KR101145751B1 - Composition for treating or preventing inflammatory bowel disease - Google Patents
Composition for treating or preventing inflammatory bowel disease Download PDFInfo
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- KR101145751B1 KR101145751B1 KR1020090098074A KR20090098074A KR101145751B1 KR 101145751 B1 KR101145751 B1 KR 101145751B1 KR 1020090098074 A KR1020090098074 A KR 1020090098074A KR 20090098074 A KR20090098074 A KR 20090098074A KR 101145751 B1 KR101145751 B1 KR 101145751B1
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- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
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Abstract
본 발명은 염증성 장 질환, 즉, 궤양성 대장염과 크론병의 치료 또는 예방에 유용한 조성물에 관한 것으로서, 나복자 추출물 또는 나복자 분말을 포함하는 것을 특징으로 한다.The present invention relates to a composition useful for the treatment or prevention of inflammatory bowel disease, ie ulcerative colitis and Crohn's disease, characterized in that it comprises a moth extract or moth powder.
Description
본 발명은 염증성 장 질환의 치료 또는 예방을 위한 조성물에 관한 것이다.The present invention relates to a composition for the treatment or prevention of inflammatory bowel disease.
염증성 장 질환(Inflammatory bowel disease, IBD)은 일반적으로 궤양성 대장염(Ulcerative colitis)과 크론병(Crohn's disease)을 일컬으며, 지난 수십 년간 전 세계적으로 많은 연구자들에 의해 연구되어 오고 있다. 그러나 이러한 노력에도 불구하고 이 질환의 병인과 병태생리 및 효과적인 치료제는 여전히 불분명한 상태이다. 특히 궤양성 대장염은 대장 점막에 염증성 궤양이 생기며, 증상은 다양하지만 설사, 장출혈, 복통 등이 주된 증상이고, 발열, 식욕부진, 체중감소 등도 흔히 올 수 있다. 또 궤양성 대장염 환자의 3% 내지 13%정도가 대장암으로 진행된다고 보고되고 있다. 이러한 염증성 잘 질환은 증상의 악화와 회복단계를 반복하는 특징을 지닌 만성 질환으로 장기간 치료를 요하는 반면, 만족할 만한 치료제는 없는 상태이다.Inflammatory bowel disease (IBD) is commonly referred to as ulcerative colitis and Crohn's disease and has been studied by many researchers around the world for decades. However, despite these efforts, the etiology, pathophysiology and effective treatment of the disease are still unclear. In particular, ulcerative colitis causes inflammatory ulcers on the colon mucosa, and symptoms vary, but diarrhea, intestinal bleeding, and abdominal pain are the main symptoms, and fever, anorexia, and weight loss may also occur frequently. It is also reported that about 3% to 13% of ulcerative colitis patients progress to colorectal cancer. The inflammatory well disease is a chronic disease characterized by aggravation of symptoms and a recovery phase, and requires long-term treatment, while there is no satisfactory treatment.
따라서 본 발명이 이루고자 하는 기술적 과제는 염증성 장 질환의 치료 또는 예방에 효과적인 조성물을 제공하는 것이다.Therefore, the technical problem to be achieved by the present invention is to provide a composition effective for the treatment or prevention of inflammatory bowel disease.
상기 기술적 과제를 달성하기 위하여, 본 발명은 나복자 분말 또는 나복자 추출물을 유효 성분으로 포함하는 것을 특징으로 하는 염증성 장 질환의 치료 또는 예방용 조성물을 제공한다.In order to achieve the above technical problem, the present invention provides a composition for the treatment or prophylaxis of inflammatory bowel disease, characterized in that it comprises a moth extract or moth extract as an active ingredient.
본 발명자들은 나복자의 염증성 장 질환 치료제로서의 가능성을 여러 가지 모델로 평가한 결과 나복자가 염증성 장 질환, 즉, 궤양성 대장염(Ulcerative colitis) 또는 크론병(Crohn's disease)의 치료 또는 예방에 유용하다는 놀라운 발견을 하였다.The inventors have evaluated the potential of larvae as a therapeutic agent for inflammatory bowel disease in a number of models, and as a result, the surprising finding that larvae is useful for the treatment or prevention of inflammatory bowel disease, namely Ulcerative colitis or Crohn's disease. Was done.
나복자는 내복자라고도 하며 십자화과의 무(Raphanus sativus L.) 또는 동속식물의 씨를 말하며, 본 발명에 따른 조성물은 염증성 장 질환의 치료 성분으로 이러한 나복자를 포함한다. 본 발명에 따른 조성물은 제제화가 용이하도록 나복자 분말 또는 나복자 추출물을 포함할 수 있다.The moths are also called the undergarments and refer to the seeds of Raphanus sativus L. or the same plant, and the composition according to the present invention includes such moths as a therapeutic component of inflammatory bowel disease. The composition according to the invention may comprise a moth extract or moth extract to facilitate formulation.
본 발명에 따른 나복자 추출물은 본 발명이 속한 분야에서 잘 알려진 추출, 분리 및 정제방법을 통해 얻을 수 있다. 예를 들어, 통상적인 용매추출방법으로서 물, 메탄올, 에탄올, 부탄올, 클로로포름, 헥산, 에틸아세테이트, 디클로로메탄, 이들의 혼합물 등의 적당한 용매를 이용하여 추출하거나 초임계 추출법을 통해서도 얻을 수 있으며, 또한 추출물을 다시 정제함으로써 더욱 고순도의 나복자 추출물을 얻을 수 있다.Nachojak extract according to the present invention can be obtained through extraction, separation and purification methods well known in the art. For example, as a conventional solvent extraction method, it can be extracted using a suitable solvent such as water, methanol, ethanol, butanol, chloroform, hexane, ethyl acetate, dichloromethane, a mixture thereof, or obtained through supercritical extraction. By further purifying the extract, it is possible to obtain a higher purity nabokja extract.
본 발명의 염증성 장 질환의 치료 또는 예방용 조성물은 의약품 및 기능성 식품의 형태로 제조될 수 있다. 이러한 의약품 및 기능성 식품은 약제학적으로 허용되는 부형제 또는 첨가제를 포함할 수 있다. 본 발명의 조성물은 단독으로 혹은 어떤 편리한 운반체, 부형제 등과 함께 혼합하여 투여될 수 있고, 그러한 투여 제형은 단회투여 또는 반복투여 제형일 수 있다. Compositions for the treatment or prevention of inflammatory bowel disease of the present invention may be prepared in the form of pharmaceuticals and functional foods. Such medicines and functional foods may include pharmaceutically acceptable excipients or additives. The compositions of the present invention may be administered alone or in admixture with any convenient carrier, excipient, and the like, and such dosage forms may be single or repeated dose formulations.
본 발명의 조성물을 포함하는 의약품 또는 기능성 식품은 고형 제제 또는 액상 제제일 수 있다. 고형 제제는 산제, 과립제, 정제, 캅셀제, 좌제 등이 있으나, 이에 한정되는 것은 아니다. 고형 제제에는 부형제, 착향제, 결합제, 방부제, 붕해제, 활택제, 충진제 등이 포함될 수 있으나 이에 한정되는 것은 아니다. 액상 제제로는 물, 프로필렌 글리콜 용액 같은 용액제, 현탁액제, 유제 등이 있으나, 이에 한정되는 것은 아니며, 적당한 착색제, 착향제, 안정화제, 점성화제 등을 첨가하여 제조할 수 있다.The pharmaceutical or functional food comprising the composition of the present invention may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, excipients, flavors, binders, preservatives, disintegrants, lubricants, fillers and the like. Liquid formulations include, but are not limited to, solutions such as water, propylene glycol solutions, suspensions, emulsions, and the like, and may be prepared by adding suitable colorants, flavors, stabilizers, viscosity agents, and the like.
예를 들어, 산제는 본 발명의 나복자 분말 또는 나복자 추출물과 유당, 전분, 미결정셀룰로오스 등 약제학적으로 허용되는 적당한 부형제를 단순 혼합함으로써 제조될 수 있다. 과립제는 본 발명의 나복자 추출물 또는 나복자 분말; 약제학적으로 허용되는 적당한 부형제; 및 폴리비닐피롤리돈, 히드록시프로필셀룰로오스 등의 약제학적으로 허용되는 적당한 결합제를 혼합한 후, 물, 에탄올, 이소프로판올 등의 용매를 이용한 습식과립법 또는 압축력을 이용한 건식과립법을 이용하여 제조될 수 있다. 또한 정제는 상기 과립제를 마그네슘스테아레이트 등의 약제학적으로 허용되는 적당한 활택제화 혼합한 후, 타정기를 이용하여 타정함으로써 제조될 수 있다.For example, powders may be prepared by simply mixing the nachos powder or nachos extract of the present invention with a suitable pharmaceutically acceptable excipient such as lactose, starch, microcrystalline cellulose. Granules may be used in the moth extract or moth powder of the present invention; Pharmaceutically acceptable excipients; And a pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropyl cellulose, and then prepared by using a wet granulation method using a solvent such as water, ethanol, isopropanol, or a dry granulation method using a compressive force. Can be. Tablets may also be prepared by mixing the granules with a suitable pharmaceutically acceptable glidant such as magnesium stearate and then tableting using a tableting machine.
본 발명의 조성물은 치료해야할 질환 및 개체의 상태에 따라 경구제, 주사제(예를 들어, 근육주사, 복강주사, 정맥주사, 주입(infusion), 피하주사, 임플란트), 흡입제, 비강투여제, 질제, 직장투여제, 설하제, 트랜스더말제, 토피칼제 등으로 투여될 수 있으나, 이에 한정되는 것은 아니다. 투여경로에 따라 통상적으로 사용되고 비독성인, 약제학적으로 허용되는 운반체, 첨가제, 비히클을 포함하는 적당한 투여 유닛 제형으로 제제화될 수 있다. 일정 시간 동안 약물을 지속적으로 방출할 수 있는 데포(depot) 제형 또한 본 발명의 범위에 포함된다.The compositions of the present invention may be administered orally, injectable (eg, intramuscular, intraperitoneal, intravenous, infusion, subcutaneous, implants), inhalants, nasal administrations, vaginal agents depending on the condition to be treated and the condition of the individual. It may be administered as a rectal agent, a sublingual agent, a transdermal agent, a topical agent, but is not limited thereto. It may be formulated into a suitable dosage unit dosage form comprising a pharmaceutically acceptable carrier, excipient, vehicle, conventionally used and nontoxic, depending on the route of administration. Depot formulations capable of continually releasing the drug for a period of time are also within the scope of the present invention.
염증성 장 질환의 개선이라는 본 발명의 목적을 달성하기 위하여 나복자 분말 또는 나복자 추출물은 매일 약 0.001 mg/kg 내지 약 10 g/kg이 투여될 수 있으며, 약 0.01 mg/kg 내지 약 1 g/kg의 1일 투여 용량이 바람직하다. 그러나 상기 투여량은 나복자 분말 또는 나복자 추출물의 정제 정도, 환자의 상태(연령, 성별, 체중 등), 치료하고 있는 상태의 심각성 등에 따라 다양할 수 있다. 필요에 따라 편리성을 위하여 1일 총 투여량이 나누어지고 하루 동안 여러 번 나누어 투여될 수 있다.In order to achieve the object of the present invention to improve inflammatory bowel disease, the moth extract or moth extract may be administered daily from about 0.001 mg / kg to about 10 g / kg, and from about 0.01 mg / kg to about 1 g / kg Daily doses are preferred. However, the dosage may vary depending on the degree of purification of the moth powder or moth extract, the condition of the patient (age, sex, weight, etc.), the severity of the condition being treated. If desired, the total daily dose may be divided for convenience and divided several times throughout the day.
본 발명은 염증성 장 질환, 보다 바람직하게는, 궤양성 대장염과 크론병의 치료 및 예방에 유용한 조성물을 제공한다.The present invention provides compositions useful for the treatment and prevention of inflammatory bowel disease, more preferably ulcerative colitis and Crohn's disease.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석돼서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<< 실시예Example 1> 아세트산 유발 대장염 개선 평가 1> Evaluation of Acetic Acid-Induced Colitis Improvement
시험 샘플의 준비 및 실험동물Preparation of Test Samples and Laboratory Animals
하기 표 1과 같은 시험 샘플을 준비하였다. 나복자 추출물의 경우 30% 에탄올 수용액을 이용하여 추출하고 감압농축한 후 건조하여 제조하였다.A test sample was prepared as shown in Table 1 below. In the case of nabokjak extract was extracted using 30% ethanol aqueous solution, concentrated under reduced pressure and dried.
6주령 Male Sprague-Dawley 쥐를 오리엔트사에서 구입하여 일반 동물실험실에서 사육하였다. 사육환경은 온도 23±2℃, 습도 55±15%, 조명시간 12시간의 조건을 유지하였으며, 동물은 케이지 당 한 마리씩 분리하여 수용하였다.Six-week-old Male Sprague-Dawley rats were purchased from Orient and bred in a general animal laboratory. The breeding environment was maintained at a temperature of 23 ± 2 ° C., a humidity of 55 ± 15% and an illumination time of 12 hours. Animals were separated and housed one per cage.
상기 쥐를 24시간 절식시킨 후, 길이 8㎝의 폴리에틸렌 튜브(직경0.76㎜)를 항문으로 밀어 넣은 후 아세트산 용액을 주입하여 대장염을 유발하였다. 아세트산은 7% 수성용액을 한 마리당 1ml의 용량으로 0.1 ml씩 천천히 점적 주사하여 주입하였다. 아세트산 주입 후 24시간 후부터 4일 동안 1일 1회씩 쥐용 경구존데를 이용하여 약물을 경구로 투여하였다.After the rats were fasted for 24 hours, colitis was induced by pushing an 8 cm long polyethylene tube (diameter 0.76 mm) into the anus and injecting an acetic acid solution. Acetic acid was injected by slow dropwise injection of 0.1 ml of a 7% aqueous solution at a dose of 1 ml per animal. The drug was orally administered once a day for 4 days from 24 hours after acetic acid injection using a rat oral sonde.
체중 평가Weight evaluation
7% 아세트산으로 대장염 유발을 하기 직전과 투여가 끝난 후 각 동물의 체중을 측정하여 각 동물의 체중감소량을 확인하였다. 체중감소량은 대장염 유발을 하기 직전의 체중에 대한 그 날의 체중의 감소(g)로 나타내었다. 그 결과를 하기 표 2에 나타내었다.The weight loss of each animal was confirmed by measuring the weight of each animal immediately before and after the administration of colitis with 7% acetic acid. Weight loss is expressed as the weight loss (g) of the day relative to the weight immediately before inducing colitis. The results are shown in Table 2 below.
상기 표 2에 나타나는 바와 같이, 7% 아세트산 용액으로 대장염을 유발한 음성 대조군들은 유발 직후부터 체중이 감소되는 경향을 보였지만 프레드니솔론을 투여한 양성 대조군과 시험군인 나복자 추출물을 투여한 군에서는 약물의 투여 4일까지 꾸준한 체중증가를 보여주었다.As shown in Table 2, the negative control group that induced colitis with 7% acetic acid solution showed a tendency to lose weight immediately after the induction, but the administration of the drug in the positive control group administered prednisolone and the nasopharyngeal extract, a
설사발생률 평가Diarrhea Incidence Assessment
7% 아세트산으로 대장염 유발한 후 24시간 후 매일 경구투여하기 직전 각 동물의 설사의 유무를 확인하였다. 각 군의 전체 동물 수에 대한 설사발생 수의 비율로 나타내었다.After the induction of colitis with 7% acetic acid, each animal was checked for the presence of diarrhea immediately before oral administration daily. It is expressed as the ratio of diarrhea number to the total number of animals in each group.
나복자 투여군은 투여 후 심한 설사에서 회복되어 정상변에 가까워 졌으며, 양성 대조군의 경우에는 대장염 유발 후 설사 정도가 심한 대변을 보았다가 점차적으로 묽은 변의 형태를 보였다. 음성 대조군의 경우에는 설사의 정도가 매우 심하여 항문 주위에 염증이 발생하였으며 그중 1마리가 혈변증상을 나타내었다. The moth-treated group recovered from severe diarrhea after administration and approached normal stools. In the positive control group, diarrhea was severe after colitis-induced diarrhea and gradually diminished. In the negative control group, diarrhea was severe and inflammation occurred around the anus. One of them showed bloody stools.
대장의 길이, 무게 및 형태 변화 평가Assessment of changes in length, weight, and morphology of the colon
마지막 약물투여 24시간 후에 각 군의 동물들을 경추 탈골시킨 후 부검을 실시하고, 대장과 비장, 간을 적출하였으며 대장의 길이는 직장 쪽으로부터 3.5cm를 제외한 부분의 대장의 길이를 측정하고, 대장과, 비장, 간의 무게를 측정하였다. 그 결과를 하기 표 3 및 도 1에 나타내었다.24 hours after the last drug administration, the animals of each group were dislocated from the cervical spine and necropsied. The colon, spleen and liver were removed. The length of the large intestine was measured except for 3.5 cm from the rectum. The weights of the spleens and livers were measured. The results are shown in Table 3 and FIG. 1.
상기 표 3 및 도 1에 나타나는 바와 같이, 정상 대조군과 비교했을 때 나복자 투여군의 경우가 대장(colon)의 길이, 무게에서 가장 정상 대조군과 가까운 수치를 나타냄을 알 수 있어 대장의 병변이 정상으로 완화됨을 알 수 있었다. 형태적인 면에서 볼 때 나복자 투여군은 궤양 및 염증이 없었고, 약간의 충혈만 있었다. 양성 대조군도 궤양이나 염증의 소견이 적었으나 음성 대조군은 염증과 궤양의 정도가 심했고 장벽은 두꺼웠다. As shown in Table 3 and Figure 1, when compared with the normal control group can be seen that the case of the moths administered group shows the value closest to the normal control in the length of the colon (colon), weight, so that the lesion of the colon is alleviated to normal And it was found. In terms of morphology, the moths administered group had no ulcers and inflammation, and only a little hyperemia. In the positive control group, there was little ulcer or inflammation, but the negative control group had severe inflammation and ulcers, and the barrier was thick.
대장 병변 소견 및 조직학적 염색 소견Colon lesion and histological staining
현미경 관찰을 위하여 실험동물을 마취한 후 복부를 정중 절개하고 소화관을 전체적으로 적출하였다. 적출한 내장 중 대장을 채취하여 실체 현미경으로 대장 병변 소견을 관찰하였다. 그 후 실온에서 10% 포르말린 용액에 고정하고 H&E 염색을 실시하였다. 그 결과를 도 2 및 도 3에 나타내었다.After anesthetizing the experimental animals for microscopic observation, the abdomen was dissected midway and the digestive tract was removed as a whole. The colon was collected from the viscera and the colon lesions were observed under a stereoscopic microscope. It was then fixed in 10% formalin solution at room temperature and subjected to H & E staining. The results are shown in FIGS. 2 and 3.
도 2에 나타나는 바와 같이, 대장을 적출하여 실체현미경으로 관찰한 결과 음성 대조군의 경우는 대장의 병변이 확인 가능하여 IBD가 유발됨을 확인할 수 있었으나, 시험군인 나복자 투여군의 경우는 병변의 정도가 완화되었음을 관찰할 수 있었다. 또한 도 3의 H&E 염색에서 관찰할 수 있는 것처럼 궤양성 대장염(IBD)가 유발된 음성 대조군은 점막하 조직상의 염증세포 침윤, 상피세포의 파괴, 점액세포의 파괴 등의 염증 소견이 관찰되었지만, 양성 대조군 및 실험대조군인 나복자 투여군에서 점액세포와 선와 등이 정상적인 형태를 보여 치료 효과를 나타내는 것으로 사료되었다 As shown in FIG. 2, the colon was extracted and observed with a stereomicroscope. As a result, the negative control group was able to confirm the lesion of the colon and confirmed that IBD was induced. It could be observed. In addition, ulcerative colitis (IBD) -induced negative control group showed inflammatory findings such as inflammatory cell infiltration, destruction of epithelial cells, and destruction of mucous cells, as observed in H & E staining of FIG. 3. The mucosa and crypts showed normal morphology in the treatment group of Nabokja, which was the experimental control group.
대장(Leader( coloncolon )의 형태 평가) Form evaluation
대장의 벽 두께 정도를 측정하고 대장을 절개하여 반으로 자른 후, 궤양 및 염증상태의 육안병변을 하기 표 4(Wallace의 기준)에 따라 점수화하였다. 그 결과를 도 4에 나타내었다.After measuring the wall thickness of the large intestine and cutting in half, the ulcer and inflammatory gross lesions were scored according to Table 4 (Wallace criteria). The results are shown in FIG.
도 4에 나타나는 바와 같이, 나복자는 한, 두곳에서 염증성을 가진 궤양이 관찰되었고 장벽의 두께와 길이가 정상군의 대장과 거의 유사한 모양을 나타냄을 확인할 수 있었으며, 양성 대조군의 경우에는 궤양의 범위가 상대적으로 넓었다. IBD가 유발된 군의 경우에는 장벽이 너무 얇아져서 장이 파열되어 변들이 흘러나올 정도였으며, 색깔 역시 변하여 누런 색으로 변해 있었다.As shown in FIG. 4, the moths were found to have inflammatory ulcers in one or two places, and the thickness and length of the barrier showed almost the same shape as the colon of the normal group. It was relatively wide. In the IBD-induced group, the barrier was so thin that the intestines ruptured and stools flowed out, and the color changed to yellow.
<< 실시예Example 2> 2> TNBSTNBS 유발 궤양성 대장염 개선 평가 Induced Ulcerative Colitis Improvement Assessment
실험동물은 실시예 1과 동일한 조건의 실험동물을 사용하였다. 아세트산 대신 실제 인간 대장염과 가장 가까운 모델인 트리니트로벤젠 설포닉 산(trinitrobenzene sulfonic acid, TNBS) 유발 대장염 모델을 사용하여 나복자 추출물의 대장염 개선 효과를 평가하였다. 접촉성 감작 알레르겐인 TNBS를 에탄올과 함께 대장에 관장 투여하면 대장염을 유발할 수 있다. 이때 에탄올은 점막장벽(mucosal barrier)을 깨서 TNBS를 장벽으로 들어가게 하는 역할을 한다. TNBS가 장에 저촉되게 되면 산화적 손상에 의해 급성 전층성 괴사를 일으키게 되며, 관장 후 수일 내에 급성 괴사와 염증이 생기고 그 후로는 단핵구 침윤 등과 함께 만성 염증 소견을 보인다. 이러한 TNBS 유발 대장염 모델은 반복성이 있으며, 전벽성 궤양을 얻을 수 있고, 면역 기전을 통한 병변이므로 만성염증까지 유발할 수 있어 사람의 염증성 장질환과 매우 유사하다.Experimental animals were used as experimental animals in the same conditions as in Example 1. Trinitrobenzene sulfonic acid (TNBS) -induced colitis model, which is the closest model to the actual human colitis instead of acetic acid, was used to evaluate the colitis improvement effect of the moth extract. TNBS, a contact sensitizing allergen, with ethanol in the large intestine can lead to colitis. Ethanol breaks the mucosal barrier and serves to enter TNBS into the barrier. When TNBS enters the intestine, acute plexus necrosis is caused by oxidative damage. Acute necrosis and inflammation occur within a few days after enema, and chronic inflammation is observed after monocyte infiltration. This model of TNBS-induced colitis is repetitive, can be obtained ulcerative wall ulcer, and because it is a lesion through the immune mechanism can cause chronic inflammation is very similar to human inflammatory bowel disease.
TNBSTNBS 유도 궤양성 대장염 유발 Induced Ulcerative Colitis
유발 물질로는 TNBS 용액(5 w/v%, Sigma)을 사용하였다. 24시간 절식시킨 6주령의 수컷 Sprague-Dawley 쥐(rat)에 폴리에틸렌 튜브(직경 0.76mm)를 항문으로 밀어 넣은 후 TNBS 용액을 주입하여 대장염을 유발하였다. TNBS 용액을 한 마리당 0.6 ml의 용량으로 0.1 ml씩 천천히 점적 주사하여 주입하였으며, 주입 후 24시간 후부터 7일 동안 1일 1회씩 같은 시간에 시험 약물을 경구 투여하였다. 시험 약물은 하기 표 5와 같으며, 추출물은 나복자 약 1 kg 당 추출용매 약 3 리터를 이용하여 제조하였다.TNBS solution (5 w / v%, Sigma) was used as the trigger. Colitis was induced by injecting a polyethylene tube (0.76 mm in diameter) into the anus into a 6-week-old male Sprague-Dawley rat fasted for 24 hours. TNBS solution was injected by slow dropwise injection of 0.1 ml at a dose of 0.6 ml per animal, and the test drug was orally administered at the same time once daily for 24 days after the injection. The test drugs are shown in Table 5 below, and the extract was prepared using about 3 liters of the extraction solvent per about 1 kg of moths.
체중 변화 평가Weight change assessment
TNBS 5 w/v%로 대장염을 유발하기 직전과 매일 경구투여하기 직전 각 동물의 체중을 측정하여 각 동물의 체중감소량을 확인하였다. 체중감소량은 대장염 유발을 하기 직전의 체중에 대한 그날의 체중의 비율(퍼센트)로 나타내었다. 음성 대조군을 제외한 모든 군에서 투약 직후부터 체중은 꾸준히 증가하는 양상을 보였다. 그 결과를 표 6에 나타내었다.The weight loss of each animal was measured by measuring the weight of each animal immediately before inducing colitis and daily oral administration with TNBS 5 w / v%. Weight loss is expressed as the ratio of the weight of the day to the weight immediately before the cause of colitis. Body weight increased steadily immediately after administration in all groups except negative control group. The results are shown in Table 6.
설사발생률 평가Diarrhea Incidence Assessment
TNBS 5 w/v%로 대장염을 유발하고 난 24시간 후 매일 경구투여하기 직전 각 동물의 설사의 유무를 확인하였다. 나복자 물 추출물 군은 투약 후 2일째부터 정상변보다 약간 무른 변을 보기 시작하였고, 5일 후부터 정상변을 보았으며, 나복자 50% 주정 추출물의 경우에도 5일 후부터 정상변을 보았다. 양성 대조군 투여 후 4일째까지 피가 섞인 설사를 조금 했지만, 5일째부터는 상태가 나아졌다. 정상 대조군의 경우에는 정상 변의 형태를 나타내었다. TNBS 5 w / v% to determine whether or not diarrhea of each animal immediately before oral administration 24 hours after inducing colitis. The moth extract group began to see slightly softer stools than normal stools from 2 days after dosing. After 5 days, the moths extracts showed normal stools even after 50 days. A little bloody diarrhea was given until
대장(Leader( coloncolon )의 형태 평가) Form evaluation
대장의 벽두께를 측정하고 대장을 절개하여 궤양 및 염증상태의 육안 병변을 상기 표 4의 기준에 따라 점수화하였다. 정상군과 비교하여 보았을 때 대장의 형태는 나복자 추출물 투여 군에서 상대적으로 정상 형태를 띠고 있음을 관찰할 수 있었다. 양성 대조군은 대장의 형태가 정상군에 비하여 상대적으로 손상의 정도가 심하다는 것을 관찰할 수 있었다. 그 결과를 도 5에 나타내었다. 또한 대장의 벽 두께 정도를 측정하고 대장을 절개하여 반으로 자른 후, 궤양 및 염증상태의 육안 병변을 상기 표 4에 따라 점수화한 결과를 도 6에 나타내었다. 도 6에 나타나는 바와 같이, 나복자 물 추출물의 효과가 가장 뛰어났으며, 나복자 50% 주정 추출물의 효과는 양성 대조군과 거의 유사하였다.The wall thickness of the large intestine was measured, and the large intestine was dissected to score gross lesions of the ulcer and inflammatory state according to the criteria of Table 4 above. Compared with the normal group, the colon form was found to be relatively normal in the moth extract group. In the positive control group, the intestinal morphology was more severe than the normal group. The results are shown in Fig. In addition, after measuring the wall thickness of the large intestine and cut in half to the incision, the gross lesions of the ulcers and inflammatory state is shown in Table 6 the results of scoring. As shown in FIG. 6, the effect of the water extract of the moths was the best, and the effect of the 50% alcohol extract of the moths was almost similar to the positive control.
염색학적Dyeing 소견 평가 Opinion evaluation
대장을 적출하여 실체현미경으로 관찰한 결과 음성 대조군의 경우는 대장의 병변이 확인 가능하여 IBD가 유발됨을 확인할 수 있었으나, 시험군인 나복자 추출물 투여군의 경우는 병변의 정도가 완화되었음을 관찰할 수 있었다. 또한 H&E 염색 결과에서 관찰할 수 있는 것처럼 궤양성 대장염(IBD)이 유발된 음성 대조군은 점막하조직 상의 염증세포 침윤, 상피세포의 파괴, 점액세포의 파괴 등의 염증 소견이 관찰되었지만, 양성 대조군 및 나복자 추출물 투여군에서 점액세포, 선와 등이 정상적인 형태를 보여 치료 효과를 나타내는 것으로 사료되었다. 특히 나복자 물 추출물 투여군이 가장 우수한 효과를 나타내었으며, 양성 대조군인 메살라진 투여군의 경우에는 나복자 추출물 투여군에 비해 만족할 만한 결과를 보이지 못했는데, 이러한 결과는 메살라진의 용량 의존성에 기인한 것으로 생각된다. 그 결과를 도 7에 나타내었다. As a result of colon extraction and observation with a stereoscopic microscope, the negative control group was able to confirm the lesions of the colon and confirmed that IBD was induced. In addition, ulcerative colitis (IBD) -induced negative control showed inflammatory findings such as inflammatory cell infiltration, destruction of epithelial cells, and destruction of mucous cells as observed in H & E staining. In the extract-treated group, mucus cells, crypts, etc. showed normal morphology and showed therapeutic effect. In particular, the moth extract group showed the best effect, and the positive control group, the mesalazine group, showed no satisfactory results compared to the moth extract group, which is considered to be due to the dose dependency of mesalazine. The results are shown in Fig.
도 1은 본 발명에 따른 비교평가실험에 있어 마지막 약물투여 24시간 후에 각 군의 동물들을 경추 탈골시킨 후 부검을 실시하여 적출한 대장의 사진 결과이다.1 is a photographic result of a colon obtained by performing a necropsy after dissecting animals of each group 24 hours after the last drug administration in the comparative evaluation experiment according to the present invention.
도 2는 본 발명에 따른 비교평가실험에 있어 적출한 대장의 병변을 관찰하기 위한 현미경 사진 결과이다.Figure 2 is a micrograph result for observing the lesions of the colon extracted in the comparative evaluation experiment according to the present invention.
도 3은 본 발명에 따른 비교평가실험에 있어 적출한 대장의 H&E 염색 결과 사진이다.Figure 3 is a photograph of the result of H & E staining of the colon extracted in the comparative evaluation experiment according to the present invention.
도 4는 본 발명에 따른 비교평가실험에 있어 궤양 및 염증 상태의 육안병변을 Wallace의 기준에 따라 점수화한 결과이다(n=4).Figure 4 is a result of scoring the gross lesions of the ulcer and inflammatory state in accordance with the criteria of Wallace in the comparative evaluation experiment according to the present invention (n = 4).
도 5는 시험 물질들을 투여한 TNBS 유발 대장염 모델 동물들로부터 적출한 대장의 사진 결과이다. 5 is a photographic result of colon extracted from TNBS-induced colitis model animals administered test substances.
도 6은 시험 물질들을 투여한 TNBS 유발 대장염 모델 동물들로부터 적출한 대장의 궤양과 염증 상태의 육안 병변을 Wallace의 기준에 따라 점수화한 결과이다(n=4).FIG. 6 shows the results of scoring the ulceration of the large intestine ulcer and gross lesions of the inflammatory state from TNBS-induced colitis model animals administered with test substances according to Wallace's criteria (n = 4).
도 7은 시험 물질들을 투여한 TNBS 유발 대장염 모델 동물들로부터 적출한 대장의 H&E 염색 사진 결과이다.FIG. 7 shows H & E staining photographs of colons extracted from TNBS-induced colitis model animals administered test substances. FIG.
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