초록

○ 연구결과
- 생약(우슬)애 대한 특성조사 및 유효 성분의 생리활성 검정
= 우슬의 주요성분 추출 및 분석 및 성분 연구
- 우슬을 활용한 세포독성 및 대사활성 기능시험
- 류마티스 및 퇴행성 골관절염에서의 우슬의 항관절염 효과(in vivo, in vitro)
- 우슬을 활용한 기능성 음료 제조
- 우슬 추출물을 이용한 항관절염 경피흡수 연고제 제조
- 항관절염 경피흡수 연고제의 활성 검증

Abstract ▼

Ⅱ. Goal and Significance of the Study
The purpose of this investigation was to examine osteoarthritis and rheumatoid arthritis effects of Achyranthes Radix by cream with Archyranthes Radix extract. In addition, Development of high value-added products of Achyranthes Radix.
Ⅲ. Research and Scop

Ⅱ. Goal and Significance of the Study
The purpose of this investigation was to examine osteoarthritis and rheumatoid arthritis effects of Achyranthes Radix by cream with Archyranthes Radix extract. In addition, Development of high value-added products of Achyranthes Radix.
Ⅲ. Research and Scope
The dried root of Achyranthes japonica Leveille et Vaniot(Amaranthaceae, AJ) is one of the source plants of Achyranthes root. AJ could be activated the blood circulation in human. The active ingredients of AJ were oleanolic acid, saponin and 20-hydroxyecdysone. 20-hydroxyecdysone was a potent inhibition substance of excess secretion of Cathepsin B causing an inflammation. In this study, we tried to isolate and evaluated loss on drying, total ash, acid-insoluble ash of 20-hydroxyecdysone from Achyranthis Radix. In order to evaluate quantitative determination of 20-hydroxyecdysone in AJ HPLC method had been used.
Separation and quantitative determination of 20-hydroxyecdysone from AJ has been conducted by using HPLC method. 20-hydroxyecdysone in a methanol extract from the raw drug was separated on a reverse phase column using n-hexane : ethanol : methanol(1:3:3) solvent system. Content of 20-hydroxyecdysone in AJ showed average 2.18% in samples collected throughout the regions of Korea.
Osteoarthritis(OA) is a degenerative joint disease characterized by fibrillation and erosion in cartilage, chondrocyte proliferation and osteophyte formation at the joint margins, and sclerotis of subchondral bone. Archyranthes Radix has had extensive therapeutic application, and there has been increasing interest in its biological effects. However, the biochemical and histological effects of Archyranthes Radix on chondrocyte have never been systematically investigated.
Therefore, the objective of this study was to investigate the effects of Archyranthes Radix on role of anti-oxidant and anti-inflammation on oxidative stress induced by H₂O₂ in rat articular chondrocytes. Articular cartilage was harvested from rat articular cartilage and osteoarthritis models was induced from MIA. Cartilage explants and isolated chondrocytes from articular cartilage were treated in various conditions as monolayer culture. After Archyranthes Radix treatment, the time-dependent effects of Archyranthes Radix was evaluated by measuring cell proliferation and by analysing cell morphology and expression of cartilage apoptotic protein using XTT. Archyranthes Radix resulted in a time- and dose-dependent cell proliferation, which was largely attributed to apoptosis. Cell death caused by H₂O₂, was inhibited by Archyranthes Radix. Histological findings that are similar to those observed in human osteoarthritis, such as disorganization of chondrocytes, erosion and fibrillation of cartilage surface, and subchondral bone exposure were observed in a MIA-induced osteoarthritis model. Safranin O-fast green staining revealed that marked diffuse reduction of proteoglycans treated with MIA. The Mankin's score were closely correlated to the grade of histological findings.
This study was performed to investigate the effects in inflammatory and pain status on rheumatoid arthritis(RA) induced rats by achyranthes radix(AR) extracts ointment according to the application methods with the change of motor-behavioral and immunohistochemistry study through the change of COX-2 in the knee joint and PGE2 concentration production in serum (pre, 3^st, 7^st, 14^st, 21^st, 28^stday). The results of this study were as follows:
In result of gross & histological examination, after RA induction were observed reddening, edema and erythema. And H&E stain were observed the destruction of articular cartilage, bony erosion and the infiltration of inflammatory cells after RA induction. In result of volume change of hind paw edema and arthritis indices test and arthritic dorsal flexion & plantar flexion pain test, group Ⅱ, Ⅲ, Ⅳ were showed that significantly decrease to arthritis indices compared with group Ⅰ at 28^th day of RA induction. In result of mechanical allodynia test, After RA induction were observed lowering of the mechanical threshold when compared to the pre RA induction. Because of RA induced rats developed mechanical hypersensitivity. Especially, group Ⅱ, Ⅲ, Ⅳ were showed that significantly increase mechanical threshold compared with group Ⅰ. In result of safranin o-fast green stain were showed histological indices, group Ⅱ, Ⅲ, Ⅳ were showed that significantly decrease the scores of cellular infiltration and synovial hyperplasia, pannus formation and cartilage destruction compared with group Ⅰ at 28^th day of RA induction and group Ⅳ were the most decreased compared with group Ⅱ, Ⅲ at 28^th day of RA induction. In result of immunohistochemistric response of COX-2, group Ⅱ, Ⅲ, Ⅳ were showed that lower response effect compared with group Ⅰ at 28^th day of RA induction.
Group Ⅰ were multicentric response but group Ⅱ, Ⅲ, Ⅳ were lower local response at synovial tissue. In result of prostaglandin E₂ concentration in serum were investigated at the end of experiment. The PGE₂ concentration of the experimental group Ⅰ were increased when compared to the pre RA induction. But the PGE₂ concentration of the group Ⅱ, Ⅲ, Ⅳ were decreased compared to the group Ⅰ.
Ⅳ. Suggestion
In conclusion, Content of 20-hydroxyecdysone in Archyranthes Radix showed average 2.18% in samples collected throughout the regions of Korea. In addition, Archyranthes Radix in osteoarthritis results are consistent with the hypothesis that under conditions of oxidative stress, the H₂O₂-induced inhibition of cell proliferation in the rat chondrocyte is mediated through a modulation of Archyranthes Radix, promoting further anti-arthritis effect, indicating a potentially important role in cartilage repair and in the treatment of osteoarthritic cartilage. The above results suggest that Achyranthes radix extracts ointment effective acts were the most therapeutic intervention in inflammatory, pain control and motor-functional recovery in osteoarthritis and rheumatoid arthritis induced rat.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 4
• SUMMARY ... 15
• CONTENTS ... 19
• 목차 ... 24
• 제 1 장 연구개발과제의 개요 ... 30
• 제 1절 연구개발의 목적과 필요성 ... 30
• 제 2절 연구개발의 범위 ... 35
• 제 2 장 국내외 기술개발 현황 ... 36
• 제 3 장 연구개발수행 내용 및 결과 ... 37
• 제 1절 우슬의 항관절염 예방 및 치료제로서의 특성 조사, 물질 분리 및 생리기능성 연구 ... 37
• 1. 생약 성분(우슬)에 대한 특성 조사 ... 37
• 2. 우슬의 성분 추출 및 분석 ... 39
• 가. 우슬의 추출용매 선정 ... 39
• 나. 추출조건 확정 ... 40
• 다. 분획에 의한 성분 분리 ... 40
• 1) 분획의 순서 ... 40
• 2) Hexane층 ... 41
• 3) Chloroform층 - 3차 ... 42
• 4) Ethyl acetate층 - 7차 ... 42
• 5) Butanol층 - 2차 ... 42
• 라. 우슬의 부위에 따른 성분함량 비교 ... 43
• 3. 우슬의 기능성 물질 분리 ... 44
• 가. 재료 및 방법 ... 44
• 1) 시약 및 재료 ... 44
• 2) 기기와 검체 ... 44
• 3) 확인시험 ... 44
• 4) 검조감량과 회분시험 ... 44
• 5) 산불용성 회분시험 ... 45
• 6) 20-Hydroxyecdysone 추출 및 정제 ... 45
• 7) HPLC의 분석조건 및 검량선 작성 ... 46
• 8) 검액의 조제 및 20-Hydroxyecdysone의 함량 ... 48
• 나. 결과 및 고찰 ... 49
• 1) 확인시험 및 건조함량 ... 49
• 2) 회분시험 및 산불용성 회분시험 ... 49
• 3) 20-Hydroxyecdysone 추출 및 정제 ... 49
• 4) 20-Hydroxyecdysone의 검량선 ... 49
• 5) 20-Hydroxyecdysone의 함량 ... 50
• 4. 추출차의 제조 ... 52
• 5. 우슬의 연골 세포 독성 실험 및 대사활성 기능 실험 ... 55
• 가. 재료 및 방법 ... 55
• 1) 시약 및 재료 ... 55
• 2) 연골세포 분리 및 배양 ... 56
• 3) 관절연골세포의 자가고사 유도 ... 57
• 4) XTT를 이용한 연골세포의 증식 변화 ... 57
• 나. 결과 및 고찰 ... 58
• 1) 우슬 농도에 따른 증식률 ... 58
• 2) 우슬 처리 후 배양시간에 따른 증식률 ... 58
• 3) 과산화수소수 농도에 따른 세포 증식률 ... 60
• 4) 과산화수소수 처리 후 배양시간에 따른 세포 증식률 ... 60
• 5) 우슬이 prostaglandin E2에 미치는 영향 ... 62
• 제 2절 퇴행성 관절염과 류마티스 관절염에 우슬의 효과 ... 63
• 1. 퇴행성관절염에서 우슬이 미치는 영향 ... 63
• 가. 서설 ... 63
• 나. 재료 및 방법 ... 64
• 1) 실험동물 ... 64
• 2) 퇴행성 골관절염 모델 ... 65
• 3) 실험구성도 ... 65
• 4) 우슬 크림 제조 ... 66
• 5) 조직학적 검사 ... 67
• (1) Hematoxylin & Eosin (H & E) 염색 ... 68
• (2) Safranin O-fast green 염색 ... 68
• (3) 조직학적 평가 ... 68
• 다. 결과 및 고찰 ... 70
• 1) 퇴행성 골관절염 유발 시 병리조직학적 평가 ... 70
• 2) 치료 종료 후 병리조직학적 소견 ... 72
• 3) 치료 종료 후 병리조직학적 평가 ... 74
• 2. 류마티스 관절염에서 우슬이 미치는 영향 ... 79
• 가. 서설 ... 79
• 나. 재료 및 방법 ... 82
• 1) 실험동물 ... 82
• 2) 류마티스 관절염 모델 ... 83
• 3) 우슬추출물 경피투과제의 제조 및 연고도포방법 ... 83
• 4) 행동학적인 관찰 평가 ... 83
• (1) 족 부종의 체적변화 ... 84
• (2) 관절염 지수의 임상적 평가 ... 84
• (3) 관절염의 배측굴곡과 저측굴곡 통증 지수 ... 84
• (4) 기계적 이질통 측정(Measurement of mechanical allodynia) ... 85
• 5) 조직학적 검사(Histological assessment) ... 86
• (1) 조직 표본제작 ... 86
• (2) 류마티스 관절염 유발 전ㆍ후 슬관절의 병리조직학적인 관찰 ... 86
• (3) 실험 종료 후 슬관절의 병리조직학적인 관찰 ... 86
• 6) COX-2 단백질에 대한 면역조직화학 염색 ... 87
• 7) 혈청에서의 Prostaglandin E2 농도 측정 ... 87
• 다. 결과 ... 89
• 1) 류마티스 관절염의 육안 및 조직학적 유발 소견 ... 89
• 2) 행동학적인 관찰 평가 ... 89
• (1) 족 부종의 체적변화 ... 89
• (2) 관절염 지수의 임상적 평가 ... 91
• (3) 관절염의 배측굴곡 통증 지수 ... 92
• (4) 관절염의 저측굴곡 통증 지수 ... 94
• (5) 기계적 이질통의 변화 ... 95
• 3) 조직화학적 평가 ... 96
• (1) 슬관절의 Safranin O-fast green 염색을 통한 조직화학적 반응 ... 96
• (2) 슬관절의 Safranin O-fast green 염색을 통한 조직학적인 지수 평가 ... 97
• (3) 슬관절의 COX-2 단백질의 발현 면역조직화학적 반응 ... 98
• 4) 혈청에서의 Prostaglandin E2(PGE2)의 농도 변화 ... 99
• 5) 혈청에서의 염증성 사이토카인의 농도 변화 ... 100
• 6) 방사선학적 소견 ... 102
• 라. 고찰 ... 104
• 제 3절 우슬 함유 경피 흡수 제제 제조 ... 108
• 1. 경피흡수제 ... 108
• 2. 경구투여제 ... 114
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 116
• 제 1절 연구개발 목표의 달성도 ... 116
• 제 2절 관련 분야에의 기여도 ... 117
• 제 5장 연구개발결과의 활용계획 ... 118
• 제 1절 활용방안 ... 118
• 제 2절 산업화 추진방안 ... 120
• 제 6장 연구개발과정에서 수집한 해외과학 기술정보 ... 121
• 제 7장 참고문헌 ... 123
• 끝페이지 ... 135