KR20190010218A - Pharmaceutical composition for preventinr or treating osteoarthritis comprising Sparassis crispa - Google Patents
Pharmaceutical composition for preventinr or treating osteoarthritis comprising Sparassis crispa Download PDFInfo
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- KR20190010218A KR20190010218A KR1020170092710A KR20170092710A KR20190010218A KR 20190010218 A KR20190010218 A KR 20190010218A KR 1020170092710 A KR1020170092710 A KR 1020170092710A KR 20170092710 A KR20170092710 A KR 20170092710A KR 20190010218 A KR20190010218 A KR 20190010218A
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
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Abstract
Description
본 발명은 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating osteoarthritis, which comprises an extract of P. falciparum as an active ingredient.
류마티스 관절염과 골관절염은 관절염으로 통칭되는 대표적인 관절 질환이나, 발명 원인과 증상, 치료법에서 차이가 있기 때문에 분명한 구분이 필요하다.Rheumatoid arthritis and osteoarthritis are typical joint diseases known as arthritis. However, because there are differences in the cause, symptoms and treatment method of the invention, a clear distinction is necessary.
골관절염은 서서히 발병하여 초기에는 관절의 강직감을 호소하고 점차적으로 진행되어 운동 시 관절에 통증을 일으키고 휴식에 의해 완화되나, 부종과 기능저하를 발생시켜 일상생활에 장애를 일으키게 하는 질병으로, 골관절염의 치료는 관절의 강직감과 통증을 경감시키고, 가동 능력을 유지 및 개선시켜 장애가 최소화되도록 하는 것이 치료의 일차 목표이다.Osteoarthritis is a slowly developing disease that initially affects the sense of firmness of the joint and gradually progresses to cause pain in the joints during exercise and relaxation by relaxation. However, it causes edema and dysfunction to cause disability in daily life. Osteoarthritis Is the primary goal of treatment to alleviate the stiffness and pain of the joints, and to maintain and improve the ability to move to minimize disability.
반면 류마티스 관절염은 관절의 염증과 통증을 보이는 대표적인 전신성 만성 자가면역성 질환으로 관절 내 활액 조직의 염증 및 증식을 시작으로 주변의 연골, 뼈 등의 모든 관절 구조를 손상시켜 변형과 장애를 유발한다. 관절 이외 타 장기도 침범하여 류마티스결절, 심막염, 폐섬화유, 신경염 등 관절 외 증상을 초래하는 질환으로 관절의 연골과 주위골에 퇴행 변화가 나타나 발병하는 골관절염과는 발병의 원인과 증상에 분명한 차이가 존재하므로, 류마티스 관절염과 골관절염은 서로 다른 치료방법의 개발이 필요하다. On the other hand, rheumatoid arthritis is a typical systemic chronic autoimmune disease that shows joint inflammation and pain. It starts inflammation and proliferation of joint synovial tissue and damages all articular structures such as cartilage, bone, etc., and causes deformation and disorder. It is a disease that causes extrinsic symptoms such as rheumatoid nodule, pericarditis, pulmonary fibrosis and neuritis by invading other organs other than the joints. Degenerative changes appear in the cartilage and surrounding bone of the joints and the osteoarthritis that develops is a clear difference in causes and symptoms There is a need for development of different treatment modalities for rheumatoid arthritis and osteoarthritis.
골관절염의 가장 큰 특징은 연골 파괴로, 연골은 항상성을 유지하는 연골세포가 변화하고 사멸되어 콜라겐(Collagen), 프로테오클리칸(Proteoglycan) 등의 세포외기질 생산이 감소하고 분해효소가 활성화되면서 연골이 파괴되는 데, 이러한 연골파괴의 과정에서 IL-1β, TNF-α 등과 같은 사이토카인의 역할이 중요하기 때문에 세포외기질 분해효소나 세포성/염증성 사이토카인 상호작용을 직접 매개하고 또한 질병의 진행을 지연시키는 약학적 치료법을 포함한다. 비처방(over-the-counter) 약물 및 처방 약물이 증상 경감을 위해 제공되나, 이들은 많은 부작용을 야기한다. 예를 들어, 아스피린, 이부프로펜 또는 아세트아미노펜과 같은 비-스테로이스성 항염증 약물의 고용량의 장기간 사용은 위장 장애(upset stomachs), 위장관 출혈 및 간 손상으로 이어질 수 있다. 코르티코스테로이드와 같은 더 강력한 처방 약물은 취약골(brittle bones), 백내장 및 혈당 상승으로 이어질 수 있기 때문에 보다 안전하고 효과적인 골관절염 치료제의 개발이 필요한 실정이다.The most important feature of osteoarthritis is cartilage destruction. Cartilage cells that maintain homeostasis are changed and killed, resulting in reduction of extracellular matrix production such as collagen and proteoglycan, The role of cytokines such as IL-1β and TNF-α in the process of destruction of these cartilage is important, so it directly mediates extracellular matrix degrading enzymes and cellular / inflammatory cytokine interactions, ≪ / RTI > Over-the-counter drugs and prescription drugs are provided for symptom relief, but they cause many side effects. For example, prolonged use of high doses of nonsteroidal antiinflammatory drugs such as aspirin, ibuprofen or acetaminophen may lead to upset stomachs, gastrointestinal bleeding and liver damage. More powerful prescription drugs, such as corticosteroids, can lead to brittle bones, cataracts, and elevated blood glucose levels, thus requiring the development of safer and more effective treatments for osteoarthritis.
본 발명은 안전하고 효과적으로 골관절염 예방 또는 치료제를 제공하기 위해, 천연물질인 꽃송이버섯 추출물을 유효성분으로 함유하는 조성물을 제공하고자 한다.The present invention is intended to provide a composition containing, as an active ingredient, an extract of Zanthoxylum mushroom, a natural substance, in order to provide a preventive or therapeutic agent for osteoarthritis safely and effectively.
본 발명은 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating osteoarthritis containing an extract of Zygomycorr japonica as an active ingredient.
또한, 본 발명은 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 개선용 건강식품을 제공한다.The present invention also provides a health food for preventing or ameliorating osteoarthritis containing an extract of P. falciparum as an active ingredient.
본 발명에 따르면, 상기 꽃송이버섯 추출물은 산화스트레스 인자인 활성산소종의 발생을 저해하고, 골관절염에 작용하여 염증성 사이토카인 및 매개 인자의 활성을 저해함으로써, 조골작용의 저해를 예방하고 파골작용을 억제함으로써 관절의 연골 및 조직의 파괴를 보호하고 개선시키는 효과가 확인됨에 따라, 상기 꽃송이버섯 추출물은 효과적인 골관절염 예방 또는 치료용 조성물로 제공될 수 있다.According to the present invention, the Zygomy mushroom extract inhibits the production of reactive oxygen species, which is an oxidative stress factor, and acts on osteoarthritis to inhibit the activity of inflammatory cytokines and mediators, thereby preventing inhibition of osteoblast function and inhibiting osteoclast action Thereby confirming the effect of protecting and improving the cartilage and tissue destruction of the joints. Accordingly, the Zygomy mushroom extract can be provided as a composition for preventing or treating osteoarthritis effectively.
도 1은 MIA에 의해 골관절염이 유도된 랫에서 뒷다리 체중 부하의 상대적 변화를 확인한 결과이다. N: 정상군, Con: MIA로 골관절염이 유도된 대조군, SCM50: MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군, SCM100: MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군, SCM200: MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군임.
도 2는 각 실험군의 혈청 내 산화 스트레스 바이오마커인 ROS 수준을 확인한 결과이다(n=8).
도 3은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 SOD 단백질 발현 수준을 확인한 결과이다.
도 4는 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 카탈라아제(catalase) 단백질 발현 수준을 확인한 결과이다.
도 5는 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 GPx 단백질 발현 수준을 확인한 결과이다.
도 6은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 p-IκBα 단백질 발현 수준을 확인한 결과이다.
도 7은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 NF-κBp65 단백질 발현 수준을 확인한 결과이다.
도 8은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 COX-2 단백질 발현 수준을 확인한 결과이다.
도 9는 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 iNOS 단백질 발현 수준을 확인한 결과이다.
도 10은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 TNF-α 단백질 발현 수준을 확인한 결과이다.
도 11은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 IL-6 단백질 발현 수준을 확인한 결과이다.
도 12는 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 Nox-4 단백질 발현 수준을 확인한 결과이다.
도 13은 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 p22phox 단백질 발현 수준을 확인한 결과이다.
도 14는 골관절염이 유도된 랫에서 꽃송이버섯 추출물(SCM)에 의한 p47phox 단백질 발현 수준을 확인한 결과이다.
도 15는 골관절염이 유도된 랫에 꽃송이버섯 추출물을 투여하고 무릎 관절 조직을 사프라닌 0로 염색하여 조직학적으로 분석한 결과로(×40), (A)는 정상군이며, (B)는 골관절염이 유도된 대조군이며, (C)는 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군이며, (D)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군이며, (E)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군이다.
도 16은 골관절염이 유도된 랫에 꽃송이버섯 추출물을 투여하고 무릎 관절 조직을 사프라닌 0로 염색하여 조직학적으로 분석한 결과로(×100), (A)는 정상군이며, (B)는 골관절염이 유도된 대조군이며, (C)는 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군이며, (D)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군이며, (E)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군이다.
도 17은 골관절염이 유도된 랫에 꽃송이버섯 추출물을 투여하고 무릎 관절 조직을 헤마톡실린&에오신(H&E)으로 염색하여 조직학적으로 분석한 결과로(×40), (A)는 정상군이며, (B)는 골관절염이 유도된 대조군이며, (C)는 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군이며, (D)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군이며, (E)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군이다.
도 18은 골관절염이 유도된 랫에 꽃송이버섯 추출물을 투여하고 무릎 관절 조직을 헤마톡실린&에오신(H&E)으로 염색하여 조직학적으로 분석한 결과로(×100), (A)는 정상군이며, (B)는 골관절염이 유도된 대조군이며, (C)는 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군이며, (D)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군이며, (E)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군이다.
도 19는 골관절염이 유도된 랫에 꽃송이버섯 추출물을 투여하고 무릎 관절 조직을 메이슨 트리크롬(Masson's Trichrome; M-T)으로 염색하여 조직학적으로 분석한 결과로(×40), (A)는 정상군이며, (B)는 골관절염이 유도된 대조군이며, (C)는 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군이며, (D)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군이며, (E)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군이다.
도 20은 골관절염이 유도된 랫에 꽃송이버섯 추출물을 투여하고 무릎 관절 조직을 메이슨 트리크롬(Masson's Trichrome; M-T)으로 염색하여 조직학적으로 분석한 결과로(×100), (A)는 정상군이며, (B)는 골관절염이 유도된 대조군이며, (C)는 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 50 mg/kg가 투여된 실험군이며, (D)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 100 mg/kg가 투여된 실험군이며, (E)는 MIA로 골관절염이 유도된 랫에 꽃송이버섯 추출물(SCM) 200 mg/kg가 투여된 실험군이다.FIG. 1 shows the relative change in hindlimb weight load in rats induced by osteoarthritis by MIA. N: Normal group, Con: Control group in which osteoarthritis was induced by MIA, SCM50: Experimental group in which osteoarthritis induced rats were administered 50 mg / kg of Zygomycota extract (SCM), SCM100: Osteoarthritis- SCM200: Experimental group in which 200 mg / kg of mushroom extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
FIG. 2 shows the results of confirming the level of oxidative stress biomarker ROS in the serum of each experimental group (n = 8).
FIG. 3 shows the results of confirming the level of SOD protein expression by Zygomycetes mushroom extract (SCM) in rat osteoarthritis-induced rats.
FIG. 4 shows the results of confirming the expression level of catalase protein by Brassica juncea Extract (SCM) in rat osteoarthritis-induced rats.
FIG. 5 shows the results of confirming the expression level of GPx protein by the extract of Zygomycorr mushroom (SCM) in oat-rats-induced rats.
FIG. 6 shows the results of confirming the expression level of p-IκBα protein in the osteoarthritis-induced rats by the mushroom extract (SCM).
FIG. 7 shows the results of confirming the expression level of NF-κBp65 protein in the oat-rats-induced rats by the mushroom extract (SCM).
FIG. 8 shows the results of confirming the level of COX-2 protein expression by the extract of Brassica juncea (SCM) in osteoarthritis-induced rats.
FIG. 9 shows the results of confirming the expression level of iNOS protein in the osteoarthritis-induced rats by the mushroom extract (SCM).
FIG. 10 shows the results of confirming the expression level of TNF-α protein by the extract of Brassica juncea (SCM) in rat osteoarthritis-induced rats.
FIG. 11 shows the results of confirming the expression level of IL-6 protein by the extract of Mushroom mushroom (SCM) in a rat induced osteoarthritis.
FIG. 12 shows the results of confirming the expression level of Nox-4 protein by the extract of Mushroom mushroom (SCM) in rat osteoarthritis-induced rats.
FIG. 13 shows the results of confirming the expression level of p22phox protein in the oat-induced rat osteoarthritis-induced rats by SCM.
FIG. 14 shows the results of confirming the expression level of p47phox protein in the oat-induced rat osteoarthritis-induced rats by SCM.
15 shows the result of histological analysis (× 40), (A) is normal group, (B) is a normal group, and (C) is a control group in which osteoarthritis-induced rats were administered 50 mg / kg of Zygomycota mushroom extract (SCM), and (D) was a control group in which osteoarthritis-induced rats were treated with MIA ), And (E) was an experimental group in which 200 mg / kg of mushroom extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
FIG. 16 shows the results of histological analysis (× 100), (A) is normal group, (B) is the normal group, and (C) is a control group in which osteoarthritis-induced rats were administered 50 mg / kg of Zygomycota mushroom extract (SCM), and (D) was a control group in which osteoarthritis-induced rats were treated with MIA ), And (E) was an experimental group in which 200 mg / kg of mushroom extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
FIG. 17 shows the result of histological analysis (× 40) of the knee joint tissue treated with hematoxylin and eosin (H & E), (A) normal group, (B) is a control group in which osteoarthritis is induced, (C) is an experimental group in which 50 mg / kg of Brassica juncea Extract (SCM) is administered to osteoarthritis-induced rats, (D) (E) is an experimental group in which 200 mg / kg of Mushroom Extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
FIG. 18 shows the results of histological analysis (× 100) of the knee joint tissues treated with hematoxylin and eosin (H & E), (A) normal group, and (B) is a control group in which osteoarthritis is induced, (C) is an experimental group in which 50 mg / kg of Brassica juncea Extract (SCM) is administered to osteoarthritis-induced rats, (D) (E) is an experimental group in which 200 mg / kg of Mushroom Extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
19 shows the results of histological analysis (× 40) of the knee joint tissues treated with Mason's Trichrome (MT), (A) normal group, and , (B) is a control group in which osteoarthritis is induced, (C) is an experimental group to which 50 mg / kg of Zygomycorrus lucitum extract (SCM) was administered to osteoarthritis-induced rats and (D) (E) is an experimental group in which 200 mg / kg of mushroom extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
20 shows the results of histological analysis (× 100) of the knee joint tissues treated with Mason's Trichrome (MT), (A) normal group, and , (B) is a control group in which osteoarthritis is induced, (C) is an experimental group to which 50 mg / kg of Zygomycorrus lucitum extract (SCM) was administered to osteoarthritis-induced rats and (D) (E) is an experimental group in which 200 mg / kg of mushroom extract (SCM) was administered to rats on which osteoarthritis was induced by MIA.
본 발명은 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 치료용 약학조성물을 제공할 수 있다.The present invention can provide a pharmaceutical composition for preventing or treating osteoarthritis containing an extract of P. falciparum as an active ingredient.
상기 꽃송이버섯 추출물은 물, C1 내지 C4의 알콜 또는 이의 혼합물에 의해 추출될 수 있다.The P. falciparum extract may be extracted with water, C1 to C4 alcohols or a mixture thereof.
상기 꽃송이버섯 추출물은 약학조성물 총 100 중량부에 대하여, 0.1 내지 90 중량부로 포함될 수 있다.The mushroom extract may be contained in an amount of 0.1 to 90 parts by weight based on 100 parts by weight of the total amount of the pharmaceutical composition.
상기 꽃송이버섯(Sparassis crispa)은 여름에서 가을까지 침엽수의 자른 그루터기나 죽은 나무의 언저리에서 자생하는 버섯으로, 면역력 강화 및 항암 효과가 확인되었으며, 최근 연구에 따르면 꽃송이버섯이 내장지방을 감소시키고 콜레스테롤 흡수를 억제하는 것으로 확인되었으나, 골관절염을 예방하거나 치료하는 효과에 대해서는 보고되어 있지 않다.Sparassis crispa is a mushroom that grows from the edge of a coniferous tree or dead tree from summer to fall. It has been confirmed that the spicassis crispa has immunity and anticancer effects. Recent studies have shown that the mushroom reduces visceral fat and cholesterol absorption , But the effect of preventing or treating osteoarthritis has not been reported.
한편, 본 발명의 일실시예에 따르면, 랫에 모노소듐 아이도아세테이트(MIA)로 골관절염을 유도시킨 후 꽃송이버섯 추출물을 체중 당 50, 100 및 200 mg씩 투여한 후 골관절염이 유발된 슬관절 내 활막조직, 연골세포 및 섬유조직에 헤마톡실린&에오신(H&E), 메이슨 트리크롬(Masson's Trichrome; M-T) 및 사프라닌-O(Safranin-O) 염색을 수행하여 조직학적으로 분석한 결과, 도 15 및 16과 같이 정상군은 관절 조직과 프로테오글리칸이 정상적으로 확인되었으나, 대조군은 골관절염 유발로 인해 정상 관절 조직과 프로테오글리칸의 파괴가 확인되었다. 반면, SCM 투여군들에서는 관절 조직과 프로테오글리칸의 파괴가 억제되는 것을 확인할 수 있었다.Meanwhile, according to one embodiment of the present invention, osteoarthritis is induced by monosodium iodoacetate (MIA) in rats and 50, 100 and 200 mg of Zygomyansis mushroom extract are administered to the rats at a dose of 50, 100 and 200 mg, (H & E), Masson's Trichrome (MT) and Safranin-O staining was performed on the tissues, cartilage cells and fibrous tissues. As a result, And 16, normal tissues showed normal joint tissues and proteoglycans, but control tissues showed normal joint tissues and proteoglycan destruction due to osteoarthritis induction. On the other hand, it was confirmed that destruction of joint tissue and proteoglycan was inhibited in the SCM treated groups.
또한, 헤마톡실린&에오신 염색 결과 도 17 및 18과 같이 정상군에서는 활막 조직 및 연골 조직이 정상으로 확인되었으나, 대조군에서는 골관절염 유발로 인하여 활막 조직과 연골 조직의 심각한 손상이 나타났다. 그러나 SCM 투여군에서는 대조군과 비교하여 활막 조직과 연골 조직의 손상이 효과적으로 억제되는 것이 확인되었다.As a result of hematoxylin & eosin staining, synovial tissues and cartilage tissues were normal in the normal group as shown in FIGS. 17 and 18, but serious damage to the synovial tissue and cartilage tissue occurred in the control group due to osteoarthritis. However, it was confirmed that the SCM group effectively inhibited the synovial tissue and cartilage tissue damage as compared with the control group.
마지막으로 메이슨 트리크롬(M-T) 염색 결과, 도 19 및 20과 같이 정상군에서는 활막 조직 및 연골 조직의 콜라겐 염색 수준이 정상으로 확인된 반면, 대조군에서는 골관절염 유발로 인하여 면역세포의 침투와 활막 조직 및 연골 조직의 심각한 손상이 나타났다. 반면 SCM 투여군에서는 면역세포의 침투와 활막 조직 및 연골 조직의 손상이 효과적으로 억제되는 것을 확인할 수 있었다.Finally, as shown in Figs. 19 and 20, the mastrichl (MT) staining showed normal levels of collagen staining in synovial tissues and cartilage tissues, whereas in the control group, osteoarthritis induces immune cell penetration, Serious damage to cartilage tissue was noted. On the other hand, it was confirmed that the penetration of immune cells and the damage of synovial tissue and cartilage tissue were effectively inhibited in SCM-treated group.
상기 결과들로부터 꽃송이버섯 추출물은 골관절염에 작용하여 염증성 사이토카인 및 매개 인자의 활성을 억제하며, 관절의 연골 및 조직의 파괴에 대한 보호작용이 있는 것으로 확인되었으며, 이에 따라 상기 꽃송이버섯 추출물은 효과적인 골관절염 치료제로 제공될 수 있다.From the above results, it was confirmed that the extract of Zygomy mushroom acts on osteoarthritis to inhibit the activity of inflammatory cytokines and mediators, and protects against destruction of cartilage and tissues of joints. Thus, Can be provided as a therapeutic agent.
본 발명의 한 구체예에서, 상기 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 치료용 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition for preventing or treating osteoarthritis containing the mushroom extract of the present invention as an active ingredient may be administered orally or parenterally in the form of injections, granules, powders, tablets, pills, capsules, suppositories, , Emulsions, drops, and liquid preparations can be used.
본 발명의 다른 구체예에서, 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 치료용 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical composition for prevention or treatment of osteoarthritis containing the extract of P. falciparum is an appropriate carrier, , At least one additive selected from the group consisting of lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
본 발명의 일실시예에 따르면 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.
상기 꽃송이버섯 추출물의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.The preferred dose of the mushroom extract may vary depending on the condition and body weight of the subject, the kind and degree of the disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.
또한, 본 발명은 꽃송이버섯 추출물을 유효성분으로 함유하는 골관절염 예방 또는 개선용 건강식품을 제공할 수 있다.In addition, the present invention can provide a health food for preventing or ameliorating osteoarthritis containing an extract of Zanthoxylum mushroom as an active ingredient.
상기 꽃송이버섯 추출물은 물, C1 내지 C4의 알콜 또는 이의 혼합물에 의해 추출될 수 있다.The P. falciparum extract may be extracted with water, C1 to C4 alcohols or a mixture thereof.
상기 건강식품은 상기 꽃송이버섯 추출물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다.유효성분의 혼합양은 그의 사용 목적 예를들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food is used in combination with other food or food additives other than the above-mentioned Zygomycorrus lucitum mushroom extract, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed is suitably adjusted according to its use purpose, .
상기 건강식품에 함유된 화합물의 유효용량은 상기 치료제의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the compound contained in the above-mentioned health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range for health and hygiene purposes or for long-term intake for health control purposes, It is clear that the component can be used in an amount of more than the above range since there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제등을 들 수 있다.There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not 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.
<참고예> 시약<Reference Example> Reagent
모노소듐 아이도아세테이트(monosodium iodoacetate; MIA), PMSF(phenylmethylsulfonyl fluoride), 디티오트레이톨(dithiothreitol; DTT)는 Sigma Aldrich Co., Ltd. (St. Louis, MO, USA)에서 구입하였다. p-IκBa(Phosphorylated inhibitor of kBa), NF-κB(nuclear factor-kappa B), 수퍼옥시드 디스무타아제(superoxide dismutase; SOD), 카탈라아제(catalase), 글루타티온과산화수효소(glutathione peroxidase; GPx), iNOS(inducible nitric oxide synthase), COX-2(cyclooxygenase-2), TNF-α(tumor necrosis factor alpha), IL-6 (interleukin-6), NOX4(nicotinamide adenine dinucleotide phosphate4), p22phox, p47phox, 히스톤(histone), β-액틴과 2차 항체는 Santa Cruz Biotechnology (Santa Cruz, CA, USA)로부터 구입하였으며, 항 Protease inhibitor mixture, Ethylenediaminetetraacetic acid (EDTA)는 Wako Pure Chemical Industries, Ltd. (Osaka. Japan)에서 구입하였다. 또한, Nitrocellulose membranes는 Amersham GE Healthcare (Little. Chalfont, UK)에서 구입하였고, ECL Western Blotting Detection Reagents와 nitrocellulose membranes는 Amersham GE Healthcare로부터 구입하여 사용하였다. 단백질 정량을 위한 BCA protein assay kit는 Thermo Scientific (Rockford, IL, USA)에서 구입하였다. Monosodium iodoacetate (MIA), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT) were purchased from Sigma Aldrich Co., Ltd. (St. Louis, Mo., USA). (NF-κB), superoxide dismutase (SOD), catalase, glutathione and glutathione peroxidase (GPx), p-IκBa (phosphorylated inhibitor of kBa) (TNF-alpha), IL-6 (interleukin-6), nicotinamide adenine dinucleotide phosphate 4 (NOX4), p22phox, p47phox, histone histone), β-actin and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Antiprotease inhibitor mixture and Ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Nitrocellulose membranes were purchased from Amersham GE Healthcare (Little Chalfont, UK), and ECL Western Blotting Detection Reagents and nitrocellulose membranes were purchased from Amersham GE Healthcare. The BCA protein assay kit for protein determination was purchased from Thermo Scientific (Rockford, IL, USA).
<< 실시예Example 1> 동물실험 및 꽃송이버섯 추출물 준비 1> Animal Experiment and Preparation of Extract of Mushroom
1. 동물1. Animals
7주령의 수컷 Sprague-Dawley rat (200∼250 g)을 대한바이오 (Gyeonggi-do, Korea)에서 공급받아 실험 당일까지 고형 사료 (Samyang corporation, Korea)와 물을 충분히 공급하고, 온도 (22±2℃), 습도 (55±5%)가 조절된 사육실에서 12시간 명-암 주기 환경을 유지하며 1주간 적응시킨 후 실험에 사용하였다.A 7-week-old male Sprague-Dawley rat (200-250 g) was supplied from Gyeonggi-do, Korea and fed with solid feed (Samyang Corporation, Korea) and water until the day of experiment. ℃) and humidity (55 ± 5%) for 12 weeks.
또한, 동물실험의 윤리적, 과학적 타당성 검토 및 효율적인 관리를 위하여 대구한의대학교 동물실험윤리 위원회 (Institutional Animal Care and Use Committee : IACUC)의 승인 (승인번호: DHU2016-094)을 받았다.In addition, I received the approval of the Institutional Animal Care and Use Committee (IACUC) of the Daegu Haany University (approval number: DHU2016-094) for the ethical and scientific feasibility study and effective management of animal experiments.
2. 골관절염 유발2. Osteoarthritis induction
골관절염을 유발시키기 위해, 랫의 오른쪽 무릎 주변을 깨끗이 제모한 후 골관절염 유발물질인 모노소듐 아이도아세테이트(monosodium iodoacetate; MIA)를 0.3 mL 인슐린 주사기(BD 31G Ultra-Fine Ⅱ, USA)를 사용하여 오른쪽 무릎 관절강 내에 50μL (80 mg/mL) 씩 투여하였다. MIA 희석 시에는 0.9% 식염수를 사용하였다. To induce osteoarthritis, monoclonal anti-osteoarthritic monosodium iodoacetate (MIA) was administered to the right side of the rat using a 0.3 mL insulin syringe (BD 31G Ultra-Fine II, USA) And 50 μL (80 mg / mL) in the knee joint. For MIA dilution, 0.9% saline solution was used.
3. 꽃송이버섯 추출물(SCM) 준비 및 투여3. Preparation and administration of Zygomycetes mushroom extract (SCM)
꽃송이버섯 1kg를 메탄올 4L를 넣어 70~80℃에서 환류 냉각 추출하는 과정을 3번 반복하여 메탄올 엑스를 153g 얻었으며, 상기 방법으로 모노소듐 아이도아세테이트(MIA)로 골관절염을 유도시킨 랫에 꽃송이버섯 추출물을 체중당 50, 100 및 200 mg씩 투여하였다.The procedure of adding 1 L of methanolic mushroom to 4 L of methanol and refluxing and cooling extraction at 70 to 80 캜 was repeated 3 times to obtain 153 g of methanol extract. On the rats which induced osteoarthritis with monosodium iodoacetate (MIA) The extract was administered at 50, 100 and 200 mg / body weight.
<< 실시예Example 2> 체중 및 2> Weight and 식이섭취량Dietary intake 확인 Confirm
꽃송이버섯 추출물의 안정성을 확인하기 위해, 체중변화 및 식이 효율 변화를 확인하였다.Changes in body weight and dietary efficiency were observed to confirm the stability of P. falciparum extract.
랫의 체중을 전자체중계로 2일간 하루 1회씩 동일 시간 동일 조건에서 측정하였고, 식이섭취량은 2일 동안 제공된 사료에서 2일간 섭취하고 남은 사료량을 측정한 후 각 실험군의 하루 사료섭취량을 산출하였다.The body weight of the rats was measured with an electronic scales for 2 days at the same time for the same time. Dietary intake was calculated by taking daily feed intake for 2 days and feeding amount of remaining feed for each day.
그 결과, 표 1과 같이 MIA 유발 전과 후 4주 동안 모든 군에서 체중이 증가하였으며 대조군과 비교하였을 때 투여군들 간의 유의성은 없었다.As a result, body weight was increased in all groups for 4 weeks before and after induction of MIA as shown in Table 1, and there was no significant difference between the administration groups when compared with the control group.
또한, 식이섭취량은 정상군 25.1 ± 1.5 g, 대조군 23.9 ± 2.7 g, SCM50군 24.4 ± 2.4 g, SCM100군 24.4 ± 2.1 g 및 SCM200군은 23.3 ± 2.4 g으로 나타났으며, 각 군 간의 유의성은 없었다.The dietary intake was 25.1 ± 1.5 g in the normal group, 23.9 ± 2.7 g in the control group, 24.4 ± 2.4 g in the SCM50 group, 24.4 ± 2.1 g in the SCM100 group and 23.3 ± 2.4 g in the SCM100 group and there was no significant difference between the groups .
group
(g/day)Intake
(g / day)
<< 실시예Example 3> 뒷다리 체중 부하 확인 3> Check the back leg weight
꽃송이버섯 추출물(SCM)을 오전에 투여하고 오후에 부하 측정을 수행하였다.Mushroom extract (SCM) was administered in the morning and load measurement was performed in the afternoon.
부하 측정은 MIA 주사 전날, 주사 후 7일, 14일에 각각 수행하였으며, Incapacitance tester (Ser No. 01/45/25, Linton instrument Co., UK)를 이용하여 오른쪽, 왼쪽 발 무게를 각각 측정하였다.Load measurement was performed on the day before the MIA injection and on the 7th and 14th days after the injection. The right and left foot weights were measured using Incapacitance tester (Ser No. 01/45/25, Linton Instrument Co., UK) .
MIA에 의해 골관절염이 유발될 경우, 랫은 MIA를 투여하지 않은 발에 의지하여 테스터의 홀더 안에 서게 하였다. 랫의 배가 기기 센서에 닿지 않은 상태에서 왼쪽, 오른쪽 각각의 발무게(g)를 측정하고, 그 결과값을 하기 계산식과 같이 관절염이 유발된 오른쪽 뒷다리의 체중 부하량에 대한 정상인 왼쪽 뒷다리의 체중 부하량을 계산하여 체중 부하 비율을 계산하고, 정상군의 체중 부하 비율에 각 군의 체중 부하 비율을 계산하여 평균±표준편차로 표시하였다.When osteoarthritis was induced by MIA, the rats were placed in the holders of the tester by relying on the feet without MIA. The left and right foot weight (g) of each rat was measured without touching the sensor of the rat, and the result was calculated as shown in the following formula, and the weight load on the left hind leg of the right hind leg relative to the arthritis- We calculated the weight-bearing ratio and calculated the weight-bearing ratio of each group to the weight-bearing ratio of normal group, and expressed as the mean ± standard deviation.
[계산식][formula]
정상군의 뒷다리 체중부하 비율을 100으로 하였을 때, 각 군의 상대적 뒷다리 체중 부하의 변화를 측정한 결과, 도 1과 같이 MIA 투여 1주일 후에는 대조군 191.44 ± 95.15로 나타나 정상군에 비해 유의성 있는 증가를 나타냈으며, SCM50군 199.11 ± 87.28, SCM100군 158.47 ± 44.84, SCM200군은 195.05 ± 35.75로 대조군과 비슷한 수치를 나타냈다. MIA 투여 2주일 후에는 대조군 142.74 ± 44.71, SCM50 145.57 ± 32.61, SCM100군 141.12 ± 38.49, SCM200군 165.10 ± 26.62로 나타났다.As a result of measuring the change in the relative hindlimb weight load of each group when the hindlimb weight ratio of the normal group was 100, it was 191.44 ± 95.15 in the
상기 결과로부터 꽃송이버섯 추출물은 골관절염이 유발된 랫의 뒷다리 통증을 효과적으로 억제시키는 것을 확인할 수 있었다. From the above results, it was confirmed that the extract of Zygomycetes mushroom effectively inhibited the hind paw pain of osteoarthritis-induced rats.
<< 실시예Example 4> 혈청 내 4> Serum 산화적Oxidative 스트레스 stress 바이오마커Biomarker 확인 Confirm
산화적 스트레스 바이오마커인 활성산소종(Reactive Oxygen Species; ROS)은 연골조직을 파괴하는 물질 중 하나로 알려짐에 따라, 골관절염이 유도된 동물모델에서 ROS에 꽃송이버섯 추출물에 미치는 영향을 확인하였다.Since the reactive oxygen species (ROS), which is an oxidative stress biomarker, is known to be one of the substances destructing cartilage tissue, the effect of ROS on Zygomycetes mushroom extract in osteoarthritis-induced animal models was confirmed.
복대정맥에서 채혈한 혈액을 4,000 rpm으로 10분간 원심분리하여 혈청을 수집하였으며, 관절조직은 1mM EDTA - 50mM 인산나트륨 완충용액(pH 7.4)를 이용하여 분쇄한 후 활성산소종(Reactive Oxygen Species; ROS)을 측정하기 위하여 25 mM DCF-DA를 혼합한 후, 형광 광도계를 이용하여 0분부터 매 5분씩 530 nm 방출파장과 485nm 여기파장을 이용하여 30분간 측정하고 산출 값을 계산하였다.Blood collected from the abdominal vein was centrifuged at 4,000 rpm for 10 minutes to collect serum. The joint tissues were pulverized using 1 mM EDTA - 50 mM sodium phosphate buffer (pH 7.4), and reactive oxygen species (ROS ) Was mixed with 25 mM DCF-DA, and the fluorescence was measured for 30 minutes using 530 nm excitation wavelength and 485 nm excitation wavelength for 5 minutes starting from 0 minutes using a fluorescence spectrophotometer, and the calculated values were calculated.
혈청에서 ROS를 보고되어진 방법(Kooy NW, Royall JA, Ischiropoulos H, Beckman JS. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic Biol Med. 1994;16(2):149-56.)으로 측정하였다.ROS in serum was measured by the method reported (Kooy NW, Royall JA, Ischiropoulos H, Beckman JS, Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic Biol Med 1994; 16 (2): 149-56).
그 결과, 도 2와 같이 대조군의 ROS(5741.1 ± 758.7 fluorescence/ml)는 정상군(3875.6 ± 283.8 fluorescence/ml)과 비교하여 유의성 있게 증가한 반면, SCM50 군(4622.2 ± 424.2 fluorescence/ml), SCM100 군(4906.7 ± 266.2 fluorescence/ml) 및 SCM200 군(4470 ± 358.1 fluorescence/ml)은 대조군과 비교하여 유의하게 감소된 것이 확인되었다.As a result, as shown in FIG. 2, ROS (5741.1 ± 758.7 fluorescence / ml) of the control group was significantly increased compared with that of the normal group (3875.6 ± 283.8 fluorescence / ml), while the SCM50 group (4622.2 ± 424.2 fluorescence / ml) (4906.7 ± 266.2 fluorescence / ml) and SCM200 group (4470 ± 358.1 fluorescence / ml) were significantly reduced compared to the control group.
<< 실시예Example 5> 혈청 내 항-타입 Ⅱ 콜라겐 항체 확인 5> Serum Anti-Type II Collagen Antibody Identification
혈청 내 항-타입 Ⅱ 콜라겐 항체의 수치는 페옥시니트리트(peroxynitrite; ONOO-)와 산화적 스트레스를 형성하는데 관련이 있는 것으로 알려짐에 따라, rat anti-type Ⅱ collagen IgG ELISA kit (Chondrex Inc., Redmond, USA)를 이용하여 혈청 내 항-타입 Ⅱ 콜라겐 항체 측정하였다. The anti-type Ⅱ collagen antibody in serum is known to be involved in the formation of oxidative stress with peroxynitrite (ONOO-). Therefore, a rat anti-type Ⅱ collagen IgG ELISA kit (Chondrex Inc., Redmond, USA) was used to measure anti-type II collagen antibody in serum.
그 결과, 표 2와 같이 대조군은 정상군보다 유의성 있는 증가를 나타낸 반면, SCM100 및 200군에서는 유의한 감소가 확인되었다.As a result, as shown in Table 2, a significant increase was observed in the control group than in the normal group, but a significant decrease was observed in the SCM100 and 200 group.
<< 실시예Example 6> 6> 산화적Oxidative 스트레스 stress 바이오마커Biomarker ROS에To ROS 대한 꽃송이버섯 추출물의 효과 확인 Identification of the effect of Mushroom extract
1. One. 웨스턴Western 블롯Blot 분석 analysis
관절조직의 세포질을 얻기 위해 100 mM Tris-HCl (pH 7.4), 5 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 15 mM CaCl2, 1.5 M 수크로스, 0.1 M DTT 및 단백질분해효소 억제제 칵테일을 첨가한 버퍼 A를 넣고 tissue grinder (Bio Spec Product, USA)로 분쇄한 후 10% NP-40 용액을 첨가하였다. 아이스 위에서 20분간 정치시킨 후 12,000 rpm으로 2분간 원심분리 하여 세포질을 포함하고 있는 상층액을 분리하였다. HCl (pH 7.4), 5 mM Tris-HCl (pH 7.5), 2 mM MgCl 2 , 15 mM CaCl 2 , 1.5 M sucrose, 0.1 M DTT and protease inhibitor After adding buffer A with cocktail, the mixture was ground with tissue grinder (Bio Spec Product, USA) and 10% NP-40 solution was added. The mixture was allowed to stand on ice for 20 minutes and then centrifuged at 12,000 rpm for 2 minutes to separate the supernatant containing cytoplasm.
핵을 얻기 위해, 10% NP-40을 첨가한 버퍼 A로 두 번 세척하고 100㎕의 버퍼 C (50 mM HEPES, 50 mM KCl, 0.3 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 10% glycerol)를 첨가해 재 부유시킨 후, 10분마다 vortex을 3번 수행하였다.To obtain the nuclei, the wells were washed twice with 10% NP-40 buffer A and 100 의 of buffer C (50 mM HEPES, 50 mM KCl, 0.3 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 10% glycerol), and then vortexed three times every 10 minutes.
그 후 4℃에서 12,000 rpm으로 10분간 원심 분리한 후 핵을 포함하고 있는 상층액을 얻어 -80℃에서 각각 냉동 보관하였다. 관절조직의 세포질의 p-IκBα, COX2, iNOS, TNF-α, IL-6, SOD-1, Catalase, GPx, NOX4, p22phox, p47phox, β-actin 및 핵의 NF-κBp65, Histone 단백질의 발현을 측정하기 위해 10㎍의 단백질을 8∼15% SDS-폴리아크릴아마이드 겔을 이용하여 전기연동 후, 아크릴아마이드 겔을 니트로셀룰로스 막으로 이동시켰다. 준비된 막에 각각의 1차 항체를 처리하여 4℃에서 하룻밤 동안 반응시킨 후 PBS-T로 6분마다 5회 세척하고, 각각 처리된 1차 항체에 사용되는 2차 항체 (PBS-T로 1:3000로 희석해서 사용)를 사용하여 상온에서 1시간 반응시킨 후, PBS-T로 6분마다 5회 세척하였다. After centrifugation at 12,000 rpm for 10 min at 4 ° C, the supernatant containing the nuclei was obtained and stored at -80 ° C for freezing. Expression of NF-κBp65 and Histone protein in the cytoplasm of joint tissues by p-IκBα, COX2, iNOS, TNF-α, IL-6, SOD-1, Catalase, GPx, NOX4, p22phox, p47phox, For measurement, 10 μg of the protein was electro-intercalated using 8 to 15% SDS-polyacrylamide gel, and the acrylamide gel was transferred to the nitrocellulose membrane. Each of the prepared membranes was treated with each primary antibody, reacted overnight at 4 ° C, washed five times with PBS-T every 6 minutes, and then the secondary antibody (PBS: 1: 1: 3000) for 1 hour at room temperature, and then washed five times with PBS-T every 6 minutes.
그 후, ECL(enhanced chemiluminescence) 용액을 GE Healthcare에 노출시킨 후, Sensi-Q2000 Chemidoc에 감광시켜 단백질 발현을 확인하고, 해당 밴드를 ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan)프로그램을 사용하여 정량하였다. After that, ECL (enhanced chemiluminescence) solution was exposed to GE Healthcare, sensitized to Sensi-Q2000 Chemidoc to confirm protein expression, and the band was quantified using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) .
2. 관절조직 내 항산화 단백질 분석2. Antioxidant protein analysis in joint tissue
ROS 소거와 관련된 항산화 활성은 항산화 효소 단백질로 알려진 SOD, CAT 및 GPx에 의해서 조절되는 데, 상기 단백질들은 스트레스에 의해 유발된 ROS의 축적에 의한 세포 손상을 억제함에 따라, 꽃송이버섯 추출물이 상기 단백질 발현에 미치는 영향을 웨스턴 블롯 분석으로 확인하였다.The antioxidative activity associated with ROS elimination is regulated by SOD, CAT and GPx, which are known as antioxidant enzyme proteins. These proteins inhibit cell damage caused by stress-induced accumulation of ROS, Were confirmed by Western blot analysis.
관절 조직에서 SOD 단백질을 분석한 결과, 도 3과 같이 정상군 1.00 ± 0.05, 대조군 0.75 ± 0.02, SCM50군 0.72 ± 0.04, SCM100군 0.75 ± 0.01, SCM200군 0.89 ± 0.04으로 확인되었으며, 특히 SCM200군에서 대조군과 비교하여 유의한 증가를 나타났다.As shown in Fig. 3, the SOD protein was found to be 1.00 ± 0.05 in the normal group, 0.75 ± 0.02 in the control group, 0.72 ± 0.04 in the SCM50 group, 0.75 ± 0.01 in the SCM100 group and 0.89 ± 0.04 in the SCM200 group, Compared with the control group.
또한, 관절 조직에서 catalase 단백질을 분석한 결과, 도 4와 같이 정상군 1.00 ± 0.06, 대조군 0.86 ± 0.05, SCM50군 0.78 ± 0.04, SCM100군 0.83 ± 0.05, SCM200군 0.97 ± 0.04로 나타났으며, 특히, SCM200군에서 대조군에 비해 증가되는 것을 확인할 수 있었다.As a result of analysis of catalase protein in joint tissues, it was 1.00 ± 0.06 in the normal group, 0.86 ± 0.05 in the control group, 0.78 ± 0.04 in the SCM50 group, 0.83 ± 0.05 in the SCM100 group and 0.97 ± 0.04 in the SCM200 group as shown in FIG. 4 , And SCM200 group compared to the control group.
마지막으로 관절 조직에서 GPx 단백질을 분석한 결과, 도 5와 같이 정상군 1.00 ± 0.08, 대조군 0.83 ± 0.03, SCM50군 0.64 ± 0.03, SCM100군 0.97 ± 0.1, SCM200군 0.99 ± 0.08으로 발현이 나타났으며, 대조군과 비교하여 SCM100 및 200군에서 발현 증가가 확인되었다.Finally, the expression of GPx protein in joint tissues was 1.00 ± 0.08 in the normal group, 0.83 ± 0.03 in the control group, 0.64 ± 0.03 in the SCM50 group, 0.97 ± 0.1 in the SCM100 group and 0.99 ± 0.08 in the SCM200 group as shown in FIG. 5 , And increased expression was observed in the SCM100 and 200 groups as compared with the control group.
상기 결과로부터 골관절염 유발 동물의 조직 내 항산화 효소는 정상군과 비교하여 유의성있게 감소되는 반면, 꽃송이버섯 추출물은 항산화 효소 단백질로 알려진 SOD, CAT 및 GPx의 발현을 증가시켜 ROS에 의한 조직 손상을 효과적으로 억제할 수 있음이 확인되었다.From the above results, antioxidant enzymes in the tissues of osteoarthritis-induced animals were significantly reduced compared to the normal group, whereas the extract of Zygomycetes mushroom increased the expression of SOD, CAT and GPx, known as antioxidant enzyme proteins, It was confirmed that
3. 관절조직 내 염증 사이토카인 및 매개 인자 분석3. Analysis of inflammatory cytokines and mediators in joint tissues
골관절염이 유도된 동물모델에서 꽃송이버섯 추출물이 관절조직 내 염증성 사이토카인 및 매개 인자의 발현 수준에 미치는 영향을 웨스턴 블롯으로 확인하였다.In the animal model in which osteoarthritis was induced, the influence of the mushroom extract on the expression level of inflammatory cytokines and mediators in joint tissues was confirmed by Western blotting.
관절 조직에서 p-IκBα 발현 수준을 분석한 결과, 도 6과 같이 정상군 1.00 ± 0.13, 대조군 1.43 ± 0.1, SCM50군 1.07 ± 0.11, SCM100군 1.18 ± 0.14 및 SCM200군 1.07 ± 0.1으로 발현이 나타났으며, 특히 SCM200군은 대조군과 비교하여 유의한 감소를 나타내었다.Analysis of p-IκBα expression level in the joint tissues revealed that the expression level was 1.00 ± 0.13 in the normal group, 1.43 ± 0.1 in the control group, 1.07 ± 0.11 in the SCM50 group, 1.18 ± 0.14 in the SCM100 group and 1.07 ± 0.1 in the SCM200 group as shown in FIG. Especially in the SCM200 group, compared with the control group.
관절 조직에서 NF-κBp65 발현 수준을 분석한 결과, 도 7과 같이 정상군 1.00 ± 0.05, 대조군 1.44 ± 0.12, SCM50군 1.21 ± 0.08, SCM100군 1.20 ± 0.11, SCM200군 1.10 ± 0.05 수준으로 발현이 나타났으며, SCM200군에서 대조군과 비교하여 유의한 감소를 나타내었다.As a result of analysis of NF-κBp65 expression level in the joint tissues, expression was 1.00 ± 0.05 in the normal group, 1.44 ± 0.12 in the control group, 1.21 ± 0.08 in the SCM50 group, 1.20 ± 0.11 in the SCM100 group and 1.10 ± 0.05 in the SCM200 group as shown in FIG. And the SCM200 group showed a significant decrease compared to the control group.
관절 조직에서 COX-2 발현을 확인한 결과, 도 8과 같이 정상군 1.00 ± 0.05, 대조군 1.18 ± 0.10, SCM50군 1.04 ± 0.05, SCM100군 1.08 ± 0.07, SCM200군 1.03 ± 0.06 수준으로 나타났으며, 정상군과 비교하여 대조군에서는 발현 증가가 확인된 반면, SCM 투여군에서는 대조군보다 감소하는 것을 확인할 수 있었다.As shown in FIG. 8, COX-2 expression in the joint tissues was 1.00 ± 0.05 in the normal group, 1.18 ± 0.10 in the control group, 1.04 ± 0.05 in the SCM50 group, 1.08 ± 0.07 in the SCM100 group and 1.03 ± 0.06 in the SCM200 group. Compared with the control group, the expression level was increased in the control group, whereas the SCM group was decreased in the control group.
관절 조직에서 iNOS 발현을 확인한 결과, 도 9와 같이 정상군 1.00 ± 0.04, 대조군 1.26 ± 0.05, SCM50군 1.09 ± 0.04, SCM100군 1.08 ± 0.05, SCM200군 1.09 ± 0.05 수준으로 발현이 나타났으며, 대조군에서는 정상군과 비교하여 유의성 있게 증가된 반면, SCM 투여군 모두 대조군과 비교하여 유의한 감소를 나타내었다.Expression of iNOS in the joint tissues was 1.00 ± 0.04 in normal group, 1.26 ± 0.05 in control group, 1.09 ± 0.04 in SCM50 group, 1.08 ± 0.05 in SCM100 group and 1.09 ± 0.05 in SCM200 group as shown in FIG. 9, , But the SCM group showed a significant decrease compared with the control group.
관절 조직에서 TNF-α 발현 수준을 확인한 결과, 도 10과 같이 정상군 1.00 ± 0.05, 대조군 1.18 ± 0.03, SCM50군 1.01 ± 0.07, SCM100군 1.09 ± 0.11 및 SCM200군 1.04 ± 0.09 수준으로 발현이 확인되었다. 정상군과 비교하여 대조군에서는 발현이 증가하는 경향을 나타낸 반면, SCM 투여군에서는 발현 감소가 나타났다.Expression levels of TNF-α in the joint tissues were found to be 1.00 ± 0.05, 1.18 ± 0.03, 1.01 ± 0.07, 1.09 ± 0.11, and 1.04 ± 0.09, respectively, as shown in FIG. 10 . Expression was increased in the control group as compared to the normal group, but decreased in the SCM-treated group.
관절 조직에서 IL-6 발현 수준을 확인한 결과, 도 11과 같이 정상군 1.00 ± 0.10, 대조군 1.31 ± 0.07, SCM50군 1.21 ± 0.09, SCM100군 1.22 ± 0.04 및 SCM200군 1.26 ± 0.06 수준으로 발현이 나타났다. 정상군과 비교하여 대조군에서는 유의한 발현 증가가 확인된 반면, SCM 투여군에서는 대조군과 비교하여 발현이 감소되는 것을 확인할 수 있었다.Expression levels of IL-6 in joint tissues were 1.00 ± 0.10 in normal group, 1.31 ± 0.07 in control group, 1.21 ± 0.09 in SCM50 group, 1.22 ± 0.04 in SCM100 group and 1.26 ± 0.06 in SCM200 group as shown in FIG. In comparison with the normal group, significant increase in expression was observed in the control group, whereas in the SCM administration group, expression was decreased compared with the control group.
상기 결과로부터 꽃송이버섯 추출물은 NFkB, COX2, iNOS 발현을 효과적으로 감소시키고 골관절염 진행에 따라 발현이 증가되는 염증성 사이토카인 IL-6의 수준을 유의적으로 감소시키는 것이 확인됨에 따라, 꽃송이버섯 추출물은 골관절염에 효과적으로 사용될 수 있다.From the above results, it was confirmed that the extract of Zygomycetes mushroom effectively reduced the expression of NFkB, COX2, and iNOS and significantly decreased the level of inflammatory cytokine IL-6, which is increased in expression of osteoarthritis. Thus, Can be effectively used.
4. 관절조직 내 4. In joint tissue ROSROS 생성 관련 인자 분석 Analysis of production related factors
Nox4, p22phox 및 p47phox가 ROS 생성 관련 인자로 알려짐에 따라, 상기 인자들에 대한 꽃송이버섯 추출물의 영향을 웨스턴 블롯으로 확인하였다. As Nox4, p22phox and p47phox were known as ROS production-related factors, the influence of the mushroom extract on the factors was confirmed by Western blotting.
관절 조직에서 Nox4 발현 수준을 확인한 결과, 도 12와 같이 정상군 1.00 ± 0.06, 대조군 0.92 ± 0.06, SCM50군 1.04 ± 0.10, SCM100군 1.13 ± 0.13 및 SCM200군 0.63 ± 0.07 수준으로 발현이 나타났다. 정상군과 비교하여 대조군에서는 발현 감소가 확인된 반면, SCM50군 및 SCM100군에서는 대조군보다 발현이 증가되는 것을 확인할 수 있었다.Expression levels of Nox4 expression in the joint tissues were found to be 1.00 ± 0.06 in the normal group, 0.92 ± 0.06 in the control group, 1.04 ± 0.10 in the SCM50 group, 1.13 ± 0.13 in the SCM100 group and 0.63 ± 0.07 in the SCM200 group as shown in FIG. Compared with the normal group, expression was decreased in the control group, whereas in the SCM50 group and the SCM100 group, the expression was increased compared with the control group.
또한, 관절 조직에서 p22phox 발현 수준을 확인한 결과, 도 13과 같이 정상군 1.00 ± 0.03, 대조군 1.04 ± 0.13, SCM50군 1.69 ± 0.26, SCM100군 2.02 ± 0.14 및 SCM200군 1.57 ± 0.15 수준으로 발현이 나타났다. 대조군은 정상군과 비교하여 유사한 수준으로 확인된 반면, SCM 투여군에서는 대조군과 비교하여 유의하게 증가되는 것을 확인할 수 있었다.In addition, expression levels of p22phox in the joint tissues were found to be 1.00 ± 0.03 in the normal group, 1.04 ± 0.13 in the normal group, 1.69 ± 0.26 in the SCM50 group, 2.02 ± 0.14 in the SCM100 group and 1.57 ± 0.15 in the SCM200 group as shown in FIG. The control group was found to be similar to that of the normal group, whereas the SCM group was significantly increased compared with the control group.
마지막으로 관절 조직에서 p47phox 발현 수준을 확인한 결과, 도 14와 같이 정상군 1.00 ± 0.33, 대조군 0.79 ± 0.06, SCM50군 0.73 ± 0.13, SCM100군 0.84 ± 0.08 및 SCM200군 0.59 ± 0.05 수준으로 발현이 나타났다. 대조군은 정상군에 비해 감소를 보였으나 정상군과 유의성은 보이지 않았다. 또한, 약물 투여군의 p47phox 단백질 발현량은 대조군에 비해 유의성은 보이지 않았으나, SCM200 고농도 투여군에서 가장 크게 감소되는 것을 확인할 수 있었다.Finally, p47phox expression levels in joint tissues were found to be 1.00 ± 0.33 in normal group, 0.79 ± 0.06 in control group, 0.73 ± 0.13 in SCM50 group, 0.84 ± 0.08 in SCM100 group and 0.59 ± 0.05 in SCM200 group as shown in FIG. There was no significant difference between the control group and normal group. In addition, the expression level of p47phox protein in the drug-treated group was not significantly different from that of the control group, but it was confirmed that the dose was significantly decreased in the SCM200-treated group.
상기 결과로부터 꽃송이버섯 추출물은 관절 조직 내 산화적 스트레스 인자인 Nox4 및 p22phox를 억제하여 골관절염에 효과를 나타낼 수 있다.From these results, it is possible to inhibit Nox4 and p22phox, which are oxidative stress factors in joint tissues, and to be effective in osteoarthritis.
<< 실시예Example 7> 조직학적 분석 7> Histological analysis
골관절염이 유발된 슬관절 내 활막조직, 연골세포 및 섬유조직의 병리조직학적 분석을 위하여 헤마톡실린&에오신(H&E)과 메이슨 트리크롬(Masson's Trichrome; M-T) 및 사프라닌-O(Safranin-O) 염색을 수행하였다.(H & E), Masson's Trichrome (MT), and Safranin-O for histopathological analysis of synovial tissue, chondrocytes and fibrous tissues in osteoarthritic knee joints. Staining was performed.
실험 종료 후 슬관절 부위를 절단하고 10% EDTA가 포함된 10% 포르말린(formalin) 용액에 넣어 관절을 탈회(decalcification)시켰다. 방사선 촬영기법으로 탈회 유무를 확인한 후 파라핀 왁스에 관절을 넣고 고정한 후 coronal section을 수행하였다. 탈회 과정을 거쳐 파라핀으로 고정된 조직을 7㎛의 크기로 자른 뒤, 헤마톡실린&에오신(H&E)과 메이슨 트리크롬(Masson's Trichrome; M-T) 및 사프라닌-O(Safranin-O) 염색을 수행하여 조직의 상태를 확인하였다. After the end of the experiment, the knee joint was cut and decalcified with 10% formalin solution containing 10% EDTA. After confirming the presence of demineralization by radiography, the coronal section was performed after fixing the joints in the paraffin wax. The tissue fixed with paraffin through the demineralization process was cut into a size of 7 μm and stained with hematoxylin and eosin (H & E), Masson's Trichrome (MT) and Safranin-O And the state of the tissue was confirmed.
염증 반응 발생 유무, 활막 세포의 증식 및 염증 세포의 조직 침윤 여부는 H&E과 M-T 염색 결과에서 확인할 수 있으며, 연골 조직의 손상 여부는 프로테오글리칸 층을 염색하는 사프라닌-O 염색 결과로 확인하였다.The presence of inflammatory reaction, synovial cell proliferation and infiltration of inflammatory cells were confirmed by H & E and M-T staining, and the damage of cartilage tissue was confirmed by the results of saffrenin-O staining for staining the proteoglycan layer.
사프라닌-O 염색 결과, 도 15 및 16과 같이 정상군은 관절 조직과 프로테오글리칸이 정상적으로 확인되었으나, 대조군은 골관절염 유발로 인해 정상 관절 조직과 프로테오글리칸의 파괴가 확인되었다. 반면, SCM 투여군들에서는 관절 조직과 프로테오글리칸의 파괴가 억제되는 것을 확인할 수 있었다.As a result of saffranin-O staining, as shown in Figs. 15 and 16, joint tissues and proteoglycans were normally observed in the normal group, but the control group showed destruction of normal joint tissues and proteoglycans due to osteoarthritis induction. On the other hand, it was confirmed that destruction of joint tissue and proteoglycan was inhibited in the SCM treated groups.
또한, 헤마톡실린&에오신 염색 결과, 도 17 및 18과 같이 정상군에서는 활막 조직 및 연골 조직이 정상으로 확인되었으나, 대조군에서는 골관절염 유발로 인하여 활막 조직과 연골 조직의 심각한 손상이 나타났다. 그러나 SCM 투여군에서는 대조군과 비교하여 활막 조직과 연골 조직의 손상이 효과적으로 억제되는 것이 확인되었다.As a result of hematoxylin & eosin staining, synovial tissues and cartilage tissues were normal in the normal group as shown in Figs. 17 and 18, but serious damage of the synovial tissues and cartilage tissues occurred in the control group due to osteoarthritis induction. However, it was confirmed that the SCM group effectively inhibited the synovial tissue and cartilage tissue damage as compared with the control group.
마지막으로 메이슨 트리크롬(M-T) 염색 결과, 도 19 및 20과 같이 정상군에서는 활막 조직 및 연골 조직의 콜라겐 염색 수준이 정상으로 확인된 반면, 대조군에서는 골관절염 유발로 인하여 면역세포의 침투와 활막 조직 및 연골 조직의 심각한 손상이 나타났다. 반면 SCM 투여군에서는 면역세포의 침투와 활막 조직 및 연골 조직의 손상이 효과적으로 억제되는 것을 확인할 수 있었다.Finally, as shown in Figs. 19 and 20, the mastrichl (MT) staining showed normal levels of collagen staining in synovial tissues and cartilage tissues, whereas in the control group, osteoarthritis induces immune cell penetration, Serious damage to cartilage tissue was noted. On the other hand, it was confirmed that the penetration of immune cells and the damage of synovial tissue and cartilage tissue were effectively inhibited in SCM-treated group.
상기 결과들로부터 꽃송이버섯 추출물은 골관절염에 작용하여 염증성 사이토카인 및 매개인자의 활성을 억제하며, 조골작용의 저해를 예방하고 파골작용을 억제함에 따라, 관절의 연골 및 조직의 파괴에 대한 보호작용이 있는 것이 확인되므로 효과적인 골관절염 치료제로 제공될 수 있다.From the above results, the extract of Zygomycetes mushroom acts on osteoarthritis to inhibit the activity of inflammatory cytokines and mediators, prevent the inhibition of osteoblast function and inhibit osteoclast action, thereby protecting the cartilage and tissue from destruction It can be provided as an effective treatment for osteoarthritis.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
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