KR20000054393A - Pharmacolocial effect and extracting method for chronic osteoporosis and rhematoid arthritis treatment by constituent drugs of oriental medicine - Google Patents

Pharmacolocial effect and extracting method for chronic osteoporosis and rhematoid arthritis treatment by constituent drugs of oriental medicine Download PDF

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KR20000054393A
KR20000054393A KR1020000030608A KR20000030608A KR20000054393A KR 20000054393 A KR20000054393 A KR 20000054393A KR 1020000030608 A KR1020000030608 A KR 1020000030608A KR 20000030608 A KR20000030608 A KR 20000030608A KR 20000054393 A KR20000054393 A KR 20000054393A
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water extract
osteoporosis
rheumatoid arthritis
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신준식
김상태
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A61K36/8998—Hordeum (barley)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A61K36/815—Lycium (desert-thorn)
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

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Abstract

PURPOSE: A drug extract and an extraction method thereof are provided. The extract comprises substances which inhibit the proliferation of osteoclast and induce the osteogenesis, therefore it can be used for the prophylaxis and the treatment of chronic bone diseases and degenerative bone diseases such as chronic osteoporosis and rheumatoid arthritis. CONSTITUTION: A drug extract for the prophylaxis and the treatment of the chronic osteoporosis and the rheumatoid arthritis comprises 100g of Hordei Fructus Germinatus, 75g of Testudinis Plastrum, 150g of Lycii Fructus, 150g of Rehmanniae Radix Preparata, 100g of Cinnamomi Cortex Spissus, 100g of Okbal, 150g of Cibotii Rhizoma, 200g of Cervi Cornu, 200g of Salviae Radix, 150g of Ledebouriellae Radix, 150g of Achyranthis Radix, 150g of Acanthopanacis Cortex, 150g of Eucommiae Cortex, 100g of Scolopendra, 35g of Crataegi Fructus and 40g of Massa Medicata Fermentata. The method for the extraction comprises the steps of: mixing 2kg of the respective drugs in dry weight with 5 L of distilled water; slowly heating the mixture at 120°C for 6 hours; filtering and concentrating under vacuum; and lyophilizing the concentrate to yield the drug extract of 95g by weight.

Description

구성약제의 만성골다공증 및 류마티스 관절염 치료를 위한 물추출물 및 추출방법{Pharmacolocial effect and extracting method for chronic osteoporosis and rhematoid arthritis treatment by constituent drugs of oriental medicine}Pharmacolocial effect and extracting method for chronic osteoporosis and rhematoid arthritis treatment by constituent drugs of oriental medicine}

본 발명은 구성약제에서 추출한 물추출물에 함유한 골수세포내 조골세포의 증식촉진과 파골세포의 분해를 억제함으로 골형성에 유효성분이 함유되어 있음을 검정하는 구성약제의 만성골다공증 및 류마티스 관절염 치료를 위한 물추출물 및 추출방법에 관한 것이다.The present invention is for the treatment of chronic osteoporosis and rheumatoid arthritis of constituent drugs to test that the active ingredient is contained in bone formation by inhibiting the proliferation of osteoblasts and the decomposition of osteoclasts in bone marrow cells contained in the water extract extracted from constituents It relates to a water extract and extraction method.

주지하는 바와 같이 현재 우리나라에서 다양한 골질환으로 호소하는 환자가 매년 증가 추세인 것으로 알려져 있으나, 골질환 치료를 목적으로 사용하는 화학요법, 수술요법 등은 아직 완벽한 성과를 거두지 못한 형편이다.As is well known, patients who are currently complaining of various bone diseases in Korea are increasing every year, but chemotherapy and surgery, which are used for the treatment of bone diseases, have not yet achieved perfect results.

이러한 골질환은 노화로 인한 만성 내지 퇴행성으로 진행되고 그에 따르는 고통뿐만 아니라 의료부담이 많이 차지하고 있어 임상 분야에 전반적으로 활용할 수 있는 신물질의 개발이 절실히 요청되고 있는 실정이다.These bone diseases are progressing from chronic to degenerative due to aging and the medical burdens as well as the pain accompanying them are required to develop new materials that can be utilized in the clinical field as a whole.

특히, 상기한 골질환 중 골다공증(Osteoporosis)은 골조직 내 미세환경 하에 존재하는 조골세포와 파골세포의 불균형에 의해 유발되는데, 지금까지 알려진 보고에 의하면 노화나 여성의 갱년기 장애로 인한 산전ㆍ후로 보통 일시적으로 일어나지만 외부로부터 병원체의 유입에 따른 항원의 2차적인 T세포 자가면역이 결국 골조직의 파괴를 촉발시키는 요인이 된다고 하나 아직 명확하지 않으며, 현재로는 바이러스나 세균의 감염으로 세포 내 IL-1, IL-6, M-CSF, TGF-β, PTH, Vitamin D3 같은 요인에 의해 파골세포화가 유도된다고 보고되고 있다.In particular, osteoporosis is caused by the imbalance of osteoblasts and osteoclasts in the microenvironment of bone tissues. According to the known reports, osteoporosis is usually temporary due to premature and postpartum periods due to aging or women's menopausal disorders. Although secondary T-cell autoimmunity of antigens following the influx of pathogens from the outside is a factor that eventually triggers the destruction of bone tissue, it is not clear yet. It has been reported that osteoclastization is induced by factors such as IL-6, M-CSF, TGF-β, PTH and Vitamin D3.

또한, 에스트로젠의 고갈과 그로 인한 파골세포 주위의 IL-6의 촉진에 따라 활성화를 유도됨으로 OPG와 RANK의 불균형적인 분비로 인한 파골세포화가 진행되고 다핵세포이면서 TRAP 양성반응을 가진 파골세포가 크게 증식되면 골단백분해효소인 Cathepsin K/L 같은 것이 분비하여 골조직에 구성하는 콜라겐(Collagen)과 칼슘의 결합이 붕괴되고 그로 인해 혈액내로 유출되어진 칼슘은 소변으로 배출하는데 결국 골조직내 칼슘의 고갈이라는 골조직내 구멍이 형성된 것 같은 질환으로 유발됨으로 여러 가지 이차적인 요인에 의해 골절이나 디스크 및 골격의 탈출이완이 초래되므로 그로 인한 심한 통증 내지 신경 염증을 동반한 신체 마비를 유발하는 지경으로 진행되어지며 한편 골조직의 붕괴와 연관지어 디스크나 만성 류마티스 관절염의 병인에 골조직 주변의 자가면역이 유발되므로 해서 촉발된 NO의 형성을 유도하는 iNOS의 합성이 촉진됨과 동시에 COX-2의 발현이 증가하고 부수적으로 PGE2의 분비가 증가되어진다.In addition, activation is induced by depletion of estrogens and the promotion of IL-6 around the osteoclasts, thereby leading to osteoclastization due to the disproportionate secretion of OPG and RANK and proliferating osteoclasts with multinucleated and TRAP-positive responses. When cathepsin K / L, which is an osteoprotein enzyme, is secreted, collagen (Collagen) and calcium binding to bone tissue are broken down and calcium released into the blood is discharged into the urine, resulting in the depletion of calcium in bone tissue. It is caused by a disease, such as a hole formation, and causes secondary fractures or loosening of discs and skeletons due to various secondary factors, which leads to severe pain or physical paralysis accompanied by nerve inflammation. Disc and bone etiology in the pathogenesis of chronic rheumatoid arthritis Mask station is caused to trigger at the same time, because the synthesis of iNOS promoted to induce the formation of NO increases the expression of COX-2 and is is incidentally increased the secretion of PGE2.

그로 인한 염증이 유발되고 연골조직을 파괴하는 환경이 조성됨으로 활액막의 붕괴를 촉진시키게 되며 상대적으로 통증을 호소하는 악순환이 가속화 될 것이다.As a result, inflammation and destruction of cartilage tissue will be created, which will promote the collapse of the synovial membrane and accelerate the relatively painful vicious cycle.

현재 상기와 같은 골질환에 관한 연구가 활발히 진행되고, 이에 따라 골수 내 일련의 면역지식이 급증함에 따라 최근에 몇 가지 골다공증 치료제가 개발되어 이들 중에는 allendrate, Tamoxifen, Vitamin D3, PTH 그리고 류마티스 관절염 치료제로 COX-2 저해제가 이미 상품화에 성공하였다.Currently, research on such bone diseases has been actively conducted, and as a result of the rapid increase in the immunological knowledge in the bone marrow, several treatments for osteoporosis have recently been developed. COX-2 inhibitors have already been commercialized.

한편, 오래 전부터 한방에서 사용하는 구성약제에는 조혈, 강장, 이뇨, 골수형성, 보신, 보양, 산전ㆍ후 관절, 요통, 건위, 소염, 활혈거습, 거풍, 해독, 지혈, 갱년기 장애, 통경, 자양, 보혈기능 등 다양한 효능이 있는 것으로 동의보감과 신농본초경에 수록되어 있으며, 기타 효능에 대해 고대문헌에 기록되어 있다.On the other hand, long-term use of herbal medicines include hematopoiesis, tonic, diuresis, bone marrow formation, bonsin, rehabilitation, pre and post-joint joints, low back pain, healthy stomach, anti-inflammatory, hemolytic, vomit, detoxification, hemostasis, menopausal disorders, pain, It has various effects such as nourishment and blood donation function, and it is listed in Dongbobogam and Shinnong Bone Carbide, and other effects are recorded in ancient literature.

그러나, 상기한 기록을 바탕으로 한방에서 많이 사용하는 구성약제의 유효한 약리 효능에 대한 구체적인 연구는 미약한 수준으로, 최근에 구성약제에서 골질환에 뛰어난 약효를 나타내는 약리 효능을 파악하여 이를 치료제로 활용하는 방안이 대두되고 있다.However, on the basis of the above record, the specific research on the effective pharmacological efficacy of the constituents that are widely used in herbal medicine is a weak level, and recently, the pharmacological effects of the pharmacological agents exhibiting excellent medicinal effects on bone diseases are utilized. The way to do it is emerging.

본 발명은 상기한 구성약제에서 골질환에 뛰어난 효과를 갖는 치료제를 개발하고자 하는 노력과 그 연구 성과를 통해 나타난 결과물로써, 본 발명의 목적은 구성약제의 약리 효능 연구를 통해 얻어진 결과를 토대로 다양한 구성약제를 감압 농축한 물추출물의 동결건조 중량들을 유효성분으로 함유하는 만성골다공증 및 류마티스 관절염같은 골질환 억제 내지 골재생 촉진용 조성물을 제공하는데 있다.The present invention is a result of the efforts to develop a therapeutic agent having an excellent effect on the bone disease in the above constituents and the research results, the object of the present invention is based on the results obtained through the study of the pharmacological efficacy of the constituents in various configurations The present invention provides a composition for inhibiting bone disease or promoting bone regeneration, such as chronic osteoporosis and rheumatoid arthritis, containing lyophilized weights of water extracts concentrated under reduced pressure as an active ingredient.

또한 본 발명의 다른 목적은, 구성약제의 조성물에서 유효성분으로 사용되는 물추출물을 추출하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for extracting the water extract used as an active ingredient in the composition of the constituent.

이와 같은 목적을 달성하기 위한 본 발명은, 골다공증 및 류마티스 관절염 같은 골질환에 응용할 치료제를 발견하기 위하여 수 천년간 동양에서 골질환 및 한의학적 관점에서 사용되어진 상기 구성약제(맥아, 구판, 구기자, 숙지황, 육계, 옥발, 구척, 녹각, 단삼, 방풍, 우슬, 오가피, 두충, 오공, 산사, 신곡)을 대상으로 연구를 수행하여 상기 약제의 물추출물이 만성골다공증 및 류마티스 관절염의 치료 및 예방에 효과적인 유효물질이 있음을 확인할 수 있었으며, 이 물추출물에서 만성골다공증 및 류마티스 관절염에 유효성분을 찾아내어 그 성분을 분리, 정제하고 그 활성성분의 구조를 밝혀내도록 함을 특징으로 한다.In order to achieve the above object, the present invention has been used in the oriental medicine for thousands of years from the viewpoint of bone disease and oriental medicine in order to find a therapeutic agent for bone diseases such as osteoporosis and rheumatoid arthritis (malt, gupan, gojija, sukjihwang, Studies on broilers, octaves, gukcheol, green tea, dansam, windproof, dew, agapi, tofu, pore, hawthorn, and new song In this water extract, it was found that the active ingredient is found in chronic osteoporosis and rheumatoid arthritis, and the component is separated and purified to reveal the structure of the active ingredient.

따라서, 본 발명은 물추출물로부터 골다공증 억제제 내지 골형성 성분을 찾아내어 작용점과 유효성분을 통해 골다공증, 류마티스 관절염, 골절, 디스크, 기타 만성 및 퇴행성 골질환 같은 임상 분야에 전반적으로 활용할 수 있다.Accordingly, the present invention finds osteoporosis inhibitors or osteogenic components from water extracts and can be utilized in clinical fields such as osteoporosis, rheumatoid arthritis, fractures, discs, and other chronic and degenerative bone diseases through the functioning points and active ingredients.

도 1은 유효약물의 추출과정에 따르는 물추출물의 추출방법을 나타낸 도면.1 is a view showing the extraction method of water extract according to the extraction process of the effective drug.

도 2는 유효약물의 추출 후 동결 건조하여 물추출물의 분광광도계로 나타낸 주 흡광도.Figure 2 is the main absorbance shown by the spectrophotometer of the water extract after freeze-dried after extraction of the effective drug.

도 3은 유효약물인 물추출물을 통해 부종을 유도시 소염작용을 나타낸 도면.Figure 3 shows the anti-inflammatory effect when inducing edema through the water extract as an effective drug.

도 4는 유효약물인 물추출물을 통해 조골세포인 ST-1의 형태학적 모양을 현미경으로 관찰한 도면.Figure 4 is a microscopic observation of the morphological shape of osteoblasts ST-1 through the water extract as an effective drug.

도 5는 유효약물인 물추출물을 통해 Raw264.7 세포주에 LPS를 자극하여 NO생성을 억제하는 것을 나타낸 그래프.Figure 5 is a graph showing the inhibition of NO production by stimulating LPS to Raw264.7 cell line through the water extract as an effective drug.

도 6은 유효약물인 물추출물을 통해 파골세포 내 COX-2의 발현을 억제한 것을 나타낸 도면.6 is a view showing the inhibition of the expression of COX-2 in osteoclasts through the water extract as an effective drug.

도 7은 유효약물인 물추출물을 통해 LPS를 자극하여 COX-2 단백질 발현 유도해서 발현을 억제한 것을 RT-PCR을 통해 확인 이를 전기영동장치에서 나타낸 도면.7 is confirmed by RT-PCR that inhibited expression by inducing COX-2 protein expression by stimulating LPS through the water extract as an effective drug, which is shown in the electrophoresis device.

도 8은 유효약물인 물추출물을 통해 LPS를 자극하여 iNOS 단백질 발현 유도해서 발현을 억제한 것을 RT-PCR을 통해 확인 이를 전기영동장치에서 나타낸 도면.8 is confirmed by RT-PCR to inhibit the expression by inducing iNOS protein expression by stimulating LPS through the water extract as an effective drug This is shown in an electrophoresis device.

도 9는 유효약물인 물추출물을 통해 난소 적출 흰쥐의 골다공증 모델을 통해 골소주의 수와 길이가 증가되어 골다공증 치료에 효과적임을 나타낸 도면.Figure 9 is an effective drug water extract through the osteoporosis model of the ovary extraction rat osteoporosis model showing that the increase in the number and length of osteoporosis is effective in treating osteoporosis.

도 10은 유효약물인 물추출물을 통해 난소 적출 흰쥐의 골다공증모델을 통해 골 분해효소의 분비억제를 확인하기 위해 면역조직화학분석을 나타낸 도면.10 is an immunohistochemical analysis for confirming the inhibition of osteolytic enzyme secretion through the osteoporosis model of ovarian extraction rats through the water extract as an effective drug.

도 11은 유효약물인 물추출물을 통해 임상적으로 디스크가 흘러나온 환자를 대상으로 치료에 디스크가 거의 소실되어 원상 복귀된 것을 CT촬영을 통해 나타낸 사진을 나타낸 도면.FIG. 11 is a photograph showing a CT photograph showing that the disk is almost lost due to treatment, and the disk is returned to the patient with a clinically released disk through the water extract as an effective drug. FIG.

이하, 첨부된 도면을 참조하여 본 발명의 실시 예를 상세히 설명하도록 한다.Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

도 1은 본 발명에 따른 유효약물의 추출과정에 따르는 물추출물의 추출방법을 나타낸 도면이다.1 is a view showing a method of extracting the water extract according to the extraction process of the effective drug according to the present invention.

본 발명의 대상이 되는 구성약제는 맥아, 구판, 구기자, 숙지황, 육계, 옥발, 구척, 녹각, 단삼, 방풍, 우슬, 오가피, 두충, 오공, 산사, 신곡 등으로, 본 발명에 따르는 조성물에서 유효성분으로 사용되는 물추출물은 다음과 같은 함량으로 이루어진다.The constituent pharmaceuticals of the present invention are malt, gupan, wolfberry, hungjihwang, broiler, octane, gukcheok, green tea, dansam, windproof, dew, staphylococcus, tofu, five holes, hawthorn, new song, etc., effective in the composition according to the present invention. Water extract used as a component consists of the following contents.

즉, 본 발명의 물추출물은, 맥아 100g, 구판 140g, 구기자 140g, 숙지황 100g, 육계 100g, 옥발 100g, 녹각 200g, 단삼 200g, 방풍 200g, 우슬 240g, 오가피 120g, 두충 120g, 오공 40g, 산사 100g, 신곡 100g을 포함하여 이루어진다.That is, the water extract of the present invention, malt 100g, old plate 140g, wolfberry 140g, sucrose sulfur 100g, broiler 100g, octane 100g, green rust 200g, sweet ginseng 200g, windproof 200g, dew 20g, oopi 120g, tofu 120g, pore 40g, hawthorn 100g , Including 100g of new songs.

이와 같은 함량을 가지는 물추출물의 추출방법은 다음과 같다.Extraction method of the water extract having such a content is as follows.

즉, 맥아, 구판, 구기자, 숙지황, 육계, 옥발, 구척, 녹각, 단삼, 방풍, 우슬, 오가피, 두충, 오공, 산사, 신곡의 총 건조 중량을 2Kg으로 해서 3차 증류수 5-15배의 부피(여기서는 5,000㎖)로 배합하여 120℃ 조건 하에 6시간동안 서서히 열을 가하여 최종적인 부피가 1,000㎖이 되게 한 다음, 여과장치에서 여과 후 감압 농축하여 200㎖이 되게 한 다음, 이를 동결 건조시켜 95g의 동결건조중량을 얻었다.That is, the total dry weight of malt, gupan, goji berry, sukjihwang, broiler, oxal, gucheok, green tea, dansam, windproof, dew, agapi, tofu, goku, hawthorn, and new song is 2Kg and the volume of tertiary distilled water is 5-15 times. (5,000 ml here), and slowly heated under 120 ° C. for 6 hours to obtain a final volume of 1,000 ml, followed by filtration and concentration under reduced pressure to 200 ml, followed by freeze drying at 95 g. Lyophilized weight of was obtained.

상기한 바와 같은 일련의 건조과정에 수득되는 엑기스 물추출물은 각각 후술하는 실험 예에 의해 입증하는 바와 같이 골다공증, 류마티스 관절염 및 항소염 활성효과를 가지고 있기 때문에 물엑기스는 본 발명의 조성물에서 유효성분으로 사용될 수 있다.Since the extract water extract obtained in the series of drying processes as described above has osteoporosis, rheumatoid arthritis and anti-inflammatory activity effects, respectively, as demonstrated by the following experimental examples, the water extract is used as an active ingredient in the composition of the present invention. Can be used.

본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 자외선 분광광도계(UV-spectrophotometer)을 수행하였으며 그 결과 유효성분의 주 패턴이 280 nm와 485 nm의 피크(peak)를 포함하고 있었다.(도면 2)In order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention, an ultraviolet spectrophotometer (UV-spectrophotometer) was performed on the water extract obtained according to the method of the present invention. It contained peaks of 280 nm and 485 nm (Fig. 2).

본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 본 발명의 조성물이 가지는 부종 억제효과를 λ- 카라게닌(λ- Carrageenan) 0.2mg/㎖ 와 자이모산-A(Zymosan-A)를 5㎍/ 0.1㎖이 되게 흰쥐의 좌측 족부에 주사를 한 후 본 발명에 따른 물추출물의 건조중량을 0.5g/㎖이 되게 해서 2일간 경구 투여한 결과를 부종감소가 90%이상 감소시킴을 알 수 있다.(도면 3)In order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention to the water extract obtained according to the method of the present invention λ-carrageenan (λ- Carrageenan) After injection of 0.2mg / mL and Zymosan-A to 5μg / 0.1mL in the left foot of the rat, the dry weight of the water extract according to the present invention was 0.5g / mL for 2 days. It can be seen that the result of oral administration reduced edema reduction by more than 90% (Fig. 3).

본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 bone marrow세포인 ST-2를 통해 세포 형태적인 변화를 확인하기 위해 6 well에 세포를 104세포를 분주한 다음, 이를 5% CO2인큐베이터(Incubator)에서 37℃하에 배양한 다음 상기 물추출물을 처리하여 세포 형태적인 변화를 확인한 결과 세포증식보다는 분화를 유도하는 양상을 보임을 알 수 있었다.(도면 4).In order to identify the active constituents in the water extract used as an active ingredient in the composition of the present invention 6 water well to confirm the morphological changes through the bone marrow cells ST-2 for the water extract obtained according to the method of the present invention After dispensing 10 4 cells into the cells, the cells were cultured at 37 ° C. in a 5% CO 2 incubator, and then treated with the water extract to confirm cell morphological changes, which induces differentiation rather than cell proliferation. (Fig. 4).

본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 상기한 바와 같이 부종을 인한 혈액내의 강력한 염증 유발원인 NO의 합성억제를 확인하기 위해 macrophage인 RAW264.7을 96 microplate에 각 well마다 103세포수가 되게 분주한 다음 12시간 배양하여 LPS를 50 ng/well이 되게 각 well에 분주하고 2시간동안 자극을 가한 다음 물추출물의 최종농도가 50㎍/㎖이 되도록 가한다.Confirmation of the synthesis inhibition of NO, a potent source of inflammation in the blood due to edema, as described above for the water extract obtained according to the method of the present invention in order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention. To do this, the macrophage RAW264.7 was dispensed into 96 microplates at 10 3 cell numbers per well, and cultured for 12 hours. LPS was dispensed into each well to 50 ng / well and stimulated for 2 hours. The concentration is added to 50 µg / ml.

여기서, 상기 물추출물의 처리는 LPS의 처리 전, 후로 해서 실시한다. 그 결과에 따른 NO의 생성은 대조군에서 약 79 ~ 98 ㎛정도인데 반해 물추출물의 생성은 약 85 ~ 90% 정도 감소함을 알 수 있었다. (도면 5).In this case, the water extract is treated before and after the treatment of LPS. As a result, the production of NO was about 79 ~ 98 ㎛ in the control group, whereas the production of water extract was found to decrease by about 85 ~ 90%. (Fig. 5).

상기 결과에서처럼 본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 류마티스 관절염의 원인인자인 COX-2의 발현을 억제하는지를 면역세포화학분석법을 조사한 결과 커버 글라스(cover glass)위에 흰쥐로부터 분리된 파골세포를 분주하고 12시간 배양한 다음 LPS를 적당량 처리해서 자극하여 상기 물추출물을 처리하고 PBS로 2번 세척하여 1차 항체인 COX-2를 반응시켜 표지 한 다음, 이를 2차 항체인 FITC를 표지하여 형광 현미경하에서 조사해 보면 본 조성물의 COX-2의 발현이 억제됨을 알 수 있다.(도면 6).Immune cells whether the water extract obtained according to the method of the present invention to inhibit the expression of COX-2, a causative agent of rheumatoid arthritis, to the water extract obtained according to the method of the present invention to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention as described above As a result of the chemical analysis, the osteoclasts isolated from the rats were dispensed on the cover glass, incubated for 12 hours, and then stimulated by treatment with an appropriate amount of LPS. The water extracts were treated and washed twice with PBS. After reacting and labeling -2, the secondary antibody FITC was labeled and examined under a fluorescence microscope, indicating that COX-2 expression of the composition was inhibited (Fig. 6).

본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 류마티스 관절염의 지표 단백질인 iNOS나 COX-2 발현의 억제에 미치는 영향을 알아보기 위해 상기 방법과 같이 세포를 PBS로 2회 세척한 다음, 이를 RNA zol 용액을 가하여 이를 세포 내 핵산과 단백질을 분리한 다음, 이를 15,000rpm으로 15분간 원심 분리하여 상등액과 isopropanol 200㎕을 가해 혼합한 후 냉장고-20℃에 15분간 방치한 다음, 이를 원심 분리하여 total RNA를 분리하여 이를 RT-PCR를 실시한 다음 1% agarose gel에서 확인한 결과 대조군에서는 발현을 유도되는 양상을 보인 반면에 물추출물 처리군에서는 발현이 억제됨을 알 수 있었다.(도면 7)(도면 8).To determine the effect of inhibiting the expression of iNOS or COX-2, which is an indicator protein of rheumatoid arthritis, to the water extract obtained according to the method of the present invention in order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention In order to see the cells, the cells were washed twice with PBS as described above, followed by addition of RNA zol solution to separate the nucleic acid and proteins in the cells, followed by centrifugation at 15,000 rpm for 15 minutes, followed by mixing with supernatant and 200 μl of isopropanol. After leaving for 15 minutes in the refrigerator -20 ℃, centrifuged to separate the total RNA, RT-PCR was carried out and confirmed by 1% agarose gel, the control group showed the induction of expression, while the water extract treatment It was found that expression was suppressed in the group (Fig. 7) (Fig. 8).

본 발명의 조성물에서 유효성분으로 사용되는 물추출물 중에 활성성분을 규명하기 위하여 본 발명의 방법에 따라 수득된 물추출물에 대하여 골다공증에 미치는 영향을 조사하기 위해 난소 적출 흰쥐에 물추출물을 0.5g/1㎖이 되게 경구투여를 7일간 실시하고 2주간 사육하여 대퇴골을 적취한 다음 이를 골조직의 표본으로 제작하였다.0.5g / 1 water extract in ovarian extract rats to investigate the effect on osteoporosis of water extract obtained according to the method of the present invention in order to identify the active ingredient in the water extract used as an active ingredient in the composition of the present invention Oral administration to ㎖ was carried out for 7 days, and breeding for 2 weeks to extract the femur bones were prepared as a sample of bone tissue.

헤마토시렌/에오신(hematoxylen/eosin)으로 염색하여 현미경하에서 관찰한 결과 대조군경우 성장판 부위의 골소주 수와 길이가 상대적으로 많이 소실된 데 비해 물추출물 처리군은 경골(tibia)의 수와 길이가 정상군과 유사하였으며, 면역조직화학 분석법에서처럼 콜라겐IV(collagen IV)의 분해효소의 발현양상 또한 대조군 경우는 상대적으로 많이 존재하나 정상군과 물추출물 처리군은 거의 발현이 없음을 알 수 있다.(도면 9)(도면 10).Staining with hematoxylen / eosin (hematoxylen / eosin) and observation under a microscope showed that the number and length of bone loss in the growth plate area were relatively large, whereas the water extract treatment group had a normal number and length of tibia. As in the immunohistochemical analysis, the expression of collagen IV degrading enzyme was also relatively high in the control group, but the normal group and the water extract treatment group showed little expression. 9) (Fig. 10).

상기 발명에 따른 일련의 물추출물 활성효과에 대한 임상적인 약리 효과를 조사하기 위해 본 발명의 방법에 따라 수득된 물추출물에 대하여 디스크 환자를 대상으로 디스크가 흘러나온 실례를 통해 물추출물을 복용한 다음 치료 후 CT촬영을 실시하여 본 결과 화살표에서처럼 디스크가 거의 소실됨을 알 수 있었다.In order to investigate the clinical pharmacological effect on the series of water extract active effects according to the present invention, the water extract obtained according to the method of the present invention was administered to the disc patient by taking a water extract from the disc. After treatment, CT scans showed that the disc was almost lost as shown by the arrow.

따라서, 본 물추출물의 분석결과와 약리작용 및 골질환 환자의 실질적인 치료효과를 확인함으로 본 물추출물의 물추출물이 골질환에 유익한 치료 및 예방효과를 가짐이 입증되었다.(도면 11).Therefore, by confirming the analysis results of the water extract and the pharmacological action and the actual therapeutic effect of patients with bone disease, the water extract of the water extract proved to have a beneficial treatment and prevention effect on bone disease (Fig. 11).

이상에서 설명한 바와 같은 본 발명은 조성물의 약제조성, 추출방법, 추출조건 특히 물추출물에서 동결 건조시켜 수득되어진 분말 엑기스는 우수한 골다공증, 류마티스 관절염 및 항소염 효과를 나타낸다.The present invention as described above, the pharmaceutical composition of the composition, the extraction method, the extraction conditions, especially the powder extract obtained by freeze drying in the water extract shows excellent osteoporosis, rheumatoid arthritis and anti-inflammatory effect.

본 발명에 따라 물추출물이 엑기스보다 동결건조 상태에서의 보존기간에 따른 변형 및 약리효과의 손실이 전무하고 본 발명에 의해 밝혀진 조성물이 골다공증이나 류마티스 관절염 및 디스크 같은 골질환 증상의 임상 분야에 예방 및 치료제로서 사용될 수 있는 효과가 있다.According to the present invention, there is no loss of modification and pharmacological effects according to the shelf life in the lyophilized state than the extract, and the composition disclosed by the present invention prevents and prevents the clinical field of bone disease symptoms such as osteoporosis or rheumatoid arthritis and disc. There is an effect that can be used as a therapeutic agent.

Claims (3)

맥아 100g, 구판 75g, 구기자 150g, 숙지황 150g, 육계 100g, 옥발 100g, 구척 150g, 녹각 200g, 단삼 200g, 방풍 150g, 우슬 150g, 오가피 150g, 두충 150g, 오공 100g, 산사 35g, 신곡 40g의 유효성분을 함유하는 것을 특징으로 하는 구성약제의 만성골다공증 및 류마티스 관절염 물추출물.Malt 100g, old plate 75g, wolfberry 150g, sucrose sulfur 150g, broiler 100g, octane 100g, gukcheok 150g, green angle 200g, soybean 200g, windproof 150g, dew 150g, ogapi 150g, tofu 150g, pore 100g, hawthorn 35g, new song 40g Chronic osteoporosis and rheumatoid arthritis water extracts of constituents, characterized in that it contains. 제1항에 있어서,The method of claim 1, 상기 유효성분으로서 물추출물을 동결 건조시켜 물추출물과 동일한 활성을 함유하는 건조분말을 제조하는 것을 특징으로 하는 구성약제의 만성골다공증 및 류마티스 관절염 물추출물.Chronic osteoporosis and rheumatoid arthritis water extracts of constituents, characterized in that the lyophilized water extract as an active ingredient to produce a dry powder containing the same activity as the water extract. 맥아, 구판, 구기자, 숙지황, 육계, 옥발, 구척, 녹각, 단삼, 방풍, 우슬, 오가피, 두충, 오공, 산사, 신곡의 건조중량 약제 2Kg을 증류수 5000㎖로 배합하는 제1단계,First step of mixing 2 kg of dry weight chemicals of malt, gupan, goji berry, ripe koji, broiler, octave, gucheok, green tea, dansam, windproof, dew, scallop, tofu, goku, hawthorn and new song with 5000 ml of distilled water, 120℃ 조건 하에 6시간동안 서서히 열을 가하여 최종적인 부피가 1,000㎖이 되게 하는 제2단계,A second step of slowly heating under 120 ° C. for 6 hours to bring the final volume to 1,000 ml, 상기 제2단계 수행 후, 여과장치에서 여과하고 감압 농축하여 200㎖이 되게 하는 제3단계,After performing the second step, the third step of filtering and filtration under a reduced pressure to 200ml, 상기 제3단계에서 감압 농축된 것을 동결 건조시켜 95g의 동결 건조 중량을 얻는 제4단계로 이루어짐을 특징으로 하는 구성약제의 만성골다공증 및 류마티스 관절염 물추출물 추출방법.Chronic osteoporosis and rheumatoid arthritis water extract extraction method of the constituent pharmaceutical composition characterized in that the fourth step to obtain a freeze-dried weight of 95g by freeze drying the concentrated under reduced pressure in the third step.
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KR100380865B1 (en) * 2000-12-06 2003-04-18 한국 한의학 연구원 Extract of Sophorae Flos for the prevention and treatment of osteoporosis
KR100380864B1 (en) * 2000-12-06 2003-04-18 한국 한의학 연구원 Extract of Sophorae Radix for the prevention and treatment of osteoporosis
KR20020066242A (en) * 2001-02-09 2002-08-14 이수찬 A herb medicine for treating an arthritis
KR100472408B1 (en) * 2001-11-22 2005-03-08 주식회사 싸이클로젠 Method For Preparing Composition For Treating Osteoporosis
KR20040011748A (en) * 2002-07-30 2004-02-11 김호철 Composition comprising the rhizomas extract of Cibotium barometz L. J. SMITH having growth-promoting activity
KR100517385B1 (en) * 2003-07-30 2005-09-28 학교법인조선대학교 Tanshinone ⅡΑ inhibiting osteoclast differentiation and bone resorption

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