KR102227963B1 - Antiangiogenic composition comprising bioconverted herbal composition, and its preparation method - Google Patents
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Abstract
본 발명은 숙지황, 백작약, 천궁 및 당귀를 포함하는 생약조성물을 아스퍼질러스 카와치 배양액으로 생물전환시켜 얻은 생물전환 조성물을 유효성분으로 포함하는 혈관형성 저해용 조성물 및 이의 제조방법에 관한 것이다. 본 발명의 생물전환 조성물은 관 형성 분석, 세포 침범 분석, 생체 내 혈관형성 모델에서의 효과로 확인된 바와 같이 혈관형성을 저해하는 효과가 우수하다.The present invention relates to a composition for inhibiting angiogenesis and a method for preparing the same, comprising a bioconversion composition obtained by bioconversion of a herbal composition comprising Sukjihwang, Baekjak, Cheongung and Angelicae into Aspergillus Kawachi culture solution as an active ingredient. The bioconversion composition of the present invention is excellent in the effect of inhibiting angiogenesis as confirmed by the effect in tube formation analysis, cell invasion analysis, and in vivo angiogenesis model.
Description
본 발명은 생물전환된 생약조성물을 포함하는 혈관형성 저해용 조성물 및 이의 제조방법에 관한 것으로, 보다 상세하게는 숙지황, 백작약, 천궁 및 당귀를 포함하는 생약조성물을 아스퍼질러스 카와치 배양액으로 생물전환시켜 얻은 생물전환 조성물과 이의 제조방법에 관한 것이다.The present invention relates to a composition for inhibiting angiogenesis including a bioconverted herbal composition and a method for preparing the same, and more particularly, a bioconversion of a herbal composition including Sukjihwang, Baekjak, Chungoong and Angelicae into Aspergillus Kawachi culture solution It relates to a bioconversion composition and a method for preparing the same.
혈관형성은 새로운 혈관의 성장으로서, 주로 모세혈관 내피세포에 의한 이동, 증식 및 관 형성에 의존하는 과정이다. 혈관형성 동안에 내피세포는 정지기 상태에서 벗어나서 빠르게 증식하며, 다양한 혈관형성 인자가 혈관형성 과정에 관여한다. 병리상태 동안 전구혈관형성 인자 및 항혈관형성 인자 사이의 미세한 균형이 깨져 비자기(nonself) 제한적 내피세포 및 주위 내피세포의 증식을 유도함으로써 혈관형성이 일어나는 것으로 이해되고 있다. 혈관형성(혈관신생)은 장기 성장, 배아 발달, 상처 치유를 포함한 다양한 과정에 필수적이지만 다양한 병리학적 조건과도 연관되어 있다. 안구 신혈관형성 질환, 관절염, 피부질환, 종양 등에서는 혈관형성이 발생하는데, 혈관형성을 저해하는 방법은 이러한 질환의 진행을 억제 및 지연시킬 수 있는 효과적인 치료법이다. Angiogenesis is the growth of new blood vessels, which is a process mainly dependent on migration, proliferation and tube formation by capillary endothelial cells. During angiogenesis, endothelial cells escape from the stationary phase and proliferate rapidly, and various angiogenic factors are involved in the angiogenesis process. It is understood that angiogenesis occurs by inducing proliferation of non-self-restricted endothelial cells and surrounding endothelial cells due to a breakdown of the fine balance between pro-angiogenic factors and anti-angiogenic factors during pathology. Angiogenesis (angiogenesis) is essential for a variety of processes, including organ growth, embryonic development, and wound healing, but is also associated with a variety of pathological conditions. Angiogenesis occurs in ocular neovascularization diseases, arthritis, skin diseases, tumors, etc., and a method of inhibiting angiogenesis is an effective treatment capable of inhibiting and delaying the progression of these diseases.
이러한 악성질환을 치료하기 위하여 다수의 항혈관형성제제가 개발되었는데, 이러한 제제의 예로는 인간의 혈관형성을 조절하는 주과정인 혈관내피성장인자(VEGF)와 그 수용체(VEGFR)와의 상호작용을 조절하는 제제, 독특한 항종양 및 항혈관형성 특성을 가지고 있는 에스트라디올의 천연 대사산물인 2-메톡시에스트라디올(2-ME2)과, 각각 플라스미노겐과 콜라겐 XVIII의 단백분해적 절단 단편인 안지오스타틴과 엔도스타틴과 같은 신규 혈관형성 억제제 등이 있다. To treat such malignant diseases, a number of anti-angiogenic agents have been developed.Examples of such agents control the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR), which is the main process that controls angiogenesis in humans. Two agents, 2-methoxyestradiol (2-ME2), a natural metabolite of estradiol, which has unique anti-tumor and anti-angiogenic properties, and angiostatin, a proteolytic cleavage fragment of plasminogen and collagen XVIII, respectively. Novel angiogenesis inhibitors such as endostatin.
이러한 항혈관형성 제제는 전신투여시 성공률이 제한되고 혈소판감소증, 백혈구감소증 및 객혈을 비롯하여 상당한 독성을 나타내는 문제점이 있다. Such anti-angiogenic agents have problems in that their success rate is limited when systemically administered, and exhibits considerable toxicity, including thrombocytopenia, leukopenia, and hemoptysis.
사물탕(四物湯)은 숙지황(熟地黃), 백작약(白芍藥), 천궁(川芎) 및 당귀(當歸)를 혼합한 약재로, 숙지황, 백작약, 천궁 및 당귀 각각 5g을 한 첩으로 하여 달여 마신다. 사물탕은 혈허증과 혈병(血病)에 두루 사용하는 약으로서, 월경불순, 불임증, 갱년기장애, 임신중독, 산후증 등에 사용된다. 사물탕은 중국의 화제국방(和劑局方)에 처음 기록되었고, 한국의 동의보감, 의문보감(醫門寶鑑), 제중신편(濟衆新編) 등에 그 처방기록이 있다. Samultang is a medicinal mixture of Sukjihwang (熟地黃), Baek peony, Cheongung, and Angelica (當歸). . Samul-tang is a drug used for blood hemostatic and blood clots, and is used for menstrual impurities, infertility, menopausal disorders, addiction to pregnancy, and postpartum symptoms. Samultang was first recorded in the topic of national defense (和劑局方) in China, and there are prescription records in Donguibogam, Mimunbogam (醫門寶鑑) and Jejungsinpyeon (濟衆新編) in Korea.
본 발명은 사물탕에 사용되는 한약재들인 숙지황, 백작약, 천궁 및 당귀를 포함하는 생약조성물을 생물전환시켜 얻은, 독성이 없으면서도 혈관형성 저해능이 우수한 조성물과 이의 제조방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a composition obtained by bioconversion of a herbal composition containing herbal medicinal herbs used in Samultang, including Sukjihwang, Baekjak, Cheongung and Angelicae, and a composition having no toxicity and excellent angiogenesis inhibitory ability, and a method for preparing the same.
상기 목적을 달성하기 위하여, 본 발명에서는,In order to achieve the above object, in the present invention,
숙지황, 백작약, 천궁 및 당귀를 포함하는 생약조성물을 아스퍼질러스 카와치 배양액으로 생물전환시켜 얻은 생물전환 조성물을 유효성분으로 포함하는 혈관형성 저해용 조성물을 제공한다.It provides a composition for inhibiting angiogenesis, comprising as an active ingredient a bioconversion composition obtained by bioconversion of a herbal composition comprising Sukjihwang, Earl, Chungoong and Angelicae into Aspergillus Kawachi culture.
상기 생약조성물은 숙지황 10~40중량%, 백작약 10~40중량%, 천궁 10~40중량% 및 당귀 10~40중량%를 포함하는 것이 바람직하다. 상기 생약조성물은 숙지황, 백작약, 천궁 및 당귀를 1:1:1:1의 중량비로 혼합하는 것이 특히 바람직하다. The herbal composition is preferably containing 10 to 40% by weight of sukjihwang, 10 to 40% by weight of the earl, 10 to 40% by weight of cheonggung and 10 to 40% by weight of Angelicae. It is particularly preferred that the herbal composition is mixed with Sukjihwang, Baekjak, Cheongung and Angelicae in a weight ratio of 1:1:1:1.
상기 아스퍼질러스 카와치 배양액은,The Aspergillus Kawachi culture solution,
밀기울 10g을 기준으로 증류수 8~12㎖를 첨가하고 밀기울이 소량의 물을 흡수하도록 으깨어 혼합한 후 멸균하는 단계;Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;
상기 멸균 후 식힌 다음 아스퍼질러스 카와치를 접종하고 상온에서 2~4일간 배양하는 단계;Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating at room temperature for 2 to 4 days;
상기 배양 후, 10mM 농도의 인산 나트륨 버퍼 스탁을 밀기울 10g을 기준으로 45~55㎖씩 첨가하여 혼합한 후 4℃에서 5~10시간 동안 침지하여 배양된 시료를 얻는 단계;After the cultivation, adding and mixing 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, and immersing for 5 to 10 hours at 4° C. to obtain a cultured sample;
상기 배양된 시료를 여과하여 여과액을 분리하는 단계; 및 Filtering the cultured sample to separate the filtrate; And
상기 여과액을 원심분리한 후 상층액을 여과하여 아스퍼질러스 카와치 배양액을 얻는 단계를 포함하는 방법에 의하여 제조된 것임이 바람직하다.It is preferable that it is prepared by a method comprising the step of centrifuging the filtrate and then filtering the supernatant to obtain Aspergillus Kawachi culture.
또한, 본 발명은, In addition, the present invention,
숙지황, 백작약, 천궁 및 당귀를 혼합하여 생약조성물을 얻는 단계;Obtaining a herbal composition by mixing Sukjihwang, Earl Peony, Cheongung and Angelicae;
상기 생약조성물에 70% 에탄올을 1:2~4의 중량비로 혼합하여 추출하는 단계;Extracting the herbal composition by mixing 70% ethanol in a weight ratio of 1: 2 to 4;
상기 추출 후 여과하여 여과물을 얻는 단계;Filtering after the extraction to obtain a filtrate;
상기 여과물을 감압농축 및 동결건조하여 추출물 분말을 얻는 단계;Concentrating the filtrate under reduced pressure and freeze-drying to obtain an extract powder;
상기 추출물 분말에 증류수를 1:5~10의 중량비로 혼합하여 음파처리하는 단계;Mixing distilled water to the extract powder at a weight ratio of 1:5 to 10 and performing sound wave treatment;
상기 음파처리된 것에 아스퍼질러스 카와치 배양액을 100:50~200의 중량비로 혼합하여 진탕 배양하는 단계; 및Mixing the Aspergillus Kawachi culture solution to the sonicated one at a weight ratio of 100:50 to 200 and culturing with shaking; And
상기 배양 후 감압농축하여 생물전환 조성물을 얻는 단계를 포함하는, 혈관형성 저해를 위한 생물전환 조성물의 제조방법을 제공한다. It provides a method for preparing a bioconversion composition for inhibiting angiogenesis, comprising the step of obtaining a bioconversion composition by concentrating under reduced pressure after the culture.
상기 제조방법은 상기 감압농축 후 동결건조시켜 분말 형태의 생물전환 조성물을 얻는 단계를 더 포함할 수 있다.The manufacturing method may further include the step of obtaining a powdery bioconversion composition by concentrating under reduced pressure and then lyophilizing.
상기 제조방법에서, 상기 생약조성물은 숙지황 10~40중량%, 백작약 10~40중량%, 천궁 10~40중량% 및 당귀 10~40중량%를 포함하는 것이 바람직하다.In the above manufacturing method, the herbal composition preferably contains 10 to 40% by weight of Sukjihwang, 10 to 40% by weight of Earl, 10 to 40% by weight of cnidium and 10 to 40% by weight of Angelicae.
상기 제조방법에서, 상기 아스퍼질러스 카와치 배양액은, In the manufacturing method, the Aspergillus Kawachi culture solution,
밀기울 10g을 기준으로 증류수 8~12㎖를 첨가하고 밀기울이 소량의 물을 흡수하도록 으깨어 혼합한 후 멸균하는 단계;Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;
상기 멸균 후 식힌 다음 아스퍼질러스 카와치를 접종하고 상온에서 2~4일간 배양하는 단계;Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating at room temperature for 2 to 4 days;
상기 배양 후, 10mM 농도의 인산 나트륨 버퍼 스탁을 밀기울 10g을 기준으로 45~55㎖씩 첨가하여 혼합한 후 4℃에서 5~10시간 동안 침지하여 배양된 시료를 얻는 단계;After the cultivation, adding and mixing 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, and immersing for 5 to 10 hours at 4° C. to obtain a cultured sample;
상기 배양된 시료를 여과하여 여과액을 분리하는 단계; 및 Filtering the cultured sample to separate the filtrate; And
상기 여과액을 원심분리한 후 상층액을 여과하여 아스퍼질러스 카와치 배양액을 얻는 단계를 포함하는 방법에 의하여 제조되는 것이 바람직하다.It is preferable that the filtrate is centrifuged and then the supernatant is filtered to obtain Aspergillus Kawachi culture.
본 발명에서 숙지황, 백작약, 천궁 및 당귀를 포함하는 생약조성물을 아스퍼질러스 카와치 배양액으로 생물전환시켜 얻은 생물전환 조성물은, 관 형성 분석, 세포 침범 분석, 생체 내 혈관형성 모델에서의 효과로 확인된 바와 같이, 혈관형성을 저해하는 효과가 우수하다. 특히, 사물탕의 원료인 천궁과 당귀를 단독으로 사용한 경우에 비해 혈관형성을 저해하는 효과가 우수하며, 생물전환되지 않은 생약조성물에 비해서도 혈관형성을 저해하는 효과가 우수하다. 따라서 본 발명의 생물전환 조성물을 유효성분으로 포함하는 혈관형성 저해용 조성물은 혈관형성과 관련된 질환의 예방 및 치료에 효과적으로 사용될 수 있다.In the present invention, the bioconversion composition obtained by bioconverting the herbal composition including Sukjihwang, Earl, Chungoong, and Angelicae into Aspergillus Kawachi culture solution was confirmed by the effect of tube formation analysis, cell invasion analysis, and in vivo angiogenesis model. As described above, the effect of inhibiting angiogenesis is excellent. In particular, the effect of inhibiting angiogenesis is superior compared to the case of using the raw materials of Samultang (Chungung and Angelicae) alone, and the effect of inhibiting angiogenesis is excellent even compared to a non-bioconverted herbal composition. Therefore, the composition for inhibiting angiogenesis comprising the bioconversion composition of the present invention as an active ingredient can be effectively used for the prevention and treatment of diseases related to angiogenesis.
도 1은 생물전환 여부가 관 형성도에 미치는 효과를 확인한 결과이다.
도 2는 생물전환 여부가 피브로넥틴에 의해 유도된 세포 침범에 미치는 효과를 확인한 결과이다.
도 3은 천궁 추출물과 당귀 추출물에서 생물전환 여부가 피브로넥틴에 의해 유도된 세포침범에 미치는 효과를 확인한 결과이다.
도 4는 생물전환 여부가 생체 내 혈관형성 모델에서 미치는 효과를 확인한 결과이다.1 is a result of confirming the effect of biotransformation on the degree of tube formation.
2 is a result of confirming the effect of biotransformation on cell invasion induced by fibronectin.
3 is a result of confirming the effect of biotransformation on the cell invasion induced by fibronectin in the extract of Chunkyung and Angelicae extract.
4 is a result of confirming the effect of biotransformation on an angiogenesis model in vivo.
본 발명의 혈관형성 저해용 조성물은, 숙지황, 백작약, 천궁 및 당귀를 포함하는 생약조성물을 아스퍼질러스 카와치 배양액으로 생물전환시켜 얻은 생물전환 조성물을 유효성분으로 포함한다.The composition for inhibiting angiogenesis of the present invention comprises, as an active ingredient, a bioconversion composition obtained by bioconverting a herbal composition including Sukjihwang, Baekjak, Cheongung, and Angelicae into Aspergillus Kawachi culture solution.
본 발명에서 "생물전환(Bio Conversion)"은 미생물이 가지고 있는 효소적 기능을 이용해 기존 생체물질로부터 원하는 물질을 강화시키는 과정으로 정의된다. In the present invention, "Bio Conversion" is defined as a process of strengthening a desired substance from an existing biomaterial using an enzymatic function of a microorganism.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 혈관형성 저해용 조성물에서 상기 생약조성물은, 숙지황, 백작약, 천궁 및 당귀를 혼합하여 얻는다. 바람직하게는 숙지황 10~40중량%, 백작약 10~40중량%, 천궁 10~40중량% 및 당귀 10~40중량%를 혼합한다. 상기 생약조성물로 특히 바람직하게는 숙지황, 백작약, 천궁 및 당귀를 1:1:1:1의 중량비로 혼합한 사물탕을 사용할 수 있다. In the composition for inhibiting angiogenesis of the present invention, the herbal composition is obtained by mixing Sukjihwang, Baekjak, Chungoong, and Angelicae. Preferably, 10 to 40% by weight of Sukjihwang, about 10 to 40% by weight of Count, 10 to 40% by weight of Chungo and 10 to 40% by weight of Angelicae are mixed. As the herbal composition, particularly preferably, Samultang in which Sukjihwang, Baekjakjak, Cheongung, and Angelicae are mixed in a weight ratio of 1:1:1:1 may be used.
상기와 같은 비율로 혼합한 생약조성물에, 70% 에탄올을 1:2~4의 중량비로 혼합하여 추출한다. 추출은 80~100℃의 온도로 2~4시간 동안 가열하여 2~3회 추출하는 것이 바람직하다.In the herbal composition mixed in the same ratio as described above, 70% ethanol is mixed and extracted in a weight ratio of 1:2-4. Extraction is preferably extracted 2 to 3 times by heating for 2 to 4 hours at a temperature of 80 to 100 °C.
추출 후 여과하여 여과물을 얻는다. 이때 필터페이퍼를 사용하여 여과하는 것이 보다 바람직하다.After extraction, it is filtered to obtain a filtrate. At this time, it is more preferable to filter using filter paper.
상기 여과물을 감압농축하여 용매를 제거하고 동결건조하여 추출물 분말을 얻는다.The filtrate is concentrated under reduced pressure to remove the solvent and freeze-dried to obtain an extract powder.
상기 추출물 분말에 증류수를 1:5~20의 중량비로 혼합하여 음파처리(sonication)한다.Distilled water is mixed with the extract powder at a weight ratio of 1:5 to 20, followed by sonication.
상기 음파처리된 것에 아스퍼질러스 카와치(Aspergillus kawachii) 배양액을 100:50~200의 중량비로 혼합하여 진탕 배양한다. 배양은 진탕배양기에서 10~15 시간 동안 배양하는 것이 바람직하다.Aspergillus Kawachi ( Aspergillus kawachii ) culture solution is mixed at a weight ratio of 100:50~200 and cultured with shaking. It is preferable to incubate for 10 to 15 hours in a shaking incubator.
상기 배양 후 감압농축하여 본 발명의 생물전환 조성물을 얻는다.After the cultivation, it is concentrated under reduced pressure to obtain the bioconversion composition of the present invention.
필요에 따라 상기 생물전환 조성물을 동결건조하여 분말 형태의 생물전환 조성물을 얻을 수 있다. If necessary, the bioconversion composition may be lyophilized to obtain a powdery bioconversion composition.
상기 아스퍼질러스 카와치 배양액은 다음과 같이 제조하는 것이 바람직하다.The Aspergillus Kawachi culture solution is preferably prepared as follows.
밀기울 10g을 기준으로 증류수 8~12㎖를 첨가하고 밀기울이 소량의 물을 흡수하도록 으깨어 혼합한 후 멸균한다. 멸균된 시료를 식히고 아스퍼질러스 카와치를 접종하여 상온에서 2~4시간 동안 배양한다. 그런 다음 희석하여 최종 농도가 10mM이 되도록 제조한 인산 나트륨 버퍼 스탁(Sodium phosphate buffer stock; 1M)을 밀기울 10g을 기준으로 45~55㎖씩 첨가하여 혼합한 후 4℃에서 밤새(overnight) 배양한다. 이렇게 배양된 시료를 여과하여 여과액을 분리하고, 분리된 여과액을 원심분리한 후 다시 상층액을 여과하여 본 발명의 아스퍼질러스 카와치 배양액을 얻는다.Add 8 to 12 ml of distilled water based on 10 g of bran, crush and mix so that the bran absorbs a small amount of water, and sterilize. Cool the sterilized sample, inoculate Aspergillus kawachi, and incubate for 2 to 4 hours at room temperature. Then, diluted and prepared sodium phosphate buffer stock (1M) to a final concentration of 10 mM was added to each of 45 to 55 mL based on 10 g of bran, mixed, and incubated overnight at 4°C. The cultured sample is filtered to separate the filtrate, the separated filtrate is centrifuged, and the supernatant is filtered again to obtain the Aspergillus Kawachi culture solution of the present invention.
얻어진 아스퍼질러스 카와치 배양액은 사용 전까지 4℃에서 보관한다.The obtained Aspergillus Kawachi culture solution is stored at 4°C until use.
본 발명의 생물전환 조성물은, 아래 실시예에서 확인되는 바와 같이, 관 형성 분석, 세포 침범 분석, 생체 내 혈관형성 모델에서 혈관형성을 저해하는 효과가 우수한 것으로 나타났다. The bioconversion composition of the present invention was found to have excellent effects of inhibiting angiogenesis in tube formation analysis, cell invasion analysis, and in vivo angiogenesis model, as confirmed in the examples below.
이하 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 이들 실시예는 본 발명을 예시하는 것으로서 본 발명의 범위가 이에 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. These examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.
<실시예 1><Example 1>
당귀, 천궁, 작약 및 숙지황을 옴니허브에서 구입하였다. 각 한약재에 한약재 중량의 3배 중량의 70% 에탄올을 혼합하고 각각 3시간 동안 가열하여 2회 추출하였다. 추출 후 필터페이퍼로 여과하고, 여과물을 감압농축하여 용매를 제거하고, 동결건조하여 추출물 분말을 제조하였다.Angelica, Cheongung, Peony and Sukjihwang were purchased from Omnihub. Each herbal medicine was mixed with 70% ethanol of 3 times the weight of the herbal medicine, heated for 3 hours, and extracted twice. After extraction, it was filtered with filter paper, the filtrate was concentrated under reduced pressure to remove the solvent, and freeze-dried to prepare an extract powder.
250㎖ 삼각플라스크에 밀기울 10g과 증류수 10㎖를 첨가하고, 밀기울이 소량의 물을 흡수하도록 으깨어 혼합한 후 멸균하였다. 멸균된 시료를 냉각시키고 아스퍼질러스 카와치를 접종하여 3시간 동안 상온에서 배양하였다. 배양 후 인산 나트륨 버퍼 스탁(1M)을 희석하여 최종농도가 10mM이 되도록 제조하고 각 플라스크에 50㎖씩 첨가하여 혼합한 후 4℃에서 하룻 밤 동안 배양하였다. 배양된 시료는 부직포에 싸서 배양액을 분리하고, 분리된 배양액을 원심분리한 후 상층액을 필터에 여과하여 아스퍼질러스 카와치 배양액을 얻었다. 얻어진 아스퍼질러스 카와치 배양액은 사용 전까지 4℃에서 보관하였다.To a 250 ml Erlenmeyer flask, 10 g of bran and 10 ml of distilled water were added, crushed and mixed so that the bran absorbs a small amount of water, and then sterilized. The sterilized sample was cooled, inoculated with Aspergillus kawachi, and incubated at room temperature for 3 hours. After incubation, sodium phosphate buffer stock (1M) was diluted to obtain a final concentration of 10 mM, 50 ml each was added to each flask, mixed, and incubated overnight at 4°C. The cultured sample was wrapped in a nonwoven fabric to separate the culture solution, the separated culture solution was centrifuged, and the supernatant was filtered through a filter to obtain Aspergillus Kawachi culture solution. The obtained Aspergillus Kawachi culture was stored at 4°C until use.
상기에서 얻은 동결건조된 추출물 분말 100g에 증류수 1 L를 첨가하여 혼합한 후 음파처리하였다. 상기 음파처리된 추출물 1 L에 상기 아스퍼질러스 카와치 배양액 1 L를 첨가하여 진탕 배양기에서 밤새 진탕 배양한 후, 감압농축 및 동결건조하여 생물전환 조성물 분말을 얻었다.To 100 g of the lyophilized extract powder obtained above, 1 L of distilled water was added and mixed, followed by sonication. 1 L of the Aspergillus Kawachi culture solution was added to 1 L of the sonicated extract, followed by shaking culture overnight in a shaking incubator, concentrated under reduced pressure, and freeze-dried to obtain a bioconversion composition powder.
<< 비교예Comparative example 1> 1>
당귀, 천궁, 작약 및 숙지황을 옴니허브에서 구입하였다. 각 한약재에 한약재 중량의 3배 중량의 70% 에탄올을 혼합하고 각각 3시간 동안 가열하여 2회 추출하였다. 추출 후 필터페이퍼로 여과하고, 여과물을 감압농축하여 용매를 제거하고, 동결건조하여 추출물 분말을 제조하였다.Angelica, Cheongung, Peony and Sukjihwang were purchased from Omnihub. Each herbal medicine was mixed with 70% ethanol of 3 times the weight of the herbal medicine, heated for 3 hours, and extracted twice. After extraction, it was filtered with filter paper, the filtrate was concentrated under reduced pressure to remove the solvent, and freeze-dried to prepare an extract powder.
<비교예 2><Comparative Example 2>
천궁을 실시예 1과 동일한 방법으로 생물전환하여 생물전환된 천궁 추출물 분말을 얻었다.Chungoong was bioconverted in the same manner as in Example 1 to obtain a bioconverted Chungoung extract powder.
<비교예 3><Comparative Example 3>
천궁을 비교예 1과 동일한 방법으로 추출하여 생물전환되지 않은 천궁 추출물 분말을 얻었다.Chungoong was extracted in the same manner as in Comparative Example 1 to obtain a Chungoung extract powder that was not bioconverted.
<비교예 4><Comparative Example 4>
당귀를 실시예 1과 동일한 방법으로 생물전환하여 생물전환된 당귀 추출물 분말을 얻었다.Angelicae was bioconverted in the same manner as in Example 1 to obtain a bioconverted Angelicae extract powder.
<비교예 5><Comparative Example 5>
당귀를 비교예 1과 동일한 방법으로 추출하여 생물전환되지 않은 당귀 추출물 분말을 얻었다.Angelicae was extracted in the same manner as in Comparative Example 1 to obtain an Angelicae extract powder that was not bioconverted.
<실험예 1><Experimental Example 1>
관 형성 검사(Tube formation assay)Tube formation assay
생물전환 조성물이 혈관신생여부에 미치는 효과를 확인하기 위하여 다음과 같이 관 형성 검사를 실시하였다.In order to confirm the effect of the bioconversion composition on angiogenesis, a tube formation test was performed as follows.
세포로는 SVEC4-10 세포(ATCC에서 구입)를 사용하였고, 시료로는 실시예 1의 생물전환 조성물 분말과 비교예 1의 생물전환되지 않은 조성물 분말을 사용하였다. 대조구에는 아무것도 처리하지 않았다.SVEC4-10 cells (purchased from ATCC) were used as cells, and the bioconversion composition powder of Example 1 and the non-bioconversion composition powder of Comparative Example 1 were used as samples. No treatment was applied to the control.
96-웰 플레이트에 마트리겔(Matrigel) 50㎕씩 첨가 후 중합반응을 위해 37℃에서 30분 동안 방치하였다. SVEC4-10 세포를 3×104cells/well로 분주하고 0㎍/㎖, 25㎍/㎖, 50㎍/㎖ 및 100㎍/㎖ 농도의 추출물 시료를 첨가하여 30분 동안 전처리하였다. 전처리된 SVEC4-10 세포를 마트리겔 상에 접종(seeding)한 후 37℃, 5% CO2 조건에서 3시간 동안 배양하였다. After addition of 50 µl of Matrigel to a 96-well plate, it was allowed to stand at 37° C. for 30 minutes for polymerization. SVEC4-10 cells were aliquoted into 3×10 4 cells/well, and extract samples at concentrations of 0 μg/ml, 25 μg/ml, 50 μg/ml, and 100 μg/ml were added, followed by pretreatment for 30 minutes. The pretreated SVEC4-10 cells were seeded on Matrigel and cultured for 3 hours at 37° C. and 5% CO 2.
배양된 세포의 관 형성도를 확인한 결과를 도 1에 나타내었다. 도 1에서 C는 무처리 대조구, SMT는 비교예 1의 생물전환되지 않은 조성물을 처리한 경우, BSMT는 실시예 1의 생물전환 조성물을 처리한 경우를 나타낸다.The results of confirming the tube formation degree of the cultured cells are shown in FIG. 1. In FIG. 1, C represents an untreated control, SMT represents a case of treating the non-bioconverted composition of Comparative Example 1, and BSMT represents a case of treating the biotransformation composition of Example 1.
도 1의 결과에서와 같이, 생물전환 조성물을 처리한 경우 모든 처리농도에서 생물전환되지 않은 조성물을 처리한 경우에 비해 관 형성도가 낮은 것이 확인되었다.As shown in the results of FIG. 1, when the bioconversion composition was treated, it was confirmed that the degree of tube formation was lower than when the non-bioconverted composition was treated at all treatment concentrations.
<실험예 2><Experimental Example 2>
세포 침범 분석(Cell invasion assay, Transwell migration assay)Cell invasion assay, Transwell migration assay
생물전환 조성물이 피브로넥틴(Fibronectin, FN)에 의해 유도된 세포 침범(cell invasion)에 미치는 효과를 트랜스웰 이동 분석(Transwell migration assay)으로 다음과 같이 확인하였다.The effect of the bioconversion composition on the cell invasion induced by fibronectin (FN) was confirmed by the following transwell migration assay.
세포로는 SVEC4-10 세포(ATCC에서 구입)를 사용하였고, 시료로는 실시예 1의 생물전환 조성물 분말과 비교예 1의 생물전환되지 않은 조성물 분말을 사용하였다. SVEC4-10 cells (purchased from ATCC) were used as cells, and the bioconversion composition powder of Example 1 and the non-bioconversion composition powder of Comparative Example 1 were used as samples.
트랜스웰(Transwell)을 5㎍/㎖의 피브로넥틴으로 코팅하고 4℃에서 밤새 방치하였다. 코팅된 트랜스웰을 1% BSA로 60분 동안 블로킹(blocking)한 후 PBS로 세척하였다. 세척 후 3×104cells/well의 SVEC4-10세포를 0㎍/㎖, 50㎍/㎖ 및 100㎍/㎖ 농도의 각 추출물과 30분 동안 전처리한 후 상부 챔버(upper chamber)에 분주하여 37℃, 5% CO2 조건에서 16시간 동안 배양하였다. 배양된 트랜스웰의 세포를 4% 파라포름알데하이드(paraformaldehyde)로 고정한 후 0.1% 크리스탈 바이올렛(Crystal violet)으로 염색하였다. 대조구로는 2% BSA로 코팅하여 4℃에서 밤새 방치한 트랜스웰을 사용하였다.Transwells were coated with 5 μg/ml fibronectin and left overnight at 4°C. The coated transwells were blocked with 1% BSA for 60 minutes and washed with PBS. After washing, 3×10 4 cells/well of SVEC4-10 cells were pretreated with each extract at concentrations of 0 μg/ml, 50 μg/ml, and 100 μg/ml for 30 minutes, and then dispensed into the upper chamber. Incubated for 16 hours at ℃, 5% CO 2 conditions. The cells of the cultured transwell were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. As a control, a transwell coated with 2% BSA and left overnight at 4°C was used.
침범된 세포율을 확인한 결과를 도 2에 나타내었다.The results of confirming the invaded cell rate are shown in FIG. 2.
도 2의 결과에서와 같이, 피브로넥틴(FN)에 의해 유도된 세포 침범상에서 생물전환 조성물(BSMT)은 생물전환되지 않은 조성물(SMT)과 달리 세포 침범을 현저히 저하시켜 2% BSA로만 처리된 대조구와 유사한 양상을 나타내었다.As shown in the results of Figure 2, in the cell invasion induced by fibronectin (FN), the biotransformation composition (BSMT) significantly reduced the cell invasion, unlike the non-biotransformed composition (SMT), and thus treated with only 2% BSA. It showed a similar pattern.
<실험예 3><Experimental Example 3>
세포 침범 분석Cell invasion assay
천궁 및 당귀 추출물이 피브로넥틴(FN)에 의해 유도된 세포 침범에 미치는 효과를 다음과 같이 확인하였다.The effect of the extracts of Chunkyung and Angelicae on the cell invasion induced by fibronectin (FN) was confirmed as follows.
시료로는 비교예 2의 생물전환된 천궁 추출물, 비교예 3의 생물전환되지 않은 천궁 추출물, 비교예 4의 생물전환된 당귀 추출물 및 비교예 5의 생물전환되지 않은 당귀 추출물을 사용하였고, 실험방법은 실험예 2와 동일하게 실험하였다. As a sample, the bioconverted Cnidium extract of Comparative Example 2, the non-bioconverted Cnidium extract of Comparative Example 3, the bioconverted Angelicae extract of Comparative Example 4, and the non-bioconverted Angelicae extract of Comparative Example 5 were used. Was experimented in the same manner as in Experimental Example 2.
침범된 세포율을 확인한 결과를 도 3에 나타내었다.The results of confirming the invaded cell rate are shown in FIG. 3.
도 3의 결과에서와 같이, 피브로넥틴(FN)에 의해 유도된 세포 침범상에서 생물전환된 천궁 추출물과 당귀 추출물은 생물전환되지 않은 천궁 추출물과 당귀 추출물과 세포 침범 상에서 차이를 나타내지 않았다.As shown in the results of FIG. 3, the bioconverted Cnidium extract and Angelicae extract in the cell invasion phase induced by fibronectin (FN) did not show any difference in the cell invasion phase and the non-bioconverted Cnidium extract and Angelicae extract.
<실험예 4><Experimental Example 4>
생체 내 마트리겔 플러그 분석(In vivo Matrigel plug analysis ( in vivoin vivo Matrigel plug assay) Matrigel plug assay)
생체 내(in vivo) 혈관형성 모델에서, 본 발명의 생물전환 조성물이 헤모글로빈 함량에 미치는 효과를 다음과 같이 확인하였다.In the in vivo angiogenesis model, the effect of the bioconversion composition of the present invention on the hemoglobin content was confirmed as follows.
마우스는 7주령 C57BL/6 마우스를 ㈜코아텍에서 구입하여 사용하였고, 시료로는 실시예 1의 생물전환 조성물 분말과 비교예 1의 생물전환되지 않은 조성물 분말을 사용하였다. As for the mice, 7-week-old C57BL/6 mice were purchased and used from Coretech, Inc., and the bioconversion composition powder of Example 1 and the non-bioconversion composition powder of Comparative Example 1 were used as samples.
4℃에서 마트리겔 혼합물(마트리겔 200μg/㎖, VEGF 300ng/㎖, 헤파린 50U)에 생물전환 조성물 분말과 생물전환되지 않은 조성물 분말을 각각 100㎍/㎖의 농도로 혼합한 후 거품을 제거하기 위해 얼음 위에 방치하였다. 준비된 마우스는 마취하고 양쪽 옆구리 털을 제거한 후 상기 마트리겔 혼합물을 각 부위별로 200㎕씩 피하 삽입하였다. 대조구에는 마트리겔 혼합물을 처리하지 않았다.To remove bubbles after mixing the bioconversion composition powder and the non-bioconversion composition powder in a Matrigel mixture (Matrigel 200μg/ml, VEGF 300ng/ml, heparin 50U) at a concentration of 100µg/ml, respectively, at 4°C. It was left on ice. The prepared mice were anesthetized and hairs on both sides were removed, and then the Matrigel mixture was inserted subcutaneously at 200µl for each site. The control was not treated with the Matrigel mixture.
1주일 동안 방치한 후 마우스를 해부하여 마트리겔의 형태와 색깔을 확인하였다. After leaving for 1 week, the mouse was dissected to check the shape and color of Matrigel.
또한 마트리겔의 일부를 잘라 500㎕ DW(+ 0.2% 헤파린)가 함유된 튜브에 넣어 냉장고에서 하룻밤 방치하고, 15,000rpm에서 20분 동안 원심분리하였다. 원심분리된 상층액 100㎕를 드랩스킨 키트용액(Drabskin kit solution) 100㎕와 혼합하여 540nm에서 헤모글로빈의 농도를 측정하였다. In addition, a part of Matrigel was cut and placed in a tube containing 500 µl DW (+ 0.2% heparin), left overnight in a refrigerator, and centrifuged at 15,000 rpm for 20 minutes. 100 µl of the centrifuged supernatant was mixed with 100 µl of a Drabskin kit solution, and the concentration of hemoglobin was measured at 540 nm.
그 결과를 도 4에 나타내었다.The results are shown in FIG. 4.
도 4의 결과에서와 같이, 생체 내 혈관형성 모델에서 VEGF 처리군은 높은 헤모글로빈 함량을 나타내었고, 생물전환 조성물을 처리하였을 때에는 생물전환되지 않은 조성물을 처리하였을 때에 비해 헤모글로빈 함량이 현저히 낮은 것으로 나타났다.As shown in the results of Figure 4, in the in vivo angiogenesis model, the VEGF-treated group showed a high hemoglobin content, and when the bioconversion composition was treated, the hemoglobin content was significantly lower than when the non-bioconverted composition was treated.
Claims (9)
상기 생약조성물의 추출물은, 숙지황, 백작약, 천궁 및 당귀를 혼합하여 얻은 생약조성물에 70% 에탄올을 1:2~4의 중량비로 혼합하여 추출한 후, 여과, 농축 및 동결건조하여 추출물 분말을 얻고, 이 추출물 분말에 증류수를 1:5~20의 중량비로 혼합하여 음파처리한 것이며,
상기 생물전환은 상기 음파처리한 생약조성물의 추출물에 아스퍼질러스 카와치 배양액을 100:50~200의 중량비로 혼합하여 진탕 배양하는 것을 특징으로 하는, 혈관형성 저해용 조성물.As an active ingredient, a bioconversion composition obtained by bioconversion of an extract of a herbal composition including Sukjihwang, Earl, Cheongung, and Angelicae into Aspergillus Kawachi culture solution,
The extract of the herbal composition is extracted by mixing 70% ethanol in a weight ratio of 1: 2 to 4 in a herbal composition obtained by mixing Sukjihwang, Baekjak, Cheongung, and Angelicae, followed by filtration, concentration, and freeze-drying to obtain an extract powder, This extract powder was mixed with distilled water at a weight ratio of 1:5 to 20 and subjected to sound wave treatment,
The biotransformation is characterized in that shaking culture by mixing Aspergillus Kawachi culture solution in a weight ratio of 100:50 ~ 200 to the extract of the herbal composition subjected to the sonication treatment, the composition for inhibiting angiogenesis.
상기 생약조성물은 숙지황 10~40중량%, 백작약 10~40중량%, 천궁 10~40중량% 및 당귀 10~40중량%를 포함하는 것을 특징으로 하는 혈관형성 저해용 조성물.The method of claim 1,
The herbal composition is a composition for inhibiting angiogenesis, characterized in that it comprises 10 to 40% by weight of Sukjihwang, 10 to 40% by weight of Earl, 10 to 40% by weight of cnidium and 10 to 40% by weight of Angelicae.
상기 생약조성물은 숙지황, 백작약, 천궁 및 당귀를 1:1:1:1의 중량비로 혼합한 것임을 특징으로 하는 혈관형성 저해용 조성물.The method of claim 1,
The herbal composition is a composition for inhibiting angiogenesis, characterized in that the mixture of Sukjihwang, Baekjak, Chungoong and Angelicae in a weight ratio of 1:1:1:1.
상기 아스퍼질러스 카와치 배양액은,
밀기울 10g을 기준으로 증류수 8~12㎖를 첨가하고 밀기울이 소량의 물을 흡수하도록 으깨어 혼합한 후 멸균하는 단계;
상기 멸균 후 식힌 다음 아스퍼질러스 카와치를 접종하고 상온에서 2~4시간 동안 배양하는 단계;
상기 배양 후, 10mM 농도의 인산 나트륨 버퍼 스탁을 밀기울 10g을 기준으로 45~55㎖씩 첨가하여 혼합한 후 4℃에서 밤새 배양하여 배양된 시료를 얻는 단계;
상기 배양된 시료를 여과하여 여과액을 분리하는 단계; 및
상기 여과액을 원심분리한 후 상층액을 여과하여 아스퍼질러스 카와치 배양액을 얻는 단계를 포함하는 방법에 의하여 제조된 것임을 특징으로 하는 혈관형성 저해용 조성물.The method according to any one of claims 1 to 3,
The Aspergillus Kawachi culture solution,
Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;
Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating for 2 to 4 hours at room temperature;
After the incubation, adding 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, mixing, and incubating at 4° C. overnight to obtain a cultured sample;
Filtering the cultured sample to separate the filtrate; And
A composition for inhibiting angiogenesis, characterized in that it is prepared by a method comprising the step of obtaining an Aspergillus Kawachi culture solution by centrifuging the filtrate and filtering the supernatant.
상기 생약조성물에 70% 에탄올을 1:2~4의 중량비로 혼합하여 추출하는 단계;
상기 추출 후 여과하여 여과물을 얻는 단계;
상기 여과물을 감압농축 및 동결건조하여 추출물 분말을 얻는 단계;
상기 추출물 분말에 증류수를 1:5~20의 중량비로 혼합하여 음파처리하는 단계;
상기 음파처리된 것에 아스퍼질러스 카와치 배양액을 100:50~200의 중량비로 혼합하여 진탕 배양하는 단계; 및
상기 배양 후 감압농축하여 생물전환 조성물을 얻는 단계를 포함하는, 혈관형성 저해를 위한 생물전환 조성물의 제조방법. Obtaining a herbal composition by mixing Sukjihwang, Earl Peony, Cheongung and Angelicae;
Extracting the herbal composition by mixing 70% ethanol in a weight ratio of 1: 2 to 4;
Filtering after the extraction to obtain a filtrate;
Concentrating the filtrate under reduced pressure and freeze-drying to obtain an extract powder;
Mixing distilled water to the extract powder at a weight ratio of 1:5 to 20 and performing sound wave treatment;
Mixing the Aspergillus Kawachi culture solution to the sonicated one at a weight ratio of 100:50 to 200 and culturing with shaking; And
A method for producing a bioconversion composition for inhibiting angiogenesis, comprising the step of obtaining a bioconversion composition by concentrating under reduced pressure after the culture.
상기 생약조성물은 숙지황 10~40중량%, 백작약 10~40중량%, 천궁 10~40중량% 및 당귀 10~40중량%를 포함하는 것을 특징으로 하는, 생물전환 조성물의 제조방법. The method of claim 6,
The herbal composition is characterized in that it comprises 10 to 40% by weight of Sukjihwang, 10 to 40% by weight of the earl, 10 to 40% by weight of cheonggung and 10 to 40% by weight of Angelicae, a method for producing a bioconversion composition.
상기 감압농축 후 동결건조시켜 분말 형태의 생물전환 조성물을 얻는 단계를 더 포함하는 것을 특징으로 하는, 생물전환 조성물의 제조방법. The method of claim 6,
The method for producing a bioconversion composition, further comprising the step of obtaining a powdery bioconversion composition by lyophilizing after concentration under reduced pressure.
상기 아스퍼질러스 카와치 배양액은,
밀기울 10g을 기준으로 증류수 8~12㎖를 첨가하고 밀기울이 소량의 물을 흡수하도록 으깨어 혼합한 후 멸균하는 단계;
상기 멸균 후 식힌 다음 아스퍼질러스 카와치를 접종하고 상온에서 2~4시간 동안 배양하는 단계;
상기 배양 후, 10mM 농도의 인산 나트륨 버퍼 스탁을 밀기울 10g을 기준으로 45~55㎖씩 첨가하여 혼합한 후 4℃에서 밤새 배양하여 배양된 시료를 얻는 단계;
상기 배양된 시료를 여과하여 여과액을 분리하는 단계; 및
상기 여과액을 원심분리한 후 상층액을 여과하여 아스퍼질러스 카와치 배양액을 얻는 단계를 포함하는 방법에 의하여 제조된 것임을 특징으로 하는, 생물전환 조성물의 제조방법.The method according to any one of claims 6 to 8,
The Aspergillus Kawachi culture solution,
Adding 8 to 12 ml of distilled water based on 10 g of bran, crushing and mixing so that the bran absorbs a small amount of water, and sterilizing;
Cooling after the sterilization, inoculating Aspergillus kawachi, and incubating for 2 to 4 hours at room temperature;
After the incubation, adding 45 to 55 ml of sodium phosphate buffer stock having a concentration of 10 mM based on 10 g of bran, mixing, and incubating at 4° C. overnight to obtain a cultured sample;
Filtering the cultured sample to separate the filtrate; And
A method for producing a bioconversion composition, characterized in that it is prepared by a method comprising the step of obtaining Aspergillus Kawachi culture by filtering the supernatant after centrifuging the filtrate.
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KR100592482B1 (en) | 2004-07-19 | 2006-06-26 | 경북대학교 산학협력단 | Methods for increasing liquiritigenin content in a licorice and its extracts and the method for the isolation and extraction of liquiritigenin from them |
KR101084446B1 (en) | 2008-06-26 | 2011-11-21 | 주식회사 비피도 | Anti-angiogenic composition comprising bioconversion materials of Angelica gigas and Zizyphus jujuba water extracts transformed by Aspergillus usamii var. shirousamii |
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