 염증성 cytokine 발현량 측정Experimental Example 8. Measurement of inflammatory cytokine expression level

염증성 cytokine 발현량을 측정하기 위하여 Quantikine® ELISA kit(R&D system, USA)를 이용하여 측정하였으며, 측정한 cytokine으로는 TNF-α, interleukin-1β(IL-1β), interleukin-6(IL-6)를 확인하였고 실험과정은 해당 protocol에 따라 수행하였다. Stop solution 처리 후, 450nm에서 흡광도 값을 측정하여 발현량을 확인하였다. To measure the inflammatory cytokine expression level, it was measured using the Quantikine® ELISA kit (R&D system, USA), and the measured cytokines were TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6). was confirmed, and the experimental procedure was performed according to the corresponding protocol. After the stop solution treatment, the absorbance value was measured at 450 nm to confirm the expression level.

그 결과, 종양괴사 인자인 TNF-α의 경우 도 13에 도시된 바와 같이 400 μg/ml 농도의 유산균 발효(FHHT)와 비발효 황련해독탕(HHT)에서 1544.3 pg/ml와 1765.4 pg/ml으로 나타났으며, 유산균 발효시 TNF-α의 발현량이 221.0 pg/ml 정도 감소가 확인되었으나 유의적 차이는 거의 없는 것으로 확인하였다. As a result, in the case of TNF-α, a tumor necrosis factor, as shown in FIG. 13, 1544.3 pg/ml and 1765.4 pg/ml in lactic acid bacteria fermentation (FHHT) and non-fermented Hwangryeonhaedoktang (HHT) at a concentration of 400 μg/ml. It was confirmed that the expression level of TNF-α during lactic acid fermentation was decreased by 221.0 pg/ml, but there was no significant difference.

IL-1β의 경우 도 14에 도시된 바와 같이 400 μg/ml 농도의 FHHT와 HHT 모두 control과 유사한 수치로 감소 효과를 나타내었다. 200 μg/ml 농도에서 FHHT와 HHT 처리시 IL-1β 발현량 감소 효과를 확인한 결과, 유산균 발효와 비발효 황련해독탕에서 43.0 pg/ml와 60.2 pg/ml으로 나타났으며, 유산균 발효시 IL-1β 발현량이 17.2 pg/ml 정도 더 감소한 효과가 확인되었다. In the case of IL-1β, as shown in FIG. 14 , both FHHT and HHT at a concentration of 400 μg/ml showed a reduction effect to a value similar to that of the control. As a result of confirming the effect of reducing IL-1β expression when FHHT and HHT were treated at 200 μg/ml concentration, 43.0 pg/ml and 60.2 pg/ml were found in Hwangryeonhaedoktang with lactic acid bacteria fermentation and IL- It was confirmed that the 1β expression level was further reduced by about 17.2 pg/ml.

IL-6의 경우 도 15에 도시된 바와 같이 400 μg/ml 농도의 유산균 발효와 비발효 황련해독탕에서 506.5 pg/ml와 1129.9 pg/ml으로 나타났으며, 유산균 발효시 IL-6 발현량이 623.4 pg/ml 정도 더 감소한 효과가 확인되었다. In the case of IL-6, as shown in FIG. 15 , it was found to be 506.5 pg/ml and 1129.9 pg/ml in fermented and non-fermented Hwangryeonhaedoktang with 400 μg/ml concentration of lactic acid bacteria, and the IL-6 expression level during lactic acid fermentation was 623.4. It was confirmed that the effect was further reduced by about pg/ml.

Cytokine은 면역계와 염증에 있어서 세포사이의 신호전달을 담당하는 단백질로서 중요한 매개자 역할을 담당한다. IL-1β, IL-6, TNF-α 와 같은 염증성 사이토카인들은 급성, 만성 염증질환의 중요한 매개자로 과도한 생성 및 작용은 병리적 결과를 이끌 수 있다. 사이토카인 혹은 그 저해제를 투여하는 것은 면역 질환 및 염증 질환과 관련된 생물학적 반응을 변화시킬 잠재적인 접근 방법이 될 수 있다.Cytokine is a protein responsible for cell-to-cell signaling in the immune system and inflammation, and plays an important mediator role. Inflammatory cytokines such as IL-1β, IL-6, and TNF-α are important mediators of acute and chronic inflammatory diseases, and excessive production and action can lead to pathological consequences. Administration of cytokines or their inhibitors could be a potential approach to altering the biological responses associated with immune and inflammatory diseases.

이와 같은 결과로 보아, Lactobacillus plantarum CK9 발효를 통해 내재적 염증물질의 생산 억제능이 증가함으로서 항염증 효과에 김치유산균이 영향을 나타내는 것으로 사료되었고 이를 통해 염증성 피부질환 치료에 응용이 가능할 것으로 보인다.Based on these results, it was thought that Kimchi lactic acid bacteria had an effect on the anti-inflammatory effect by increasing the production inhibitory ability of intrinsic inflammatory substances through Lactobacillus plantarum CK9 fermentation.

Claims (4)

황련, 황금, 황백 및 치자 추출물로 이루어지는 황련해독탕을 제조하는 단계;
상기 황련해독탕에 Lactobacillus plantarum 균주를 접종하는 단계; 및
상기 균주 접종 후 발효시키는 단계를 포함하여 구성되는 것을 특징으로 하는 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법.
preparing hwangryeonhaedoktang consisting of hwangryeon, gold, hwangbaek and gardenia extract;
inoculating the Lactobacillus plantarum strain into the Hwangnyeonhaedoktang; and
Method for producing Hwangryeonhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects, characterized in that it comprises the step of fermenting after inoculation of the strain.
 청구항 1에 있어서,
상기 균주는 Lactobacillus plantarum CK9인 것을 특징으로 하는 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법.
The method according to claim 1,
The strain is Lactobacillus plantarum CK9, characterized in that the antibacterial, anti-inflammatory and antioxidant effect of the enhanced Hwangryeon Haedok-tang manufacturing method.
 청구항 1 또는 청구항 2에 있어서,
상기 발효시키는 단계는 상기 균주 접종 후 36시간 동안 발효하는 것을 특징으로 하는 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법.
The method according to claim 1 or 2,
The fermenting step is a method for producing Hwangryeonhaedoktang with enhanced antibacterial, anti-inflammatory and antioxidant effects, characterized in that it is fermented for 36 hours after inoculation of the strain.
 청구항 1 또는 청구항 2에 있어서,
상기 황련해독탕을 제조하는 단계는,
황련, 황금, 황백, 치자 및 yeast extract를 물에 넣는 단계;
상기 혼합물을 전탕하는 단계; 및
상기 전탕된 혼합물을 여과한 다음, 원심분리하여 상층액만을 분리하는 단계를 포함하여 구성되는 것을 특징으로 하는 항균, 항염 및 항산화 효과가 증진된 황련해독탕의 제조방법.
The method according to claim 1 or 2,
The step of preparing the hwangryeonhaedoktang,
Putting yellow lily, gold, yellow white, gardenia and yeast extract into water;
boiling the mixture; and
A method for producing Hwangryeon Haedok-tang with enhanced antibacterial, anti-inflammatory and antioxidant effects, characterized in that it comprises the step of filtering the soaked mixture and then centrifuging to separate only the supernatant.
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