KR102612962B1 - Pharmaceutical composition for preventing and treating osteoarthritis - Google Patents
Pharmaceutical composition for preventing and treating osteoarthritis Download PDFInfo
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- KR102612962B1 KR102612962B1 KR1020210006178A KR20210006178A KR102612962B1 KR 102612962 B1 KR102612962 B1 KR 102612962B1 KR 1020210006178 A KR1020210006178 A KR 1020210006178A KR 20210006178 A KR20210006178 A KR 20210006178A KR 102612962 B1 KR102612962 B1 KR 102612962B1
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Abstract
본 발명은 약학조성물에 관한 것으로, 더 상세하게는 골관절염 예방 및 치료에 효능이 있는 약학조성물에 관한 것이다.
본 발명의 약학조성물은 꾸지뽕 열매 추출물, 우슬추출물 및 상황버섯추출물을 포함하는 복합추출물을 유효성분으로 한다.The present invention relates to a pharmaceutical composition, and more specifically, to a pharmaceutical composition effective in preventing and treating osteoarthritis.
The pharmaceutical composition of the present invention contains a complex extract containing Cudrania fruit extract, Hyssopia extract, and Sanghwang mushroom extract as an active ingredient.
Description
본 발명은 약학조성물에 관한 것으로, 더 상세하게는 골관절염 예방 및 치료에 효능이 있는 약학조성물에 관한 것이다.The present invention relates to a pharmaceutical composition, and more specifically, to a pharmaceutical composition effective in preventing and treating osteoarthritis.
골관절염(OA)은 65세 이상의 연령에서의 유병률이 남성에서 약 60%이고 여성에서 약 70%인 가장 일반적인 형태의 관절염이다. 골관절염을 위한 현재의 치료 선택지는 제한적이다. 이들은 단순 진통제, 비스테로이드성 항염증 약물(NSAID) 또는 관절내(IA) 주사 글루코코르티코이드 및 하이알루론산(HA) 제제에 의한 대증적 치료를 포함한다. 비약리학적 척도는 신체 운동 및 체중 손실부터 관절 세척액, 및 궁극적으로는 외과적 관절 대체에 이르기까지의 범위이다.Osteoarthritis (OA) is the most common form of arthritis with a prevalence of approximately 60% in men and approximately 70% in women over the age of 65 years. Current treatment options for osteoarthritis are limited. These include symptomatic treatment with simple analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) or intra-articular (IA) injected glucocorticoid and hyaluronic acid (HA) preparations. Non-pharmacological measures range from physical exercise and weight loss to joint irrigation fluids and ultimately surgical joint replacement.
골관절염 치료에 있어서는, 여전히 이용 불가능한 질병-조절성(disease-modifying) 골관절염 약물에 대한, 그리고 또한 장시간 지속되는 효과를 갖는 효능이 있는 통증 치료제에 대한 충족되지 않은 큰 요구가 있다. 더욱이, 연장된 지속기간의 치료를 종종 필요로 하는 골관절염 질병의 만성 성질로 인해, 어떠한 주요 부작용도 갖지 않는 골관절염 치료제에 대한 충족되지 않은 상당한 요구가 있다. 골관절염 통증의 완화를 위한 현재 이용 가능한 전신 약물, 예를 들어 비선택적 NSAID 및 선택적 사이클로옥시게나제 2(COX-2) 억제제는 OA의 초기 내지 중기 병기에서는 효과적이지만 관절이 악화됨에 따라 적절한 통증 완화를 제공하는 데에는 종종 실패한다. In the treatment of osteoarthritis, there is a great unmet need for disease-modifying osteoarthritis drugs that are still unavailable, and also for efficacious pain treatments with long-lasting effects. Moreover, due to the chronic nature of osteoarthritis disease, which often requires treatment of extended duration, there is a significant unmet need for treatments for osteoarthritis that do not have any major side effects. Currently available systemic medications for relief of osteoarthritis pain, such as non-selective NSAIDs and selective cyclooxygenase 2 (COX-2) inhibitors, are effective in early to intermediate stages of OA but do not provide adequate pain relief as the joint deteriorates. It often fails to provide
게다가, NSAID는 상당수의 환자에서 위장 합병증을 야기하고, COX-2 억제제는 최근에 심혈관 부작용/위험에 관한 우려를 증가시켜 왔으며, 그 결과 미국 시장으로부터 COX-2 억제제인 Vioxx 및 Bextra가 퇴출되게 되었다. 골관절염을 치료 또는 예방하기 위한 약물은 여전히 대다수의 환자에 의해 요구되고 있으며, 고령화 사회로 진입함에 따라, 더욱 요구도가 높아질 것으로 예상된다. 이에, 인체 안전성을 확보할 수 있는 천연원료 유래의 골관절염 예방 및 치료제에 대한 기술 개발이 요구되는 실정이다.In addition, NSAIDs cause gastrointestinal complications in a significant number of patients, and COX-2 inhibitors have recently raised concerns about cardiovascular side effects/risks, resulting in the withdrawal of the COX-2 inhibitors Vioxx and Bextra from the U.S. market. . Drugs to treat or prevent osteoarthritis are still required by the majority of patients, and as we enter an aging society, the need is expected to increase further. Accordingly, there is a need to develop technology for the prevention and treatment of osteoarthritis derived from natural ingredients that can ensure human safety.
본 발명의 목적은 천연재료 추출물을 포함하여 골관절염 예방 및 치료 효능이 있는 약학조성물 제공하는 것이다.The purpose of the present invention is to provide a pharmaceutical composition effective in preventing and treating osteoarthritis, including extracts of natural ingredients.
본 발명은 골관절염 예방 및 치료용 약학조성물로, 꾸지뽕 열매 추출물, 우슬추출물 및 상황버섯추출물을 포함하는 복합추출물을 유효성분으로 한다.The present invention is a pharmaceutical composition for preventing and treating osteoarthritis, and the active ingredient is a complex extract containing Cudrania fruit extract, Hyssop extract, and Sanghwang mushroom extract.
본 발명의 일 실시예에 따른 골관절염 예방 및 치료용 약학조성물에 있어서, 상기 상황버섯추출물은 꾸지뽕나무에 배양된 상황버섯을 추출한 것을 특징으로 한다.In the pharmaceutical composition for preventing and treating osteoarthritis according to an embodiment of the present invention, the Sanghwang mushroom extract is characterized by extracting Sanghwang mushrooms cultured on the Cudrania mulberry tree.
본 발명의 일 실시예에 따른 골관절염 예방 및 치료용 약학조성물에 있어서, 상기 복합추출물은 상기 꾸지뽕 열매추출물 100중량부에 대하여, 상기 우슬추출물 50 내지 120 중량부, 상기 상황버섯추출물 50 내지 100 중량부를 혼합하라 수 있다.In the pharmaceutical composition for preventing and treating osteoarthritis according to an embodiment of the present invention, the complex extract is 50 to 120 parts by weight of the Argentine root extract and 50 to 100 parts by weight of the Sanghwang mushroom extract, based on 100 parts by weight of the Cudrania fruit extract. You can mix it.
본 발명의 일 실시예에 따른 골관절염 예방 및 치료용 약학조성물에 있어서, 상기 복합추출물은 감압건조 후, 동결건조된 분말로 수득될 수 있다.In the pharmaceutical composition for preventing and treating osteoarthritis according to an embodiment of the present invention, the complex extract can be obtained as a freeze-dried powder after drying under reduced pressure.
본 발명의 일 실시예에 따른 골관절염 예방 및 치료용 약학조성물에 있어서, 상기 꾸지뽕 열매 추출물, 상기 우슬추출물 및 상황버섯추출물은 열수에 환류추출될 수 있다.In the pharmaceutical composition for preventing and treating osteoarthritis according to an embodiment of the present invention, the Cudrania fruit extract, the Hyssop extract, and the Phellodendron chinensis extract may be refluxed and extracted in hot water.
본 발명의 일 실시예에 따른 골관절염 예방 및 치료용 약학조성물에 있어서, 상기 복합추출물은 -70℃ 내지 -90℃에서 동결건조될 수 있다.In the pharmaceutical composition for preventing and treating osteoarthritis according to an embodiment of the present invention, the complex extract may be freeze-dried at -70°C to -90°C.
본 발명은 꾸지뽕 열매 추출물, 우슬추출물 및 상황버섯추출물을 포함하는 복합추출물을 유효성분으로 하며, 상기 상황버섯추출물은 꾸지뽕나무에 배양된 상황버섯을 추출한 것을 특징으로 하는 건강식품조성물이다.The present invention is a health food composition that uses a composite extract containing Cudrania fruit extract, Hyssop extract, and Sanghwang mushroom extract as an active ingredient, and the Sanghwang mushroom extract is a health food composition characterized by extracting Sanghwang mushrooms cultured from Cudrania mulberry trees.
본 발명에 따른 약학조성물은 골관절염 예방 및 치료에 효능이 있으며, 천연추출물을 포함하는 것으로, 인체 안정성이 확보되어 예방 또는 치료제로서 유용하게 사용될 수 있다.The pharmaceutical composition according to the present invention is effective in preventing and treating osteoarthritis, and since it contains natural extracts, its safety in the human body is ensured and it can be usefully used as a preventive or therapeutic agent.
도 1 내지 도 5는 본 발명의 약학조성물의 투여에 따른 세포활성 평가 결과,
도 6 내지 도 10은 본 발명의 약학조성물의 투여에 따른 in-vitro 대식세포 유전량 발현량 평가 결과,
도 11 내지 도 14는 본 발명은 본 발명의 약학조성물의 투여에 따른 in-vitro 단백질 발현량 평가 결과, 혈당 감소 효과 결과,
도 15는 본 발명의 약학조성물의 투여에 따른 체중부하 측정 결과,
도 16 내지 도 19은 본 발명의 약학조성물 투여에 따른 in-vivo 연골조직 유전량 발현량 평가 결과,
도 20 내지 도 25은 본 발명의 약학조성물 투여에 따른 in-vivo 혈청 내 바이오마커 생성량 평가 결과,
도 26은 본 발명의 약학조성물 투여에 따른 Mico-CT 결과,
도 27 내지 28은 본 발명의 약학조성물 투여에 따른 조직학적 검사 결과이다.
1 to 5 show cell activity evaluation results following administration of the pharmaceutical composition of the present invention,
Figures 6 to 10 show the results of in-vitro macrophage gene expression level evaluation according to administration of the pharmaceutical composition of the present invention;
11 to 14 show the results of in-vitro protein expression level evaluation according to administration of the pharmaceutical composition of the present invention, the results of blood sugar reduction effect,
Figure 15 shows the results of measuring weight bearing following administration of the pharmaceutical composition of the present invention;
Figures 16 to 19 show in-vivo cartilage tissue genetic expression level evaluation results according to administration of the pharmaceutical composition of the present invention;
Figures 20 to 25 show the results of evaluating the amount of biomarkers produced in in-vivo serum according to administration of the pharmaceutical composition of the present invention;
Figure 26 shows Mico-CT results according to administration of the pharmaceutical composition of the present invention;
Figures 27 and 28 show histological examination results following administration of the pharmaceutical composition of the present invention.
본 명세서에서 사용되는 기술 용어 및 과학 용어에 있어서 다른 정의가 없다면, 이 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 통상적으로 이해하고 있는 의미를 가지며, 하기의 설명 및 첨부 도면에서 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대한 설명은 생략한다. Unless otherwise defined, the technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art to which this invention pertains, and the gist of the present invention is summarized in the following description and accompanying drawings. Descriptions of known functions and configurations that may unnecessarily obscure are omitted.
또한, 본 명세서에서 사용되는 단수 형태는 문맥에서 특별한 지시가 없는 한 복수 형태도 포함하는 것으로 의도할 수 있다.Additionally, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly dictates otherwise.
또한, 본 명세서에서 특별한 언급 없이 사용된 단위는 중량을 기준으로 하며, 일 예로 % 또는 비의 단위는 중량% 또는 중량비를 의미하고, 중량%는 달리 정의되지 않는 한 전체 조성물 중 어느 하나의 성분이 조성물 내에서 차지하는 중량%를 의미한다.In addition, units used without special mention in this specification are based on weight, and as an example, the unit of % or ratio means weight % or weight ratio, and weight % refers to the amount of any one component of the entire composition unless otherwise defined. It refers to the weight percent occupied in the composition.
또한, 본 명세서에서 사용되는 수치 범위는 하한치와 상한치와 그 범위 내에서의 모든 값, 정의되는 범위의 형태와 폭에서 논리적으로 유도되는 증분, 이중 한정된 모든 값 및 서로 다른 형태로 한정된 수치 범위의 상한 및 하한의 모든 가능한 조합을 포함한다. 본 발명의 명세서에서 특별한 정의가 없는 한 실험 오차 또는 값의 반올림으로 인해 발생할 가능성이 있는 수치범위 외의 값 역시 정의된 수치범위에 포함된다. In addition, the numerical range used in this specification includes the lower limit and upper limit and all values within the range, increments logically derived from the shape and width of the defined range, all double-defined values, and the upper limit of the numerical range defined in different forms. and all possible combinations of the lower bounds. Unless otherwise specified in the specification of the present invention, values outside the numerical range that may occur due to experimental error or rounding of values are also included in the defined numerical range.
본 명세서의 용어, '포함한다'는 '구비한다', '함유한다', '가진다' 또는 '특징으로 한다' 등의 표현과 등가의 의미를 가지는 개방형 기재이며, 추가로 열거되어 있지 않은 요소, 재료 또는 공정을 배제하지 않는다. The term 'comprise' in this specification is an open description with the same meaning as expressions such as 'comprising', 'contains', 'has' or 'characterized by', and includes elements that are not additionally listed, Does not exclude materials or processes.
본 명세서의 용어. “골관절염”은 점진적인 관절 연골의 소실 및 그와 관련된 이차적인 증상을 동반하는 질환을 의미한다.Terms in this specification. “Osteoarthritis” refers to a disease accompanied by gradual loss of joint cartilage and associated secondary symptoms.
본 명세서의 용어, "예방"은 본 발명 조성물의 투여로 내분비계 질환의 발병을 억제 또는 지연시키는 모든 행위를 의미한다. As used herein, the term “prevention” refers to any action that inhibits or delays the onset of an endocrine disease by administering the composition of the present invention.
본 명세서의 용어, "치료"란 본 발명 조성물의 투여로 내분비계 질환의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any action that improves or beneficially changes the symptoms of an endocrine disease by administering the composition of the present invention.
본 명세서의 용어, "식품"이란 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 식품, 식품 첨가제, 기능성식품 및 음료를 모두 포함하는 의도이다.The term "food" in this specification refers to a natural product or processed product containing one or more nutrients, preferably in a state ready to be eaten directly after a certain degree of processing, and has the usual meaning. As such, it is intended to include all foods, food additives, functional foods, and beverages.
본 명세서의 용어, "기능성식품"이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 생체 조절 기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미하며, 특히 "건강기능식품"을 포함한다.As used herein, the term "functional food" refers to a food group or food composition that provides added value so that the food functions for a specific purpose using physical, biochemical, biotechnological methods, etc., to control the biological defense rhythm, It refers to foods that have been designed and processed to fully express the bioregulating functions related to disease prevention and recovery in the living body, and especially includes “health functional foods.”
본 명세서의 용어, "건강보조식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 가공한 식품으로서, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 건강보조의 목적으로 특정성분을 원료로 하거나 식품원료에 들어있는 성분을 추출, 농축, 정제, 혼합 등의 방법으로 제조, 가공한 식품을 말한다. 종래, 내분비계 질환은 수술로 치료가 어려움에 따라, 증세의 개선과 합병증의 예방이 치료에 있어서 중요하다. 치료방법으로 약물요법, 식이요법 및 운동 요법이 있으나, 흔히 사용되는 약물요법은 약물복용에 따른 부작용과 환자의 내성이 끊임없는 문제가 되고 있어, 천연물을 이용한 내분비계 치료제의 개발이 필요하다.As used herein, the term "health supplement" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body, and has useful health effects such as adjusting nutrients to the structure and function of the human body or physiological effects. It refers to foods manufactured and processed using specific ingredients as raw materials or by extracting, concentrating, refining, or mixing ingredients contained in food ingredients for the purpose of health supplementation. Conventionally, endocrine diseases are difficult to treat with surgery, so improving symptoms and preventing complications are important in treatment. Treatment methods include drug therapy, diet, and exercise therapy, but the side effects and patient resistance caused by commonly used drug therapy are constant problems, so the development of endocrine treatment using natural products is necessary.
본 발명은 꾸지뽕 열매 추출물, 우슬추출물 및 상황버섯추출물을 포함하는 복합추출물을 유효성분으로 하는 골관절염 예방 및 치료용 약학조성물로, 천연유래 재료로부터 추출된 추출물들을 함유함에 따라 안전성이 확보됨과 동시에, 골관절염 예방 및 치료 효능을 가질 수 있다.The present invention is a pharmaceutical composition for preventing and treating osteoarthritis, which contains a complex extract containing Cudrania fruit extract, Hyssop extract, and Sanghwang mushroom extract as an active ingredient. As it contains extracts extracted from natural materials, safety is ensured and osteoarthritis is prevented. It may have preventive and therapeutic effects.
구체적으로, 꾸지뽕나무(cudrania tricuspidata)는 구지뽕나무, 굿가시 나무, 활뽕 나무라 불리우며, 뽕과 나무에 속하는 낙엽 교목이다. 이러한 꾸지뽕나무의 뿌리껍질, 나무껍질 및 잎에는 인체에 유효한 다양한 성분이 포함되어 있어서 부위에 따라 혈압강하제, 결핵치료제, 해열제, 이뇨제, 지형제 등의 약제로 이용되었다.Specifically, Cudrania tricuspidata , also called Guji mulberry tree, Gut thorn tree, and live mulberry tree, is a deciduous tree belonging to the Mulberry family. The root bark, bark, and leaves of the Cudrania mulberry tree contain various ingredients effective for the human body, and have been used as medicines such as blood pressure lowering agents, tuberculosis treatments, antipyretics, diuretics, and antihypertensives depending on the area.
특히, 꾸지뽕나무 열매는 가을에 재배되는 것으로, 쓴맛과 단맛이 어우러진 맛을 낸다. 그 효능으로는 열을 내리고 혈분에서 남은 열사(熱邪)를 없애며 근육과 힘줄을 풀어주고 경락을 잘 통하게 함으로써, 타박상으로 상처가 나고 멍이 들었을 때에는 약으로서 복용할 수도 있으며, 꾸지뽕나무 열매를 술로 담가 양기 보충을 비롯하여스테미너 음식으로도 사용한다.In particular, the fruits of the Cudrania mulberry tree are grown in the fall and have a taste that combines bitterness and sweetness. Its efficacy is to reduce fever, remove residual heat from the blood, relax muscles and tendons, and improve meridian circulation. It can be taken as medicine for bruises and bruises, and the fruits of the Cudrania tree can be used as alcohol. It is soaked and used as a stamina food as well as to replenish energy.
본 발명의 꾸지뽕 열매 추출물은 꾸지뽕나무의 열매를 추출한 것으로, 꾸지뽕나무 열매의 일반성분은 수분 76.0~80.1%, 조단백질 2.2~3.5%, 조지방 1.7~2.9%, 조회분 0.8~1.2%, 탄수화물 14.5~16.4%를 포함하며, 무기성분으로는 Ca, Fe, K, Mg, Na, P 등이 포함되고, 불용성 식이섬유가 수용성 식이섬유 보다 월등히 많은 것으로 알려져 있다. The Cudrania fruit extract of the present invention is obtained from the fruit of the Cudrania Mulberry tree. The general components of the Cudrania Mulberry fruit are 76.0-80.1% moisture, 2.2-3.5% crude protein, 1.7-2.9% crude fat, 0.8-1.2% ash, and 14.5-14.5 carbohydrates. It contains 16.4%, and inorganic components include Ca, Fe, K, Mg, Na, P, etc., and insoluble dietary fiber is known to be much more abundant than soluble dietary fiber.
우슬(Achyranthes japonica)은 비름과에 속하는 다년생 식물로 한국, 중국, 일본 등에 분포한다. 높이는 50츠 내지 100cm 정도이고 가지가 많이 갈라지며, 잎은 마주난다. 꽃은 꽃받침이 5개, 수술이 5개이다. 식물 줄기에있는 마디의 형상이 소의 무릎과 유사하다고 하여 쇠무릎이라 호칭되기도 한다. 우슬은 쇠무릎의 뿌리에 해당하는 약재이다. 우슬은 한방 약용 재료이면서도 식품 원재료로 사용가능하며, 세포 독성 및 유전 독성이 없다고 보고되어 있고, 신장과 간장에 작용하여 어혈을 제거하는 효능이 있다고 알려져 있다. Achyranthes japonica is a perennial plant belonging to the Achyrantheceae family and is distributed in Korea, China, and Japan. The height is about 50 to 100 cm, the branches are many, and the leaves are opposite each other. The flower has 5 calyxes and 5 stamens. It is also called iron knee because the shape of the nodes on the plant stem is similar to that of a cow's knee. Useul is a medicinal herb that corresponds to the root of the iron knee. In addition to being an oriental medicinal ingredient, it can also be used as a food raw material. It is reported to have no cytotoxicity or genotoxicity, and is known to have the effect of removing stagnant blood by acting on the kidneys and liver.
본 발명의 우슬추출물은 우슬의 잎, 어린 새싹, 본체, 본체 껍질, 줄기, 줄기 껍질, 뿌리, 뿌리 껍질, 뿌리줄기, 종자, 열매, 미숙과, 완숙과, 과육, 과피, 꽃, 수술군(androecium), 암술군(gynoecium), 꽃받침, 수술, 꽃잎, 꽃받침 조각, 심피 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나로부터 수득될 수 있다. The Hyssop extract of the present invention includes the leaves, young shoots, body, body bark, stem, stem bark, root, root bark, rhizome, seed, fruit, immature fruit, ripe fruit, pulp, pericarp, flower, and stamen group ( It can be obtained from any one selected from the group consisting of androecium, gynoecium, sepals, stamens, petals, sepal pieces, carpels, and combinations thereof.
상황버섯은 담자균문(Basidiomycokina)의 민주름버섯 목(Aphyllophorales)의 소나무비늘 버섯과 (Hymenochaetaceae)의 진흙버섯 속에 속하는 버섯으로 뽕나무의 줄기에 자생하며, 표면은 흑갈색이지만 아랫면은 황색을 띠므로 뽕나무상(桑), 누르황(黃)자를 써서 상황(桑黃)이라고 부른다. 상황버섯은 박달나무, 자작나무, 물박달나무, 개회나무, 황철나무, 소나무, 참나무, 병꽃나무, 전나무, 분비나무, 거제수, 단풍나무, 뽕나무 등 다양한 나무에 배양된 것을 사용할 수 있으나, 본 발명은 꾸지뽕나무(cudrania tricuspidata)에 배양된 상황버섯을 사용하는 것을 특징으로 한다. 꾸지뽕나무에서 배양된 상황버섯을 추출한 상황버섯추출물을 포함하는 본 발명은 종래, 천연추출물 대비 현저히 우수한 골관절염 예방 및 치료효과를 가질 수 있다. Sanghwang mushroom is a mushroom belonging to the genus Hymenochaetaceae of the pine scale mushroom family ( Hymenochaetaceae ) of the Aphyllophorales order of the Basidiomycokina phylum. It grows naturally on the stems of mulberry trees. The surface is dark brown, but the underside is yellow, so it is similar to the mulberry tree. It is called Sanghwang (桑黃) using the characters (桑) and Nurhwang (黃). Sanghwang mushrooms can be cultured on various trees, such as birch, birch, water birch, ash tree, cypress tree, pine tree, oak tree, bottle flower tree, fir tree, fir tree, geoje tree, maple tree, mulberry tree, etc., but according to the present invention It is characterized by using Sanghwang mushrooms cultured on cudrania tricuspidata . The present invention, which includes Sanghwang mushroom extract obtained from Sanghwang mushrooms cultured from the Cudrania mulberry tree, can have significantly superior osteoarthritis prevention and treatment effects compared to conventional, natural extracts.
꾸지뽕나무에서 상황버섯을 배양하는 방법은 벌채된 꾸지뽕나무 원목을 살균하는 단계, 원목에 상황버섯 종균을 접종하고 균사 배양하는 단계, 균사 배양된 원목을 토양에 식재하고 광도, 온도 및 습도를 조절하여 재배 및 수확하는 단계를 포함할 수 있으나, 이에 한정되지 않는다. 바람직하게는 꾸지뽕나무 원목 살균하는 단계 이후, 원목에 상황버섯 종균을 접종하는 단계 이전에, 꾸지뽕나무 원목을 기능성액에 침지한 후 건조하는 단계를 더 포함할 수 있다. 상기 기능성액은 바람직하게 꾸지뽕나무 열매를 추출한 꾸지뽕 열매 추출물 및 우슬추출물을 포함하는 것으로, 꾸지뽕 열매 추출물 : 우슬추출물은 중량비 1: 1.5 내지 3비율로 혼합될 수 있다.The method of cultivating Sanghwang mushrooms from a Cudrania mulberry tree includes the steps of sterilizing felled Cudrania mulberry logs, inoculating the logs with Sanghwang mushroom spawn and cultivating mycelia, planting the mycelium-cultured logs in soil, and controlling the light intensity, temperature, and humidity. It may include, but is not limited to, cultivation and harvesting steps. Preferably, after the step of sterilizing the Cudrania tree logs and before the step of inoculating the logs with Sanghwang mushroom spawn, the step of immersing the Cudrania logs in a functional liquid and then drying them may be further included. The functional liquid preferably contains Cudrania fruit extract and Columbus root extract obtained from Cudrania mulberry fruit, and Cudrania fruit extract: Columnarium extract can be mixed at a weight ratio of 1: 1.5 to 3.
꾸지뽕 열매 추출물, 우슬추출물, 상황버섯추출물은 각각의 원료로부터 분리된 활성성분 즉, 목적하는 활성을 보이는 물질의 혼합물을 의미할 수 있다. 추출물은 일 양태에 있어서, 물, 유기용매 또는 이들의 혼합용매의 추출액일 수 있으며 이와 달리 이의 건조 분말 또는 이를 이용하여 제형화된 모든 형태를 포함할 수 있다. 다른 일 양태로, 추출물은 추출과정을 거친 추출액을 분획한 것도 포함될 수 있다. 추출물은 예를 들면, 초음파 추출법, 환류 냉각 추출법, 열수 추출법 또는 상기 유기용매를 추출용매로 하여 공지의 방법에 의해 추출된 추출물일 수 있다. Cudrania fruit extract, Hyssop extract, and Sanghwang mushroom extract may refer to active ingredients separated from each raw material, that is, a mixture of substances showing the desired activity. In one embodiment, the extract may be an extract of water, an organic solvent, or a mixed solvent thereof, or alternatively, it may include a dry powder thereof or any form formulated using the same. In another aspect, the extract may also include fractionated extract liquid that has undergone an extraction process. The extract may be, for example, an extract extracted by an ultrasonic extraction method, a reflux cooling extraction method, a hot water extraction method, or a known method using the organic solvent as an extraction solvent.
바람직하게, 각 추출물은 열수에 환류추출된 열수추출물일 수 있다. 열수추출물은 원료내 영양성분을 다량 함유할 수 있어 골관절염 치료 및 예방제 사용 시 더욱 효능을 가질 수 있다. 일 예로, 각 원료(꾸지뽕 열매, 우슬, 상황버섯)를 100중량부를 1000 내지 2000중량부의 열수에 넣고 2 내지 3시간 동안 환류추출 할 수 있다. 열수는 40 내지 70℃, 바람직하게는 50 내지 60℃의 온도일 수 있다. 상기 범위보다 열수가 낮은 온도일 경우 원료의 추출이 거의 이루어지지 않으며, 상기 범위보다 열수가 높은 온도일 경우, 원료의 탄화가 일어나 매우 혼탁한 복합추출물을 수득하게 된다.Preferably, each extract may be a hot water extract refluxed in hot water. Hot water extracts can contain a large amount of nutrients in the raw materials, so they can be more effective when used as a treatment and preventive agent for osteoarthritis. As an example, 100 parts by weight of each raw material (cultivating mulberry fruit, lysul, and Sanghwang mushrooms) can be placed in 1000 to 2000 parts by weight of hot water and refluxed for 2 to 3 hours. The hot water may have a temperature of 40 to 70°C, preferably 50 to 60°C. If the temperature of the hot water is lower than the above range, extraction of the raw material is almost impossible, and if the temperature of the hot water is higher than the above range, carbonization of the raw material occurs, resulting in a very turbid complex extract.
복합추출물은 상술한, 꾸지뽕 열매 추출물, 우슬추출물 및 상황버섯추출물을 포함하는 것으로, 골관절염 치료 및 예방 효능을 가질 수 있다.The complex extract includes the above-described Cudrania fruit extract, Hyssopia extract, and Sanghwang mushroom extract, and may have efficacy in treating and preventing osteoarthritis.
구체적으로, 복합추출물은 상기 꾸지뽕 열매추출물 100중량부에 대하여, 상기 우슬추출물 50 내지 120 중량부, 상기 상황버섯추출물 50 내지 100 중량부가 혼합된 것일 수 있다. 상기와 같은 범위에서 재료 투입량 대비 실질적인 골관절염 예방 및 치료 효과에 중요한 의미를 가지는 다량의 유효성분을 포함하여 경제성을 지닐 수 있다.Specifically, the complex extract may be a mixture of 50 to 120 parts by weight of the Argentine root extract and 50 to 100 parts by weight of the Sanghwang mushroom extract, based on 100 parts by weight of the Cudrania mulberry fruit extract. Within the above range, it can be economically feasible by including a large amount of active ingredients that are important for the actual prevention and treatment of osteoarthritis compared to the amount of material input.
본 발명의 복합추출물은 rotary vacuum evaporator를 통해 감압건조 된 후, -70℃ 내지 -90℃, 구체적으로 -75℃ 내지 -85℃의 초저온에서 동결 건조 및 보관될 수 있다. 동결건조의 압력은 3 내지 80torr, 바람직하게는 40 내지 60torr 일 수 있으며, 시간은 5 내지 48시간, 바람직하게는 20 내지 30시간 일 수 있다. 상기한 범위에서 건조가 원활하게 진행될 수 있다.The complex extract of the present invention can be dried under reduced pressure using a rotary vacuum evaporator, then freeze-dried and stored at ultra-low temperatures of -70°C to -90°C, specifically -75°C to -85°C. The pressure of freeze-drying may be 3 to 80 torr, preferably 40 to 60 torr, and the time may be 5 to 48 hours, preferably 20 to 30 hours. Drying can proceed smoothly within the above range.
이상에서 설명한 본 발명의 골관절염 예방 및 치료용 약학조성물은 천연 성분으로서 안전성이 확보되어 골관절염 예방 및 치료에 유용하게 사용될 수 있다.The pharmaceutical composition for preventing and treating osteoarthritis of the present invention described above has guaranteed safety as a natural ingredient and can be usefully used for preventing and treating osteoarthritis.
본 발명의 복합추출물은 실제 임상투여 시에 경구 및 비경구의 여러가지 제형으로 될 수 있는데 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며 이러한 고형제제는 위의 생약재 추출물에 하나 이상의 부형제, 예를 들면 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(sucrose) 또는 락토오스(loctose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스트레이트, 탈크 같은 윤활제들을 더 포함할 수 있으며, 경구를 위한 액상 제제로는 현탁액, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.The complex extract of the present invention can be formulated into various oral and parenteral formulations during actual clinical administration. When formulated, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. It can be. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain the above herbal extracts with one or more excipients, such as starch, calcium carbonate, and sucrose. ) or it can be prepared by mixing lactose, gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium straight and talc may be included. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients are included. , for example, humectants, sweeteners, fragrances, preservatives, etc. may be included.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성 용제, 현탁용제로는 프로필렌 글리콜(propylene glycol), 폴리에틸렌글리콜, 올리브오일과 같은 식물성기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있으나 이에 제한되는 것은 아니다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, and suspensions such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. It is not limited.
본 발명에 따른 조성물은 본 발명이 속하는 기술 분야에서 공지된 통상의 방법에 따라 정제, 캡슐, 과립제, 환제, 현탁액, 시럽 등의 다양한 제형으로 제형화되어 사용될 수 있다.The composition according to the present invention can be formulated and used in various dosage forms such as tablets, capsules, granules, pills, suspensions, syrups, etc. according to conventional methods known in the technical field to which the present invention pertains.
본 발명의 복합추출물은 우수한 골관절염 치료 또는 예방 효과를 제공할 뿐 만아니라, 약물에 의한 독성 및 부작용도 없어 장기간 복용시에도 안심하고 사용할 수 있다.The complex extract of the present invention not only provides excellent osteoarthritis treatment or prevention effects, but also has no drug-related toxicity or side effects, so it can be used safely even when taken for a long period of time.
이에 따라, 복합추출물은 식품의 주, 부원료 및 식품 첨가제로서 사용이 가능하며, 본 발명은 복합추출물을 포함하는 건강식품조성물로 제공될 수 있다. 건강식품 조성물은 건강보조식품, 기능성 식품, 식품첨가제 등에 포함되는 것으로. 상기 건강보조식품, 기능성식품, 식품 첨가제는 간기능 개선 또는 내분비계 질환 예방용 작용을 가진다. 기능성식품 및 건강보조식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 더욱 포함할 수 있으며, 기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다. Accordingly, the complex extract can be used as a main, secondary ingredient and food additive for food, and the present invention can be provided as a health food composition containing the complex extract. Health food compositions include health supplements, functional foods, food additives, etc. The health supplements, functional foods, and food additives have the effect of improving liver function or preventing endocrine diseases. Functional foods and health supplements may further include food additives that are foodologically acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the production of functional foods.
복합추출물을 첨가할 수 있는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 기능성 식품 등이 있다. 추가로, 본 발명에서 식품에는 특수영양식품(예, 조제유류, 영,유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류 (예, 라면류, 국수류 등), 건강보조식품, 조미식품(예, 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예, 스넥류), 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 김치, 절임식품(각종 김치류, 장아찌 등), 음료(예, 과실,채소류 음료, 두유류, 발효음료류 등), 천연조미료(예, 라면 스프 등)을 포함하나 이에 한정되지 않는다. 식품, 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다.Foods to which complex extracts can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. Additionally, in the present invention, foods include special nutritional foods (e.g., infant formula, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplements, and seasoned foods ( (e.g., soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), beverages (e.g. Examples include, but are not limited to, fruit and vegetable beverages, soy milk, fermented beverages, etc.) and natural seasonings (e.g., ramen soup, etc.). Food, beverages, or food additives can be manufactured by conventional manufacturing methods.
본 발명에서 기능성 음료란 갈증을 해소하거나 맛을 즐기기 위하여 마시는 것의 총칭을 의미하며 기능성 음료를 포함하는 의도이다. 음료는 지시된 비율로 필수 성분으로서 복합추출물을 유효성분으로 포함하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 천연 탄수화물의 예는 모노사카라이드, 예를 들어 포도당, 과당 등 디사카라이드, 예를 들어 말토스, 수크로스 등 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 상기한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일 주스, 과일 쥬스 음료, 야채 음료의 제조를 위한 과육을 추가로 함유할 수 있다.In the present invention, functional beverage refers to a general term for drinking to quench thirst or enjoy taste, and is intended to include functional beverages. Aside from containing the complex extract as an essential ingredient in the indicated ratio, the beverage has no particular restrictions on other ingredients, and like regular beverages, it may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of natural carbohydrates include monosaccharides, such as glucose, fructose, etc., disaccharides, such as maltose, sucrose, etc., and polysaccharides, such as common sugars such as dextrin, cyclodextrin, etc., and xylitol, Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. In addition, the composition of the present invention may additionally contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.
본 발명의 발효물은 자연식품으로서 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.The fermented product of the present invention is a natural food and has almost no toxicity or side effects, so it can be safely used even when taken for a long period of time for preventive purposes.
이하, 실시예 및 비교예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당해 기술 분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다.Hereinafter, the present invention will be described in more detail through examples and comparative examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.
(실시예 1 내지 4)(Examples 1 to 4)
꾸지뽕나무 열매(홍익약초영농법인) 50g에 증류수 500㎖ 넣고 3시간 동안 환류추출하고 필터로 여과하였다.500 ml of distilled water was added to 50 g of Cudrania mulberry fruit (Hongik Medicinal Herb Farming Corporation), and the extract was refluxed for 3 hours and filtered.
그리고, 우슬(대전한약국)의 어린 잎 30g에 증류수 500㎖ 넣고 3시간 동안 환류추출하고 필터로 여과하였다.Then, 500 ml of distilled water was added to 30 g of young leaves of Woosul (Daejeon Oriental Pharmacy), extracted under reflux for 3 hours, and filtered.
또한, 꾸지뽕나무에서 배양한 상황버섯 자실체 50g에 증류수 500㎖ 넣고 3시간 동안 환류추출하고 필터로 여과하였다.In addition, 500 ml of distilled water was added to 50 g of Sanghwang mushroom fruiting bodies cultured from the Cudrania mulberry tree, extracted under reflux for 3 hours, and filtered.
각추출물을 freeze dryer로 -80℃에서 동결 건조하여 분발상의 복합추출물을 수득하였다. 복합추출물은 초저온 냉동고 (-80℃)에서 보관하였으며, 증류수에 희석해 각각 50㎍/㎖, 100㎍/㎖, 200㎍/㎖ , 400㎍/㎖ 의 농도로 제조하였다. 이하, 도면에서 가각 LPA 50, 100, 200, 400으로 표기된다.Each extract was freeze-dried at -80°C using a freeze dryer to obtain a powdered composite extract. The complex extract was stored in an ultra-low temperature freezer (-80°C) and diluted in distilled water to prepare concentrations of 50㎍/㎖, 100㎍/㎖, 200㎍/㎖, and 400㎍/㎖, respectively. Hereinafter, in the drawings, they are indicated as LPA 50, 100, 200, and 400, respectively.
(비교예 1)(Comparative Example 1)
실시예 4에서, 상황버섯을 추출한 상황버섯추출물을 투입하지 않은 것을 제외하고, 실시예 4와 동일하게 제조되었다.In Example 4, it was prepared in the same manner as Example 4, except that the Sanghwang mushroom extract obtained from the Sanghwang mushroom was not added.
(비교예 2)(Comparative Example 2)
실시예 4에서, 꾸지뽕나무가 아닌 참나무(Quercus acutissima)에 배양된 상황버섯을 추출한 상황버섯추출물을 사용한 것을 제외하고, 실시예 4와 동일하게 제조되었다.In Example 4, Quercus, not Quercus acutissima ) was prepared in the same manner as Example 4, except that Sanghwang mushroom extract obtained from cultured Sanghwang mushrooms was used.
(비교예 3)(Comparative Example 3)
실시예 4에서, 꾸지뽕나무가 아닌 산뽕나무(Morus bombycis)에 배양된 상황버섯을 추출한 상황버섯추출물을 사용한 것을 제외하고, 실시예 4와 동일하게 제조되었다.In Example 4, it was prepared in the same manner as Example 4, except that Sanghwang mushroom extract extracted from Sanghwang mushrooms cultured on Morus bombycis rather than Cudrania mulberry tree was used.
(골관절염 예방 및 치료능 평가 실험.)(Osteoarthritis prevention and treatment evaluation experiment.)
1.실험준비1. Experiment preparation
1)조골세포 배양1) Osteoblast cell culture
인간 유래 조골세포인 MG-63 세포는 10% fetal bovine serum와 1% penicillin-streptomycin으로 조성된 RPMI-1640 배지를 사용하여 37℃, 5% CO2 조건이 유지되는 세포배양기에서 배양하였다. 2-3일 주기로 계대 배양하고, 분화 유도를 위해 10 mM β-glycerolphosphate 와 50 ㎎/㎖의 ascorbic acid를 첨가하여 분화 유도 배지로 사용하였으며, 3일마다 배지를 교환하였다.MG-63 cells, which are human-derived osteoblasts, were cultured in a cell culture medium maintained at 37°C and 5% CO2 using RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Subculture was carried out in cycles of 2-3 days, and to induce differentiation, 10 mM β-glycerolphosphate and 50 mg/ml ascorbic acid were added and used as a differentiation induction medium, and the medium was changed every 3 days.
2)파골세포 배양2)Osteoclast culture
인간 유래 조골세포인 MG-63 세포는 10% fetal bovine serum와 1% penicillin-streptomycin으로 조성된 RPMI-1640 배지를 사용하여 37℃, 5% CO2 조건이 유지되는 세포배양기에서 배양하였다. 2-3일 주기로 계대 배양하고, 분화 유도를 위해 10 mM β-glycerolphosphate 와 50 ㎎/㎖의 ascorbic acid를 첨가하여 분화 유도 배지로 사용하였으며, 3일마다 배지를 교환하였다.MG-63 cells, which are human-derived osteoblasts, were cultured in a cell culture medium maintained at 37°C and 5% CO2 using RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Subculture was carried out in cycles of 2-3 days, and to induce differentiation, 10 mM β-glycerolphosphate and 50 mg/ml ascorbic acid were added and used as a differentiation induction medium, and the medium was changed every 3 days.
3)대식세포 배양3) Macrophage culture
마우스 대식세포인 RAW264.7 세포는 10% fetal bovine serum와 1% penicillin-streptomycin으로 조성된 DMEM 배지를 사용하여 37℃, 5% CO2 조건이 유지되는 세포배양기에서 배양하였으며, 2-3일 주기로 계대 배양하여 실험을 진행하였다.RAW264.7 cells, mouse macrophages, were cultured in a cell incubator maintained at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and passaged every 2-3 days. The experiment was conducted by culturing.
4)동물 및 사료4) Animals and feed
본 실험을 위하여 사용된 SD-Rat (4주령, 수컷, 170∼200g)은 라온바이오 (Korea)에서 구입하여 사용하였다. 실험동물은 1주간의 안정기를 가지면서 순화를 시켰으며, 안정기 및 실험기간에 모든 실험군에는 일반 사료 (ENVIGO, U.S.A.)를 자유식이 하며 물을 충분히 공급하였다. 1주간의 안정기 이후 5주령부터 동물 실험을 진행하였다. 동물 사육실의 조건은 conventional system으로 22±2℃, 1일 중 12시간은 200-300 Lux로 조명하고, 12시간은 모든 빛을 차단하였다. 본 실험은 대전대 동물실험윤리 위원회의 승인 (승인번호 DJUARB2020-024)을 받아 동물윤리준칙에 의거하여 실험하였다.SD-Rat (4 weeks old, male, 170-200 g) used for this experiment was purchased from Raon Bio (Korea). The experimental animals were acclimatized during a one-week stability period. During the stability period and the experimental period, all experimental groups were fed regular feed (ENVIGO, U.S.A.) ad libitum and supplied with sufficient water. After a one-week stabilization period, animal experiments were conducted starting at 5 weeks of age. The conditions of the animal breeding room were a conventional system of 22±2℃, lighting at 200-300 Lux for 12 hours a day, and blocking all light for 12 hours. This experiment was approved by the Animal Experiment Ethics Committee of Daejeon University (approval number DJUARB2020-024) and conducted in accordance with animal ethics standards.
5)관절염 유발 및 시료처리5) Arthritis induction and sample processing
안정기를 통한 순화 후 5주령이 된 SD-Rat을 실험에 사용하였다. 실험 그룹은 관절염 유발과 경구 투여를 진행 하지 않는 정상군, 관절염 유발 후 증류수만을 경구 투여하는 대조군, indomethacin 2 ㎎/㎏/day의 농도로 투여하는 실험군, LPA를 200, 400 ㎎/㎏/day 의 농도로 경구 투여하는 실험군 등 총 5개의 그룹으로 나누었다. 5주령이 된 SD-Rat을 에테르로 마취하여 우측 무릎을 제모 후, 우측 무릎 관절낭에 0.9 % saline에 용해시킨 MIA (60 ㎎/㎖)를 당뇨주사기 (BD insulin syringe)로 50 ㎕ 주입함으로써 관절염을 유발하였다. 관절염 유발 후 유발되지 않은 동물과 그룹 당 10마리를 초과한 경우는 동물윤리준칙에 맞추어 안락사를 진행하였으며, 최종적으로 그룹 당 10마리로 그룹간의 동물 수를 맞추고 MIA로 관절염을 유발하는 대조군과 실험군은 매일 1회 2 ㎖씩 oral zonde를 이용하여 2주간 각각의 시료를 경구 투여하였다.After acclimatization through the stabilization period, 5-week-old SD-Rats were used in the experiment. The experimental groups were a normal group that did not induce arthritis or undergo oral administration, a control group that administered only distilled water orally after inducing arthritis, an experimental group that administered indomethacin at a concentration of 2 mg/kg/day, and a group that administered LPA at a concentration of 200 and 400 mg/kg/day. It was divided into a total of 5 groups, including an experimental group administered orally at different concentrations. After anesthetizing a 5-week-old SD-Rat with ether and removing hair from its right knee, arthritis was treated by injecting 50 ㎕ of MIA (60 mg/ml) dissolved in 0.9% saline into the right knee joint capsule using a diabetic syringe (BD insulin syringe). It was caused. Animals that were not induced after arthritis induction and those that exceeded 10 animals per group were euthanized in accordance with animal ethics rules. Finally, the number of animals in each group was adjusted to 10 animals per group, and the control and experimental groups that induced arthritis by MIA were Each sample was administered orally at a dose of 2 ㎖ once a day for 2 weeks using an oral zonde.
6)혈액 채취 및 혈청 보관6)Blood collection and serum storage
실험종료 후 심장천자법으로 채혈한 후 혈액을 30분간 상온에서 굳혀 3,000 rpm에서 15분간 원심 분리하여 혈청을 분리한 후 혈청 바이오마커 측정에 필요한 용량만큼 1.5 ㎖ tube에 넣어 초저온 냉동고 (-80℃)에 보관하였다.After completing the experiment, blood was collected by cardiac puncture, solidified at room temperature for 30 minutes, centrifuged at 3,000 rpm for 15 minutes to separate serum, and placed in a 1.5 ml tube in the amount required for serum biomarker measurement in an ultra-low temperature freezer (-80°C). It was stored in .
7)유전자 발현량 측정7) Measurement of gene expression level
(1)RNA 추출(1) RNA extraction
세포와 관절조직에 easy blue 1 ㎖와 chloroform 200 ㎕를 넣고 vortexing 해준 후, 13000 rpm, 4℃에서 10분 동안 원심분리 하였다. 상층액 400 ㎕와 binding buffer 400 ㎕를 실온에서 1분 동안 반응시킨 뒤 반응액 700 ㎕를 column에 주입하여 13000 rpm에서 30초 동안 원심분리하였다. Column에 washing buffer A를 700 ㎕ 넣고 13000 rpm에서 30초 동안 원심분리 후, washing buffer B를 700 ㎕ 넣고 동일하게 원심분리 하였다. Column 하단을 1.5 ㎖ tube로 교체한 후, column에 elution buffer를 30 ㎕ 넣고 1분 동안 반응시킨 뒤 13000 rpm에서 1분 동안 원심분리하여 total RNA를 추출하였다.1 ml of easy blue and 200 ㎕ of chloroform were added to the cells and joint tissues, vortexed, and centrifuged at 13000 rpm at 4°C for 10 minutes. 400 ㎕ of the supernatant and 400 ㎕ of binding buffer were reacted at room temperature for 1 minute, then 700 ㎕ of the reaction solution was injected into the column and centrifuged at 13000 rpm for 30 seconds. 700 ㎕ of washing buffer A was added to the column and centrifuged at 13000 rpm for 30 seconds, then 700 ㎕ of washing buffer B was added and centrifuged in the same manner. After replacing the bottom of the column with a 1.5 ml tube, 30 ㎕ of elution buffer was added to the column, reacted for 1 minute, and then centrifuged at 13000 rpm for 1 minute to extract total RNA.
(2)cDNA 합성(2)cDNA synthesis
역전사 (reverse transcription) 반응은 RT premix kit의 mixture (reaction buffer, dNTPs mixture, RNase inhibitor, stabilizer, oligo dT15 primer) total RNA를 1 ㎍ 넣고 DEPC-DW을 최종 부피가 20 ㎕가 되도록 첨가하였다. 이 혼합액을 잘 섞은 후, 45℃에서 60분 반응시켜 first-strand cDNA를 합성하고, 95℃에서 5분 동안 방치하여 M-MLV RT를 불활성화 시켜 합성이 완료된 cDNA를 polymerase chain reaction (PCR)에 사용하였다.For the reverse transcription reaction, 1 μg of total RNA of the RT premix kit mixture (reaction buffer, dNTPs mixture, RNase inhibitor, stabilizer, oligo dT15 primer) was added, and DEPC-DW was added to a final volume of 20 μl. After mixing this mixture well, react at 45°C for 60 minutes to synthesize first-strand cDNA, leave at 95°C for 5 minutes to inactivate M-MLV RT, and perform polymerase chain reaction (PCR) on the completed cDNA. used.
(3)유전자 증폭(3) Gene amplification
합성이 완료된 cDNA를 증폭시키기 위하여 real-time PCR을 진행하였으며, real-time 전용 tube에 cDNA 1 ㎕, 각 primer 2 ㎕, SYBR Green 10 ㎕, DEPC-DW 5 ㎕씩 넣어 95℃에서 2분 동안 반응시키고 다음 95℃에서 5초, 62.5℃에서 30초를 40회 반복하여 유전자를 증폭시켰다. 유전자 발현량은 대조군에 비하여 계산하였으며, 사용된 primer의 sequence는 표 1(table 1)과 같다.Real-time PCR was performed to amplify the cDNA whose synthesis was completed. 1 ㎕ of cDNA, 2 ㎕ of each primer, 10 ㎕ of SYBR Green, and 5 ㎕ of DEPC-DW were added to a real-time dedicated tube and reacted at 95°C for 2 minutes. Then, the gene was amplified by repeating 5 seconds at 95°C and 30 seconds at 62.5°C 40 times. The gene expression level was calculated compared to the control group, and the sequences of the primers used are shown in Table 1.
2.통계처리2.Statistical processing
실험 결과는 SPSS 21.0를 이용하여 mean±standard error of mean으로 나타내었으며, ANOVA를 사용하여 다중 비교하였고 Duncan test를 통해 p<0.05, p<0.01 및 p<0.001 수준에서 유의성을 검정하였다.The experimental results were expressed as mean ± standard error of mean using SPSS 21.0, multiple comparisons were made using ANOVA, and significance was tested at the p < 0.05, p < 0.01, and p < 0.001 levels through the Duncan test.
(실험예 1) in-vitro 세포활성 분석(Experimental Example 1) In-vitro cell activity analysis
1) 세포생존률 1) Cell survival rate
세포를 24 well plate에 4×104 cells/well로 분주하여 24시간 동안 배양하였다. 24시간 후, LPA을 50, 100, 200, 400 ㎍/㎖의 농도로 처리하여 다시 24시간 동안 배양 후 세포배양액 100 ㎕당 10 ㎕의 EZ-Cytox 용액을 첨가하여 세포배양기에서 30분간 반응시켰다. 반응 후 450 ㎚에서 흡광도의 변화를 측정하여 대조군에 대한 세포생존율을 백분율로 표시하였다.Cells were distributed at 4 × 104 cells/well in a 24 well plate and cultured for 24 hours. After 24 hours, the cells were treated with LPA at concentrations of 50, 100, 200, and 400 ㎍/㎖ and cultured for another 24 hours. Then, 10 ㎕ of EZ-Cytox solution per 100 ㎕ of cell culture was added and incubated for 30 minutes in a cell incubator. After reaction, the change in absorbance was measured at 450 nm, and the cell viability relative to the control group was expressed as a percentage.
하기 도 1 내지 도 3에는 각각 조골세포, 파골세포, 대식세포의 생존율 결과가 도시되어 있다. 도 1을 참조하면, 조골세포의 생존율을 측정한 결과, LPA는 400 ㎍/㎖ 이상의 농도에서 90% 이하의 세포생존율이 나타났다. 도 2를 참조하면, 파골세포의 생존율을 측정한 결과, LPA는 400 ㎍/㎖ 이상의 농도에서 90% 이하의 세포생존율이 나타났다. 도 3을 참조하면, 대식세포의 생존율을 측정한 결과, LPA는 400 ㎍/㎖ 이상의 농도에서 90% 이하의 세포생존율이 나타났다. Figures 1 to 3 below show the survival rate results of osteoblasts, osteoclasts, and macrophages, respectively. Referring to Figure 1, as a result of measuring the survival rate of osteoblasts, LPA showed a cell survival rate of less than 90% at a concentration of 400 μg/ml or more. Referring to Figure 2, as a result of measuring the survival rate of osteoclasts, LPA showed a cell survival rate of less than 90% at a concentration of 400 μg/ml or more. Referring to Figure 3, as a result of measuring the survival rate of macrophages, LPA showed a cell survival rate of less than 90% at a concentration of 400 μg/ml or more.
2)ALP활성2)ALP activity
MG-63 세포를 96 well plate에 5×103 cells/well로 분주하여 24시간 동안 배양하였다. 24시간 후 유도배지로 교환하고 LPA을 50, 100, 200 ㎍/㎖의 농도로 처리하여 4일 동안 배양한 뒤 배양액을 제거하고 증류수로 세척하였다. 이후 0.1 % Triton X-100을 20 ㎕씩 첨가하여 37 ℃에서 30분간 용해시킨후, 세포 상층액에 0.1 N glycine과 100 mM의 p-NPP를 첨가한 후 37 ℃에서 30분간 반응 시켰다. 반응 후 0.1 N NaOH을 넣어 반응을 정지시킨 다음, 405 ㎚에서 흡광도를 측정하였다.MG-63 cells were distributed at 5 × 103 cells/well in a 96 well plate and cultured for 24 hours. After 24 hours, the induction medium was replaced, treated with LPA at concentrations of 50, 100, and 200 μg/ml, and cultured for 4 days. The culture medium was removed and washed with distilled water. Afterwards, 20 ㎕ of 0.1% Triton After the reaction, 0.1 N NaOH was added to stop the reaction, and then the absorbance was measured at 405 nm.
도 4에는 ALP활성 측정 결과가 도시되어 있다. 도 4를 참조하면, LPA 처리군은 모두 대조군에 비해 증가하였다.Figure 4 shows the results of ALP activity measurement. Referring to Figure 4, all LPA-treated groups increased compared to the control group.
3) TRAP 활성3) TRAP activity
RAW264.7 세포를 96 well plate에 5×103 cells/well로 분주하여 24시간 동안 배양하였다. 배지를 제거 후, DMEM 배지에 분화 인자인 RANKL 50 ng/㎖, 10 uM PD98059을 넣고 LPA 50, 100, 200 ㎍/㎖의 농도별로 처리하여 4일간 배양하였다. 이후 배지를 제거 하고 PBS로 세척한 후 고정용액 (citrate 용액 + acetone + formaldehyde)로 세포를 고정하였고 기질 용액으로는 1.36 ㎎/㎖ 4-nitrophenyl phosphate disodium salt, 10 mM tartrate를 포함하는 50 mM citrate buffer (pH 4.6)를 제조하여 고정한 세포에 분주하였다. 37 ℃에서 30분간 반응 후 효소 반응액을 0.1 N NaOH로 반응을 정지시킨 다음, 405 ㎚에서 흡광도를 측정하였다. TRAP 활성은 시료의 흡광도를 대조군에 대한 백분율로 표시하였다.RAW264.7 cells were distributed at 5 × 103 cells/well in a 96 well plate and cultured for 24 hours. After removing the medium, differentiation factors RANKL 50 ng/ml and 10 uM PD98059 were added to DMEM medium, and the cells were treated with LPA at different concentrations of 50, 100, and 200 μg/ml and cultured for 4 days. After removing the medium and washing with PBS, the cells were fixed with a fixative solution (citrate solution + acetone + formaldehyde), and the substrate solution was 50 mM citrate buffer containing 1.36 mg/ml 4-nitrophenyl phosphate disodium salt and 10 mM tartrate. (pH 4.6) was prepared and dispensed onto fixed cells. After reaction at 37°C for 30 minutes, the enzyme reaction solution was stopped with 0.1 N NaOH, and the absorbance was measured at 405 nm. TRAP activity was expressed as a percentage of the absorbance of the sample relative to the control.
도 5에는 파골세포의 TRAP 활성을 측정한 결과가 도시되어 있다 도 5를 참조하면 파골세포의 TRAP활성 측정 결과, LPA의 모든 농도에서 대조군에 비해 유의성 있는 (** ; p<0.01, *** ; p<0.001)감소가 나타났다.Figure 5 shows the results of measuring TRAP activity of osteoclasts. Referring to Figure 5, the results of measuring TRAP activity of osteoclasts showed significant (**; p<0.01, ***) compared to the control group at all concentrations of LPA. ; p<0.001) decreased.
(실험예 2) in-vitro 유전자 발현량 측정(Experimental Example 2) Measurement of in-vitro gene expression level
도 6 내지 도 10에는 마우스 대식세포의 NOS2 발현량, IL1B 발현량, L6 발현량, TNFA 발현량, PTGS2 발현량 측정 결과가 각각 도시되어 있다.Figures 6 to 10 show the measurement results of NOS2 expression level, IL1B expression level, L6 expression level, TNFA expression level, and PTGS2 expression level of mouse macrophages, respectively.
도 6을 참조하면, 마우스 대식세포의 NOS2 발현량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (* : p<0.05, ** : p<0.01, *** : p<0.001)감소가 나타났다. 도7을 참조하면, 마우스 대식세포의 IL1B 발현량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (* : p<0.05, ** : p<0.01, *** : p<0.001)감소가 나타났다. 도 8을 참조하면, 마우스 대식세포의 IL6 발현량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (* : p<0.05, ** : p<0.01)감소가 나타났다. 도 9를 참조하면 마우스 대식세포의 TNFA 발현량을 측정한 결과, LPA의 50 ㎍/㎖ 농도를 제외한 모든 농도에서 대조군에 비해 유의성 있는 (** ; p<0.01, *** ; p<0.001)감소가 나타났다. 도 10을 참조하면 마우스 대식세포의 PTGS2 발현량을 측정한 결과, LPA의 50 ㎍/㎖ 농도를 제외한 모든 농도에서 대조군에 비해 유의성 있는 (* ; p<0.05, ** ; p<0.01)감소가 나타났다.Referring to Figure 6, as a result of measuring the NOS2 expression level in mouse macrophages, LPA was significantly higher than the control group at all concentrations (*: p < 0.05, **: p < 0.01, ***: p < 0.001). A decrease was observed. Referring to Figure 7, as a result of measuring the IL1B expression level of mouse macrophages, LPA was significantly higher than the control group at all concentrations (*: p < 0.05, **: p < 0.01, ***: p < 0.001). A decrease was observed. Referring to Figure 8, as a result of measuring the IL6 expression level of mouse macrophages, LPA showed a significant (*: p < 0.05, **: p < 0.01) decrease compared to the control group at all concentrations. Referring to Figure 9, as a result of measuring the expression level of TNFA in mouse macrophages, there was significant (** ; p<0.01, *** ; p<0.001) compared to the control group at all concentrations except the 50 ㎍/ml concentration of LPA. A decrease was observed. Referring to Figure 10, as a result of measuring the expression level of PTGS2 in mouse macrophages, there was a significant (* ; p<0.05, ** ; p<0.01) decrease compared to the control group at all concentrations except the 50 ㎍/ml concentration of LPA. appear.
(실험예 3) in-vitro 단백질 발현량(Experimental Example 3) In-vitro protein expression level
RAW264.7 세포를 6 well plate에 2×106 cells/welll로 분주하여 24시간 동안 배양하고 50, 100, 200 ㎍/㎖ 농도의 LPA를 처리하고 2시간 후, 100 ng/㎖의 LPS를 하여 다시 24시간 동안 배양하였다. 이 후, 1200 rpm에서 5분 동안 원심분리하여 얻은 세포를 D-PBS로 2회 세척하고 세포 pellet에 protease inhibitor cocktail Ⅰ, phosphatase inhibitor Ⅱ,Ⅲ가 포함된 RIPA buffer를 넣어 단백질을 추출하였다. 추출한 단백질은 BCA proein assay kit를 이용하여 정량하였으며, sample loading buffer와 섞어 95℃에서 5분간 반응시켜 준비하였다. 준비된 단백질은 10% acrylamide gel을 통해 SDS-PAGE하여 크기별로 분리하였으며, PVDF membrane에 이동시켰다. 단백질이 옮겨진 membrane을 3% BSA에 담가 상온에서 2시간동안 반응시켰다. TBS-T buffer를 이용하여 세척하고 primary antibody를 넣어 4℃에서 16시간동안 반응시켰다. 다시 3회 세척하고 secondary antibody를 넣어 상온에서 1시간동안 반응시켰으며, 다시 세척하고 ECL solution을 통해 단백질을 발색시켰다. 이후, chemidoc fusion FX를 통해 단백질 발현량을 분석하였다.RAW264.7 cells were distributed in a 6 well plate at 2 Cultured for 24 hours. Afterwards, the cells obtained by centrifugation at 1200 rpm for 5 minutes were washed twice with D-PBS, and the protein was extracted by adding RIPA buffer containing protease inhibitor cocktail I and phosphatase inhibitors II and III to the cell pellet. The extracted protein was quantified using the BCA proein assay kit, and was prepared by mixing with sample loading buffer and reacting at 95°C for 5 minutes. The prepared proteins were separated by size by SDS-PAGE through a 10% acrylamide gel and transferred to a PVDF membrane. The membrane onto which the protein was transferred was soaked in 3% BSA and reacted at room temperature for 2 hours. After washing using TBS-T buffer, primary antibody was added and reacted at 4°C for 16 hours. It was washed again three times, secondary antibody was added, and reacted at room temperature for 1 hour. After washing again, the protein was developed using ECL solution. Afterwards, the protein expression level was analyzed through chemidoc fusion FX.
1) ERK1) ERK
도 11에는 ERK 단백질 발현량 측정 결과가 도시되어 있다. 도 11을 참조하면, ERK 단백질 발현량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (*** ; p<0.001)감소가 나타났다.Figure 11 shows the results of measuring ERK protein expression level. Referring to Figure 11, as a result of measuring the ERK protein expression level, LPA showed a significant (***; p<0.001) decrease compared to the control group at all concentrations.
2) JNK2) JNK
도 12에는 JNK 단백질 발현량 측정 결과가 도시되어 있다. 도 12를 참조하면, JNK 단백질 발현량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (*** ; p<0.001)감소가 나타났다.Figure 12 shows the results of measuring JNK protein expression level. Referring to Figure 12, as a result of measuring the expression level of JNK protein, LPA showed a significant (***; p<0.001) decrease compared to the control group at all concentrations.
3) JNK3) JNK
도 13에는 JNK 단백질 발현량 측정 결과가 도시되어 있다. 도 13을 참조하면, p38 단백질 발현량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (*** ; p<0.001)감소가 나타났다Figure 13 shows the results of measuring JNK protein expression level. Referring to Figure 13, as a result of measuring p38 protein expression, LPA showed a significant (***; p<0.001) decrease compared to the control group at all concentrations.
4) NO 4) NO
도 14에는 JNK 단백질 발현량 측정 결과가 도시되어 있다. 도 14를 참조하면, 마우스 대식세포의 NO 생성량을 측정한 결과, LPA는 모든 농도에서 대조군에 비해 유의성 있는 (*** ; p<0.001)감소가 나타났다Figure 14 shows the results of measuring JNK protein expression level. Referring to Figure 14, as a result of measuring NO production in mouse macrophages, LPA showed a significant (***; p<0.001) decrease compared to the control group at all concentrations.
(실험예 4) in-vivo 체중부하 측정(Experimental Example 4) In-vivo weight bearing measurement
체중부하 측정은 실험종료 전날에 incapacitance test meter를 이용하여 측정하였으며, 플라스틱 방에 비스듬히 세운 후 각 뒷발에 가해지는 세기를 10초에 걸쳐 평균 산출하였다. 처치된 우측 뒷발에 분포된 체중의 백분율은 다음과 같은 방정식을 이용하여 계산하으며, 결과를 도 15에 도시하였다.Weight bearing was measured using an incapacitance test meter the day before the end of the experiment. After standing at an angle in a plastic room, the force applied to each hind foot was averaged over 10 seconds. The percentage of body weight distributed to the treated right hind paw was calculated using the following equation, and the results are shown in Figure 15.
Weight bearing (%) = (유발된 하지의 무게/정상 하지의 무게)× 100Weight bearing (%) = (Weight of induced lower extremity/Weight of normal lower extremity)×100
도 15를 참조하면, 체중부하를 측정한 결과, 대조군은 정상군에 비해 체중부하 비율이 감소하였으며, LPA 투여군 모두 대조군에 비해 유의성 있는 (** ; p<0.01, *** ; p<0.001)증가가 나타났다Referring to Figure 15, as a result of measuring weight bearing, the weight bearing ratio of the control group decreased compared to the normal group, and both LPA administered groups showed significant (** ; p<0.01, *** ; p<0.001) compared to the control group. an increase was seen
(실험예 5) in-vivo 유전자 발현량(Experimental Example 5) In-vivo gene expression level
1) IL1B1) IL1B
도 16에는 연골조직 IL1B 발현량을 측정한 결과가 도시되어 있다. 도 16을 참조하면, LPA 400 ㎎/㎏ 투여군은 대조군에 비해 유의성 있는 (** ; p<0.01)감소가 나타났다Figure 16 shows the results of measuring the expression level of IL1B in cartilage tissue. Referring to Figure 16, the LPA 400 mg/kg administration group showed a significant (**; p<0.01) decrease compared to the control group.
2) IL62) IL6
도 17에는 연골조직 IL6 발현량을 측정한 결과가 도시되어 있다. 도 17을 참조하면, LPA 400 ㎎/㎏ 투여군에서 대조군에 비해 유의성 있는 (* ; p<0.05)감소가 나타났다.Figure 17 shows the results of measuring IL6 expression level in cartilage tissue. Referring to Figure 17, a significant (*; p<0.05) decrease was observed in the LPA 400 mg/kg administration group compared to the control group.
3) TNFA3) TNFA
도 18에는 연골조직 TNFA 발현량을 측정한 결과가 도시되어 있다. 도 18을 참조하면, LPA 모든 투여군에서 대조군에 비해 유의성 있는 (* ; p<0.05, ** ;p<0.01)감소가 나타났다.Figure 18 shows the results of measuring the expression level of TNFA in cartilage tissue. Referring to Figure 18, a significant (* ; p<0.05, ** ;p<0.01) decrease was observed in all LPA administration groups compared to the control group.
4) PTGS24) PTGS2
도 19에는 연골조직 PTGS2 발현량을 측정한 결과가 도시도어 있다. 도 19를 참조하면, LPA 400 ㎎/㎏ 투여군에서 대조군에 비해 유의성 있는 (* ; p<0.05, *** ; p<0.001)감소가 나타났다Figure 19 shows the results of measuring the expression level of PTGS2 in cartilage tissue. Referring to Figure 19, a significant (* ; p<0.05, *** ; p<0.001) decrease was observed in the LPA 400 mg/kg administration group compared to the control group.
(실험예 6) in-vivo 혈청 바이오마커 생성량 평가(Experimental Example 6) Evaluation of in-vivo serum biomarker production
96 well plate에 분리한 혈청과 측정하고자 하는 standard를 100 ㎕씩 넣고 37℃에서 90분간 반응시켰다. 반응 후 washing buffer를 이용하여 3회 세척 작업을 진행한 후 100 ㎕의 detection antibody를 넣어 다시 37℃에서 60분간 반응시키고 세척하였다. 세척 후 HRP cunjugate를 100 ㎕씩 넣어 37℃에서 30분간 반응시키고 세척한 뒤 substrate reagent를 90 ㎕씩 넣어 37℃에서 15분간 반응시키고 50 ㎕의 stop solution을 추가하여 ELISA reader기를 통해 450 ㎚에서 흡광도를 측정하였으며, standard curve를 기준으로 절대 값으로 표시하였다. 100 ㎕ each of the separated serum and the standard to be measured were added to a 96 well plate and reacted at 37°C for 90 minutes. After the reaction, the reaction was washed three times using washing buffer, then 100 ㎕ of detection antibody was added, reacted again at 37°C for 60 minutes, and washed. After washing, add 100 ㎕ of HRP cunjugate and react at 37℃ for 30 minutes. After washing, add 90 ㎕ of substrate reagent and react at 37℃ for 15 minutes. Add 50 ㎕ of stop solution and measure the absorbance at 450 ㎚ using an ELISA reader. Measurements were made and expressed as absolute values based on the standard curve.
1) COMP1) COMP
도 20에는 혈청 내 COMP 생성량을 측정한 결과가 도시도어 있다. 도 20을 참조하면, LPA 투여군 모두 대조군에 비해 유의성 있는 (* ; p<0.05, *** ; p<0.001) 감소가 나타났다.Figure 20 shows the results of measuring the amount of COMP production in serum. Referring to Figure 20, both LPA administration groups showed a significant (*; p<0.05, ***; p<0.001) decrease compared to the control group.
2) CTXⅡ2) CTXⅡ
도 21에는 혈청 내 CTXⅡ 생성량을 측정한 결과가 도시되어 있다. 도 21을 참조하면, LPA 400 ㎎/㎏ 투여군에서 대조군에 비해 유의성 있는 (** ; p<0.01) 감소가 나타났다Figure 21 shows the results of measuring the amount of CTXII produced in serum. Referring to Figure 21, a significant (**; p<0.01) decrease was observed in the LPA 400 mg/kg administration group compared to the control group.
3) PGE2 3) PGE 2
도 22에는 혈청 내 PGE2 생성량을 측정한 결과가 도시되어 있다. 도 22를 참조하면, LPA 투여군 모두 대조군에 비해 유의성 있는 (*** ; p<0.001) 감소가 나타났다.Figure 22 shows the results of measuring the amount of PGE 2 produced in serum. Referring to Figure 22, both LPA administration groups showed a significant (***; p<0.001) decrease compared to the control group.
4) IL-1β4) IL-1β
도 23에는 혈청 내 IL-1β 생성량을 측정한 결과가 도시되어 있다. 도 23을 참조하면, LPA 투여군 모두 대조군에 비해 유의성 있는 (*** ; p<0.001) 감소가 나타났다.Figure 23 shows the results of measuring the amount of IL-1β produced in serum. Referring to Figure 23, both LPA administration groups showed a significant (***; p<0.001) decrease compared to the control group.
5) IL-65) IL-6
도 24에는 혈청 내 IL-6 생성량을 측정한 결과가 도시되어 있다. 도 24를 참조하면, LPA 투여군 모두 대조군에 비해 유의성 있는 (*** ; p<0.001) 감소가 나타났다Figure 24 shows the results of measuring the amount of IL-6 produced in serum. Referring to Figure 24, both LPA administration groups showed a significant (***; p<0.001) decrease compared to the control group.
6) TNF-α6) TNF-α
도 25에는 혈청 내 TNF-α 생성량을 측정한 결과가 도시되어 있다. 도 25를 참조하면, LPA 투여군 모두 대조군에 비해 유의성 있는 (*** ; p<0.001) 감소가 나타났다Figure 25 shows the results of measuring the amount of TNF-α production in serum. Referring to Figure 25, both LPA administered groups showed a significant (***; p<0.001) decrease compared to the control group.
(실험예 7)(Experimental Example 7) Micro-CT 및 조직학적 검사Micro-CT and histological examination
실험 종료 후 우측 대퇴골과 경골 부위를 절단하고 피부 및 근육을 제거하여 10% 포르말린에 고정시킨 조직을 시험검사 기관인 KPNT (Korea)에 Micro-CT 및 조직염색 H&E 염색을 의뢰하였다. 이후, Micro-CT 촬영 결과를 3D로 변환하여 연골 부위를 판독하였으며, 염색된 조직은 광학현미경을 통해 관찰하여 조직학적 검사를 실시하였다.After completing the experiment, the right femur and tibia were cut, the skin and muscles were removed, and the tissue fixed in 10% formalin was submitted to KPNT (Korea), a test laboratory, for Micro-CT and tissue staining, H&E staining. Afterwards, the Micro-CT imaging results were converted to 3D to read the cartilage area, and the stained tissue was observed through an optical microscope and histological examination was performed.
1) Mico-CT1) Mico-CT
도 26을 참조하면, 무릎 관절을 micro CT를 이용하여 촬영한 결과, 정상군에 비해 대조군과 LPA 투여군의 연골의 양(Cartilage volume, 보라색 표기)이 감소하였으며, LPA 400㎎/㎏투여군은 대조군에 비해 유의성 있는 (** : p<0.01) 증가가 나타났다.Referring to Figure 26, as a result of imaging the knee joint using micro CT, the amount of cartilage (cartilage volume, purple notation) in the control and LPA administration groups was decreased compared to the normal group, and the LPA 400 mg/kg administration group was compared to the control group. A significant (**: p<0.01) increase was observed.
2) 조직학적 검사2) Histological examination
도 27에는 H&E 염색을 실시한 결과가 도시되어 있다. 도 27을 참조하면 대조군 프로테오글리칸이 소실된 반면, LPA 투여군은 대조군에 비해 연골 주변과 활막 내 프로테오글리칸의 분포 증가를 확인할 수 있었다.Figure 27 shows the results of H&E staining. Referring to Figure 27, while the control group's proteoglycans were lost, the LPA-administered group showed an increase in the distribution of proteoglycans around the cartilage and within the synovium compared to the control group.
도 28에는 MT 염색을 실시한 결과, 대조군은 프로테오글리칸이 소실된 반면, LPA 투여군은 대조군에 비해 연골 주변과 활막 내 프로테오글리칸의 분포 증가를 확인할 수 있었다In Figure 28, as a result of MT staining, proteoglycans were lost in the control group, while the distribution of proteoglycans around the cartilage and within the synovium was confirmed to be increased in the LPA-administered group compared to the control group.
(실험예 8) 종합평가 (Experimental Example 8) Comprehensive evaluation
실시예 4 및 비교예 1 내지 3에서 제조한 약학조성물의 골관절염 예방 또는 치료제로서 효과를 비교하기 위하여, 종합평가를 실시하였다. 실험예 1 내지 실험예 7에서 실시예의 약학조성물 대신 비교예 1 내지 3의 약학조성물을 시험군으로 각각 사용하여 시험한 다음, 실시예의 약학조성물과 효능도를 비교하였다. 효능도는 5점 척도법에 의해 평가하도록 하였으며, 효능도 결과는 하기의 표 4에 나타내었다. 우수한 효능을 가질수록 5점에 가깝다.In order to compare the effectiveness of the pharmaceutical compositions prepared in Example 4 and Comparative Examples 1 to 3 as agents for preventing or treating osteoarthritis, a comprehensive evaluation was conducted. In Experimental Examples 1 to 7, the pharmaceutical compositions of Comparative Examples 1 to 3 were used as test groups instead of the pharmaceutical compositions of the Examples, and the efficacy was compared with the pharmaceutical compositions of the Examples. Efficacy was evaluated using a 5-point scale, and the efficacy results are shown in Table 4 below. The better the efficacy, the closer it is to 5 points.
상기 표 2의 결과를 살펴보면, 본 발명의 복합추출물을 함유하는 실시예는 골관절염 예방 및 치료제로서 효과를 가짐을 확인할 수 있었다. Looking at the results in Table 2, it was confirmed that the examples containing the complex extract of the present invention were effective as a preventive and therapeutic agent for osteoarthritis.
이상과 같이 본 발명에서는 특정된 사항들과 한정된 실시예 및 도면에 의해 설명되었으나 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐, 본 발명은 상기의 실시예에 한정되는 것은 아니며, 본 발명이 속하는 분야에서 통상의 지식을 가진 자라면 이러한 기재로부터 다양한 수정 및 변형이 가능하다. As described above, the present invention has been described with specific details, limited embodiments, and drawings, but these are provided only to facilitate a more general understanding of the present invention, and the present invention is not limited to the above embodiments, and the present invention Anyone skilled in the art can make various modifications and variations from this description.
따라서, 본 발명의 사상은 설명된 실시예에 국한되어 정해져서는 아니되며, 후술하는 특허청구범위뿐 아니라 이 특허청구범위와 균등하거나 등가적 변형이 있는 모든 것들은 본 발명 사상의 범주에 속한다고 할 것이다. Accordingly, the spirit of the present invention should not be limited to the described embodiments, and the scope of the patent claims described below as well as all modifications that are equivalent or equivalent to the scope of this patent claim shall fall within the scope of the spirit of the present invention. .
Claims (7)
상기 상황버섯 추출물은 꾸지뽕나무에 배양된 상황버섯을 추출한 것을 특징으로 하고,
상기 복합추출물은 상기 꾸지뽕 열매추출물 100중량부에 대하여, 상기 우슬추출물 50 내지 120 중량부, 상기 상황버섯추출물 50 내지 100 중량부를 혼합한 것인, 골관절염 예방 및 치료용 약학조성물.The active ingredient is a complex extract containing Cudrania fruit extract, Hyssop extract, and Sanghwang mushroom extract,
The Sanghwang mushroom extract is characterized by extracting Sanghwang mushrooms cultured on the Cudrania mulberry tree,
The composite extract is a pharmaceutical composition for the prevention and treatment of osteoarthritis, which is a mixture of 50 to 120 parts by weight of the Argentine root extract and 50 to 100 parts by weight of the Sanghwang mushroom extract with respect to 100 parts by weight of the Cudrania fruit extract.
상기 복합추출물은 감압건조 후, 동결건조된 분말로 수득된 것인 골관절염 예방 및 치료용 약학조성물.
According to paragraph 1,
A pharmaceutical composition for preventing and treating osteoarthritis, wherein the complex extract is obtained as a freeze-dried powder after being dried under reduced pressure.
상기 꾸지뽕 열매 추출물, 상기 우슬추출물 및 상황버섯추출물은 열수에 환류추출된 것인 골관절염 예방 및 치료용 약학조성물.
According to paragraph 1,
A pharmaceutical composition for preventing and treating osteoarthritis, wherein the Cudrania fruit extract, the Hyssop extract, and the Sanghwang mushroom extract are refluxed and extracted in hot water.
상기 복합추출물은 -70℃ 내지 -90℃에서 동결건조된 것인 골관절염 예방 및 치료용 약학조성물.According to paragraph 1,
The complex extract is a pharmaceutical composition for preventing and treating osteoarthritis, which is freeze-dried at -70°C to -90°C.
상기 상황버섯추출물은 꾸지뽕나무에 배양된 상황버섯을 추출한 것을 특징으로 하는 건강식품조성물
The active ingredient is a complex extract containing Cudrania fruit extract, Hyssop extract, and Sanghwang mushroom extract.
The Sanghwang mushroom extract is a health food composition characterized in that it is extracted from Sanghwang mushrooms cultured on the Cudrania mulberry tree.
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