KR102001730B1 - A composition comprising Extract of Rhus verniciflua for preventing or treating Multiple Sclerosis - Google Patents
A composition comprising Extract of Rhus verniciflua for preventing or treating Multiple Sclerosis Download PDFInfo
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- KR102001730B1 KR102001730B1 KR1020170153063A KR20170153063A KR102001730B1 KR 102001730 B1 KR102001730 B1 KR 102001730B1 KR 1020170153063 A KR1020170153063 A KR 1020170153063A KR 20170153063 A KR20170153063 A KR 20170153063A KR 102001730 B1 KR102001730 B1 KR 102001730B1
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- multiple sclerosis
- rhus verniciflua
- eae
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Abstract
본 발명은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 치료용 약학적 조성물 및 건강기능식품에 관한 것이다. The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
Description
본 발명은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 치료용 약제학적 조성물 및 건강기능식품에 관한 것이다. The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
다발성경화증은 인체 내 면역계의 결손에 의해 유발되는 질환으로 뇌와 척수의 백질부분의 신경을 덮는 지방질로 된 수초(myelin sheath)를 침범하여 탈수초화(demyelination)를 산발적으로 일으키는 질환으로 수초가 손상을 받아 수초층의 두께가 감소하면 신경자극은 전달되지만, 효율성이 떨어지고 이에 정상적인 신경자극 전달이 방해를 받아 발병되는 질병이다. 쉽게 설명하자면, 전선을 둘러싸고 있는 절연체가 손상되면 전기의 전도가 느려지는 것과 마찬가지로 신경세포 간의 전도 속도가 느려지는 현상을 초래하여 증상을 일으킨다. 초기에는 수초가 회복되면서 완화증상이 나타나지만, 결국 악화가 반복되며 손상이 영구화된다. 이 같은 수초의 손상으로 인해 신경이 적절히 작용을 하지 못하여 시야장애나 균형감 상실 등의 증상이 야기되고 일부 환자에서는 마비가 올 수 있다.Multiple sclerosis is a disease caused by the deficiency of the immune system in the human body. It is a disease that sporadically causes demyelination by invading the myelin sheath, which covers the nerves of the brain and spinal cord. When the thickness of the supercell is decreased, the nerve stimulation is transmitted, but the efficiency is lowered and the nerve stimulation transmission is disturbed. If the insulator surrounding the wire is damaged, the conduction rate between the nerve cells is slowed down as well as the conduction of electricity is slowed. In the early days, the recovery of a few seconds causes symptoms of relief, but eventually the deterioration is repeated and the damage is permanent. Such a few seconds of damage may cause nerves to fail to function properly, causing visual disturbances or loss of balance, and in some patients, paralysis.
다발성경화증의 치료는 크게 급성기 치료와 장기적인 질병완화 치료, 그리고 증상완화 치료로 나눌 수 있다. 급성기에는 일반적으로 고용량 스테로이드(steroid)의 정맥주사 요법을 사용한다. 스테로이드는 급성기에 증상을 완화시키고 회복 기간을 줄여주지만, 장기적으로 투여할 경우에는 다양한 부작용이 나타날 수 있으므로 사용에 주의를 요한다. 여러 가지 원인에 의해 질병완화 치료하기 어려운 환자들은 저용량 스테로이드 유지 요법을 사용하기도 한다. 대표적인 질병완화 치료제로는 인터페론(beta-interferon)과 글라티라머 아세테이트(glatiramer acetate)가 있다. 이들은 재발완화형 다발성경화증(relapsing-remitting multiple sclerosis, RRMS)에서 재발하는 횟수를 줄이고, 재발 시에 나타나는 증상을 완화하는 효과를 가지고 있다. 그러나 이차진행형으로 전환된 환자에서는 뚜렷한 효과가 나타나지 않기 때문에 이 경우에는 환자의 장애를 예방하기 위한 조기 치료가 가장 중요하다. 다발성경화증으로 인해 나타나는 여러 증상과 신경학적 장애를 줄여주기 위한 증상완화 치료도 시행한다. 이러한 치료들은 재발율이 높으며, 재발을 확실하게 방지하는 방법은 없다.Treatment of MS is largely divided into acute phase treatment, long-term disease relief treatment, and symptom relief treatment. The acute phase generally uses intravenous therapy with high-dose steroids. Steroids alleviate symptoms at the acute phase and reduce the duration of recovery, but long-term administration can cause a variety of side effects and should be used with caution. Patients who are difficult to cure by various causes may use low-dose steroid maintenance therapy. Therapeutic agents such as interferon (beta-interferon) and glatiramer acetate. They have the effect of reducing the number of recurrences in relapsing-remitting multiple sclerosis (RRMS) and relieving the symptoms of relapsing-remitting multiple sclerosis (RRMS). However, in the case of patients who have been converted to secondary progressive type, there is no obvious effect, so in this case, early treatment is most important to prevent the patient's disability. Symptomatic treatment to reduce symptoms and neurological disorders caused by multiple sclerosis is also performed. These treatments have a high recurrence rate, and there is no way to reliably prevent recurrence.
이러한 배경 하에 본 발명자들은 다발성 경화증에 효과가 있는 천연 추출물에 관한 연구개발을 수행하여 본 발명을 완성하였다. Under these circumstances, the present inventors completed the present invention by carrying out research and development on natural extracts effective for multiple sclerosis.
본 발명의 목적은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 치료용 약제학적 조성물을 제공하는 것이다. It is an object of the present invention to provide a pharmaceutical composition for preventing or treating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
또한, 본 발명의 목적은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 개선용 건강기능식품을 제공하는 것이다. It is also an object of the present invention to provide a health functional food for preventing or ameliorating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
본 발명자들은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 치료용 약제학적 조성물 또는 건강기능식품의 유효 성분으로 유용하게 사용될 수 있음을 밝힘으로써 본 발명을 완성하였다. The present inventors have completed the present invention by disclosing that they can be effectively used as an active ingredient of a pharmaceutical composition or health functional food for preventing or treating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
이하, 본 명세서에 대하여 더욱 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 명세서에서 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다. Whenever a component is referred to as "comprising ", it is to be understood that the component may include other components as well, without departing from the scope of the present invention.
본 명세서에서 상기 "예방"이란 상기 약학적 조성물의 투여로 다발성 경화증을 억제 또는 지연시키는 모든 행위를 의미한다. 또한, 상기 "치료"란 상기 약학적 조성물의 투여로 다발성 경화증의 증세가 호전되거나 이롭게 변경하는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that inhibits or delays MS by administration of the pharmaceutical composition. The term "treatment" as used herein means any action that improves or alleviates symptoms of multiple sclerosis upon administration of the pharmaceutical composition.
본 발명은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 치료용 약제학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing or treating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
한편, 옻나무는 옻나무과(Anacardiaceae)에 속하는 낙엽활엽수목으로 학명은 Rhus verniciflua STOKES이다. 수액은 옻 또는 건칠이라 하여 공업용과 약용에 쓰이고, 특히 한방과 민간에서 주독, 해열, 구충, 학질, 복통, 통경, 변비 등에 약재로 쓰이고 옻닭과 같이 식품으로 이용되어 왔다. On the other hand, Rhus verniciflua STOKES is a deciduous broad-leaved tree belonging to Anacardiaceae (Rhus verniciflua). The sap is used for industrial and medicinal purposes because it is called lacquer or dry lacquer. Especially, it has been used as a medicinal product for gastrointestinal, paralytic, antiparasitic medicine, abdominal pain, constipation, constipation, etc.
한편, 옻나무는 옻칠을 위한 칠액을 생산하며, 그 껍질은 칠피라고 하여 한약재로서 이용된다. 칠액은 이를 건조하여 건칠이라는 이름으로 역시 한약재로 사용된다. 동의보감, 광제비급 등 한의서에 따르면 칠피를 제거한 옻나무의 목질부는 칠목 또는 칠수라고 하여 부종, 옹저 등의 증상에 사용되는 것으로 알려져 있다. 칠목은 옻나무의 목질부에 해당하는 것으로 추출물의 함량이 극히 적고, 푸스틴, 피세틴과 같은 플라보노이드 성분을 함유하고 있으며 알러지 유발성분인 우루시올의 함량은 극히 적어 손쉽게 옻이 오르는 성분을 제거하여 옻닭 등에 이용된다. 옻나무의 껍질을 제거할 경우 대부분의 경우 옻이 오르는 성질을 제거할 수 있다. 그러나, 칠피는 옻나무의 칠액을 생산하는 껍질부에 해당하는 것으로 칠액(건칠)에는 미치치 못하지만, 다수의 우루시올을 함유하고 있어 쉽게 옻이 오를 수 있다. 칠피에는 레진성분 및 소량의 플라보노이드 성분들이 함유되어 있다. 건칠은 옻나무에서 칠액으로 얻어진 것을 건조한 것으로, 칠액에는 알러지 유발성분인 우루시올을 다량 함유하고 있으며 그 복용에 있어서도 조심을 기해야 한다. 또한 건칠에는 다수의 폴리페놀 성분이 다량 함유되어 있으며 우루시올의 중합체로서의 레진들도 다량 함유되어 있다.On the other hand, lacquer tree produces lacquer for lacquer and its shell is used as herbal medicine called "chili". It is dried and used as herbal medicine under the name of dry skin. According to the Dong-bo-bo-gyung, the pseudo-ritual, etc., the woody part of the lacquer tree which has been removed from the chili is said to be used for symptoms such as edema and necrosis. The main component is the woody part of the lacquer tree. The content of the extract is extremely small. It contains flavonoids such as fostin and picetin. The content of urushiol, which is an allergen component, is very small. do. Removing the shell of Rhus verniciflua can remove the nature of poison ivy in most cases. However, chili pepper is a shell part that produces chrysanthemum lacquer. It does not cover chili (lacquer), but it contains a lot of urushiol, so it can easily climb lacquer. Chili pepper contains a resin component and a small amount of flavonoids. It is dried by the lacquer tree in dry lacquer, and it contains a large amount of urushiol which is an allergen inducing ingredient in the cholesterol, and care should be taken in its taking. In addition, it contains a large amount of polyphenol components and also contains a large amount of resins as polymers of urushiol.
본 발명의 일 구현예에 있어서, 상기 옻나무 추출물은 옻나무의 나무껍질을 추출한 것이다. 더욱 구체적으로 상기 옻나무 추출물은 옻나무의 나무껍질을 말린 것을 추출한 것이다. In one embodiment of the present invention, the Rhus verniciflua extract is a bark extract of Rhus verniciflua. More specifically, the Rhus verniciflua extract is obtained by extracting the dried bark of Rhus verniciflua.
본 발명의 일 구현예에 있어서, 상기 옻나무 추출물은 열수 추출물이다. 더욱 구체적으로 상기 옻나무 추출물은 옻나무의 나무껍질을 열수 추출한 것이다. In one embodiment of the present invention, the Rhus verniciflua extract is a hot-water extract. More specifically, the Rhus verniciflua extract is a hot-water extract of the bark of Rhus verniciflua.
본 발명의 일 구현예에 있어서, 상기 옻나무의 열수 추출 시 추출 온도는 90℃ 내지 100℃이다. In one embodiment of the present invention, the extracting temperature in hot water extraction of Rhus verniciflua is 90 ° C to 100 ° C.
본 발명의 일 구현예에 있어서, 상기 옻나무 추출물은 상기 열수추출물의 n-헥산 분획물이다. 상기와 같이 n-헥산을 이용하여 분획하는 경우에 옻 나무의 알레르기를 일으키는 우르시올(urushiol)을 제거할 수 있다. In one embodiment of the present invention, the Rhus verniciflua extract is a n-hexane fraction of the hot-water extract. In the case of fractionation using n-hexane as described above, urushiol which causes allergy of the lacquer tree can be removed.
본 발명의 약학적 조성물은 약효를 증가시키지는 않으나 약학적 조성물에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 성분을 추가로 포함할 수 있다. 또한, 본 발명의 약학적 조성물은 약학적으로 허용 가능한 첨가제를 추가적으로 포함할 수 있다. 약학적으로 허용 가능한 첨가제는 예컨대, 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 있으나, 이를 한정하지 않는다. 추가로, 상기 약학적 조성물은 단독 사용하거나 기존에 사용된 다발성 경화증에 대한 예방 또는 치료 활성을 가지는 물질을 포함할 수 있다.The pharmaceutical composition of the present invention does not increase the drug efficacy but may further include components that are commonly used in pharmaceutical compositions and can improve odor, taste, and visual appearance. In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, gum, arabic gum, pregelatinized starch, cornstarch, powdered cellulose, But are not limited to, hydroxypropylcellulose, opaques, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc. In addition, the pharmaceutical composition may include a substance that has a prophylactic or therapeutic activity against MS or MS that is used alone or in the past.
본 발명의 상기 약학적 조성물은 약학적으로 허용 가능한 담체를 포함하고 경구 또는 비경구용의 인체 또는 수의용으로 제형화될 수 있다. 본 발명의 약학적 조성물을 제제화하는 경우 충진제, 증량제, 결합제, 습윤제, 붕해제 및 계면활성제 등의 희석제 또는 부형제를 사용할 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제 및 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 화합물을 포함하는 약학적 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose) 또는 락토오스(Lactose) 및 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘, 스티레이트, 탈크 같은 윤활제를 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물 및 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 및 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제가 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜 및 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and may be formulated for oral or parenteral administration for human or veterinary use. When the pharmaceutical composition of the present invention is formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, which may be prepared by mixing the pharmaceutical composition comprising the compound of the present invention with at least one excipient such as starch, calcium carbonate Calcium carbonate, Sucrose or Lactose, and Gelatin. In addition to simple excipients, lubricants such as magnesium, styrene, and talc may be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances and preservatives in addition to commonly used simple diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여할 수 있으며, 비경구 투여시 피부 외용 또는 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사 주입방식을 선택하는 것이 바람직하다. The pharmaceutical composition of the present invention may be administered orally or parenterally in accordance with the desired method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, It is preferable to select the injection method.
본 발명의 상기 약학적 조성물은 개체에 투여하여 다발성 경화증을 예방 또는 치료할 수 있다. 본 발명에서 사용된 용어, "개체"는 다발성 경화증 또는 이의 직, 간접적 원인에 의해 유발된 질환을 가지고 있으며, 본 발명의 상기 약학적 조성물을 투여하여 증상이 호전될 수 있는 질환을 가진 인간을 포함한 말, 양, 돼지, 염소, 개 등의 포유동물을 의미하나, 바람직하게는 인간을 의미한다. The pharmaceutical composition of the present invention can be administered to an individual to prevent or treat multiple sclerosis. As used herein, the term "individual" refers to a human having a disease caused by multiple sclerosis or a direct or indirect cause thereof, and including a human having a disease whose symptoms may be alleviated by administering the pharmaceutical composition of the present invention Refers to mammals such as horses, sheep, pigs, goats, dogs and the like, preferably humans.
본 발명에서 사용된 용어, "투여"는 어떠한 적절한 방법으로 개체에 본 발명의 약학적 조성물을 도입하는 것을 의미한다. 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 본 발명의 약학적 조성물이 표적 세포로 이동할 수 있도록 하는 임의의 장치에 의해 투여될 수 있다.As used herein, the term "administering" means introducing a pharmaceutical composition of the invention into a subject by any suitable method. The route of administration may be oral or parenteral administration via any conventional route so long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows it to migrate to a target cell.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용된 용어, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 체중, 성별, 연령, 건강상태, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있으며, 1일 1회 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 다만, 상기 투여량의 본 발명의 범위를 한정하는 것은 아니다. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term, pharmaceutically effective amount, as used herein, means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary with the weight, sex, age, , The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. May be administered singly or multiply. It is important to take the above factors into consideration and to administer an amount capable of achieving the maximum effect in a minimal amount without side effects, and it can be easily determined by a person skilled in the art, and can be administered once a day or divided into several doses . However, the scope of the present invention is not limited to these dosages.
또한, 본 발명은 옻나무 추출물을 유효성분으로 포함하는 다발성 경화증의 예방 또는 개선용 건강기능식품을 제공한다. The present invention also provides a health functional food for preventing or ameliorating multiple sclerosis comprising Rhus verniciflua extract as an active ingredient.
본 발명에 따른 건강기능 식품은 본 발명의 옻나무 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. The health functional food according to the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.
상기 옻나무 추출물은 전술한 바와 동일하다. The Rhus verniciflua extract is the same as described above.
본 명세서에서 상기 "기능 식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 본 명세서의 상기 "기능"이란 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적인 작용으로 보건 용도에 유용한 효과를 의미할 수 있다. The term " functional food "as used herein means a food prepared and processed by using raw materials or ingredients having useful functions in the human body. The" function " Which may be useful for health use.
상기 식품의 종류에는 특별한 제한은 없다. 상기 옻나무 추출물을 첨가할 수 있는 식품의 예로는 각종 스프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함할 수 있다. 상기 외에 본 발명의 옻나무 추출물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강기능식품은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. There is no particular limitation on the kind of the food. Examples of the food to which the Rhus verniciflua extract can be added include various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and may include all health foods in a conventional sense. In addition to the above, the Rhus verniciflua extract of the present invention may further contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination.
본 발명의 옻나무 추출물은 척수에서 탈수초화(demyelination), T 세포의 유주/침윤, 염증성 사이토카인/케모카인의 mRNA의 발현을 억제한다. 또한, 본 발명의 옻나무 추출물은 MAPK 및 NF-KB의 신호 기전을 억제한다. The Rhus verniciflua extract of the present invention inhibits demyelination, migration / invasion of T cells, and expression of inflammatory cytokine / chemokine mRNA in the spinal cord. In addition, the Rhus verniciflua extract of the present invention inhibits the signaling mechanism of MAPK and NF-KB.
따라서, 본 발명에 따른 옻나무 추출물은 다발성 경화증을 예방하거나 치료 또는 개선용의 약제학적 조성물 또는 건강기능식품으로 활용할 수 있다. Therefore, the Rhus verniciflua extract according to the present invention can be utilized as a pharmaceutical composition or health functional food for preventing, treating or improving multiple sclerosis.
도 1은 다발성경화증 동물모델(EAE)에서 행동학적인 검사를 통한 옻나무 추출물의 신경 보호 효과를 확인한 도이다.
도 2는 본 발명의 혼합추출물(RVS)을 처리를 통한 EAE가 유도된 동물 모델의 허리 척수 절편에서 탈수초화 및 면역세포 침윤 정도를 확인한 도이다.
도 3은 다발성경화증 동물모델(EAE)의 척수 내에 분포하는 미세아교세포의 활성도를 확인한 도이다.
도 4는 다발성경화증 동물모델(EAE)의 척수에서 RT-PCR 분석을 시행하여 염증매개인자의 활성도를 검사하고 정량화를 한 결과를 나타낸 도이다.
도 5는 다발성경화증 동물모델(EAE)의 척수에서 혈액뇌장벽의 변화를 조사하고 정량화한 결과를 나타낸 도이다.
도 6은 다발성경화증 동물모델(EAE)의 척수에서 CD4+ T 세포(T helper 세포; Th)의 활성도를 조사하고 정량화한 결과를 나타낸 도이다.
도 7은 다발성경화증 동물모델(EAE)의 척수에서 웨스턴 블롯 분석을 통해 MAPKs와 NF-kB 신호기전의 활성화 정도를 나타낸 도이다. Fig. 1 shows the neuroprotective effect of Rhus verniciflua extract by behavioral examination in an animal model of multiple sclerosis (EAE).
FIG. 2 is a graph showing the degree of dehydration and immune cell infiltration in the spinal cord section of EAE-induced animal model through treatment with the mixed extract of the present invention (RVS).
FIG. 3 is a graph showing the activity of microglial cells distributed in the spinal cord of an animal model of multiple sclerosis (EAE).
FIG. 4 is a graph showing the results of quantitative analysis of the activity of inflammatory mediators by RT-PCR analysis of the spinal cord of an animal model of multiple sclerosis (EAE).
FIG. 5 is a graph showing the results of investigation and quantification of changes in the blood brain barrier in the spinal cord of the multiple sclerosis animal model (EAE).
6 is a graph showing the results of quantitative analysis of the activity of CD4 + T cells (T helper cells; Th) in the spinal cord of an animal model of multiple sclerosis (EAE).
FIG. 7 is a graph showing the degree of activation of MAPKs and NF-kB signaling via Western blot analysis in spinal cord of an animal model of multiple sclerosis (EAE).
이하, 하기 실시예 및 실험예에 의해 본 발명을 보다 상세하게 설명한다. 그러나, 하기 실시예는 본 발명의 범위를 제한하는 것은 아니며, 이는 본 발명의 이해를 돕기 위한 것으로 해석되어야 할 것이다.Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples should not be construed as limiting the scope of the present invention, but should be construed to facilitate understanding of the present invention.
실시예 1. 옻나무 추출물의 제조 Example 1. Preparation of Rhus verniciflua extract
옻나무(Rhus verniciflua Stokes; RVS)의 껍질(건칠)을 열수 추출하여 동결 건조하였다. 구체적으로, 건조된 옻나무의 껍질 200 g을 약 3 cm의 크기로 자르고, 환류 추출 시스템에 1 L의 증류수와 함께 넣고, 실온에서 60분간 방치한 후, 90-100℃ 에서 2 시간 동안 추출하였다. 남은 찌꺼기를 동일한 조건으로 4회 반복하여 추출하고, 모든 추출액을 여과지(Whatman International Ltd, 3MM Chr, #3030 917, UK)를 사용하여 여과하고, 농축기(EYELA N-1200A, EYELA, Rikakikai Co. Ltd., Tokyo, Japan)를 이용하여 60℃ 이하에서 감압 농축한 다음, 이를 동결 건조하여 얻은 짙은 갈색의 분말상태의 옻나무 추출물 10 g (평균 수득율 5.0%)을 얻었다. 상기 분말은 증류수에 녹인 뒤, 알레르기를 일으키는 우르시올(urshinol) 성분을 제거하기 위해 n-헥산을 이용하여 분획한다. 얻어진 분획물을 농축하고 동결건조하여 하기 실험의 시료로 사용하였다. Rhus verniciflua Stokes (RVS) peel (dried) was hot-water extracted and lyophilized. Specifically, 200 g of dried lacquer husk was cut into a size of about 3 cm, placed in a reflux system together with 1 L of distilled water, allowed to stand at room temperature for 60 minutes, and then extracted at 90-100 ° C for 2 hours. The remaining residue was extracted four times with the same conditions repeatedly. All the extract was filtered using a filter paper (Whatman International Ltd, 3MM Chr, # 3030 917, UK) and concentrated in a concentrator (EYELA N-1200A, EYELA, Rikakikai Co. Ltd , Tokyo, Japan), and 10 g of a dark brown powdery Rhus verniciflua extract (average yield 5.0%) obtained by lyophilization was obtained. The powder is dissolved in distilled water and then fractionated using n-hexane to remove the urshinol component causing allergy. The obtained fractions were concentrated and lyophilized to be used as a sample in the following experiment.
이하 상기의 방법으로 추출된 옻나무 추출물을 'RVS'로 칭한다. Hereinafter, the Rhus verniciflua extract extracted by the above method is referred to as 'RVS'.
실시예 2. 동물모델(실험적 자가 면역성 뇌척수염, experimental autoimmune encephalomyelitis; EAE)의 제조Example 2. Preparation of animal models (experimental autoimmune encephalomyelitis (EAE))
실험적 자가 면역성 뇌척수염(EAE)은 T 세포 매개성 자가면역성 질병으로써, 중추신경계에서 염증세포의 침윤과 탈수초화의 특성을 가지는 사람의 다발성경화증(multiple sclerosis, MS)의 동물모델이다. 본 발명의 옻나무 추출물의 효과를 알아보기 위해 하기와 같은 방법으로 동물모델(실험적 자가 면역성 뇌척수염, experimental autoimmune encephalomyelitis, EAE)을 제조하였다.Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated autoimmune disease, an animal model of multiple sclerosis (MS) in humans with the characteristics of inflammatory cell infiltration and dehydration in the central nervous system. In order to examine the effect of the Rhus verniciflua extract of the present invention, an animal model (experimental autoimmune encephalomyelitis, EAE) was prepared by the following method.
구체적으로, 암컷 성체 C57BL/6 마우스 (Narabiotec Co., Ltd., Seoul, Republic of Korea; weight, 23-25 g; Seed mice were originated from Taconic Biosciences Inc., Cambridge, IN, USA)를 명암주기(light on 08:00 to 20:00)에서 23±2℃의 일정한 온도를 유지하였으며, 음식과 물을 자유롭게 섭취시켰다. 상기 동물들은 실험 1주일 전 실험환경에 적응시켰다. 행동학적인 검사를 하기 위하여, 도 1에 나타낸 바와 같이 RVS를 EAE 유도 후 질병이 나타나기 시작하는 시점(8~9일)부터 매일 1회씩 구강으로 투여하였다 (그룹당 n = 3-6):Specifically, female C57BL / 6 mice (Narabiotec Co., Seoul, Republic of Korea; weight: 23-25 g; Seed mice were originated from Taconic Biosciences Inc., Cambridge, IN, USA) light on 08:00 to 20:00) was maintained at a constant temperature of 23 ± 2 ° C and food and water were freely consumed. The animals were adapted to the experimental environment one week prior to the experiment. For behavioral testing, RVS was administered orally once daily (n = 3-6 per group) from the time point when the disease began to appear after EAE induction (8-9 days) as shown in Figure 1:
EAE를 유도하기 위해, 상기 마우스의 뒷발에 200 μg의 MOG35-55 (Sigma-Aldrich, St. Louis, MO, USA), 불완전한 Freund's adjuvant (Difco, Detroit, MI, USA), 및 550 μg의 결핵균(mycobacterium tuberculosis) H37Ra (Difco)을 함유하는 100 ㎕의 에멀젼을 피하로 주사하여 면역화시켰다(Choi et al., 2015; Lee et al., 2016a; Lee et al., 2016b; Lee et al., 2016c). 마우스는 면역화 0일 및 면역화 후 2일에 250 ng의 백일해 독소(PTX; List Biologic, Campbell, CA, USA)를 복강 내 주사하였다. 허위대조군의 마우스는 MOG35-55 펩티드 또는 PTX 대신에 식염수로 단독 처리하였다. EAE의 임상 징후는 다음과 같이 매일 점수를 매겼다: (Sigma-Aldrich, St. Louis, MO, USA), incomplete Freund's adjuvant (Difco, Detroit, MI, USA), and 550 μg of Mycobacterium tuberculosis (Lee et al., 2016a; Lee et al., 2016b; Lee et al., 2016c) were injected subcutaneously by intraperitoneal injection of 100 쨉 l of emulsion containing mycobacterium tuberculosis H37Ra (Difco) . Mice were intraperitoneally injected with 250 ng pertussis toxin (PTX; List Biologic, Campbell, CA, USA) on
증상 없음(단계 0); 꼬리 긴장도의 부분적 상실(단계 1); 꼬리의 마비(단계 2); 한쪽 뒷다리의 약한 마비(paraparesis)(단계 3); 양쪽 뒷다리의 마비 (paraplegia)(단계 4); 한쪽 앞 다리까지 마비(tetraparesis)(5 단계); 앞쪽 양쪽 다리까지 마비(tetraplegia)(6 단계); 죽음(death)(7 단계).No symptoms (stage 0); Partial loss of tail strain (step 1); Paralysis of the tail (stage 2); Weak paraparesis of one hind limb (step 3); Paraplegia of both hind limbs (step 4); Tetraparesis up to one leg (5 steps); Tetraplegia to the anterior both legs (stage 6); Death (Step 7).
실시예 3. RVS의 투여 Example 3. Administration of RVS
본 발명의 RVS (25, 50, 100 및 200 mg/kg)를 EAE를 유도한 다음 임상증상이 나타나기 시작하는 시점(8-9일째)부터 30일째까지 매일 1회씩 구강으로 투여하였다(onset treatment, 온셋투여). RVS를 생리식염수에 희석하였으며 개체 당 투여량이 100 ㎕가 되도록 하였다. RVS (25, 50, 100, and 200 mg / kg) of the present invention was administered to the oral cavity once daily from the time point when the clinical symptoms started to appear (8-9 days to 30 days after induction of EAE) Onset administration). RVS was diluted in physiological saline and the dose per patient was adjusted to 100 μl.
다발성 경화증 동물 모델을 이용한 실험의 실험군 분류는 다음과 같다. Experimental groupings of experiments using multiple sclerosis animal models are as follows.
- Nor군 : MOG 펩타이드와 RVS를 투여하지 않은 정상 대조군- Nor group: Normal control group without MOG peptide and RVS
- EAE군 : MOG 펩타이드로 EAE를 유도한 군- EAE group: EAE induced by MOG peptide
- EAE + RVS 군 : MOG 펩타이드로 EAE를 유도하고 RVS (25, 50, 100 및 200 mg/kg)를 구강으로 투여한 군- EAE + RVS group: EAE was induced with MOG peptide and oral administration of RVS (25, 50, 100 and 200 mg / kg)
- RVS군 : MOG 펩타이드로 EAE를 유도하지 않은 정상 마우스에 RVS (100 mg/kg)를 구강으로 투여한 군- RVS group: Oral administration of RVS (100 mg / kg) to normal mice not inducing EAE with MOG peptide
실시예 4. 다발성경화증 동물모델(EAE)에서 행동학적인 검사를 통한 RVS의 신경보호 효과Example 4. Neuroprotective effect of RVS through behavioral examination in an animal model of multiple sclerosis (EAE)
도 1은 다발성경화증 동물모델(EAE)에서 행동학적인 검사를 통한 옻나무 추출물의 신경 보호 효과를 확인한 도이다. Fig. 1 shows the neuroprotective effect of Rhus verniciflua extract by behavioral examination in an animal model of multiple sclerosis (EAE).
구체적으로, 도 1A는 행동학적 발명 정도를 나타낸 도이고, 도 1B는 도1A를 정량적으로 나타낸 도이다. Specifically, FIG. 1A is a diagram showing the degree of behavioral invention, and FIG. 1B is a diagram quantitatively showing FIG. 1A.
EAE군의 행동학적인 증상은 EAE 유도 후, 8일째에서 9일째 사이에 나타나기 시작하여 20일째에서 22일째 사이에 최고조(3.6 ± 0.2)에 도달하였으며, 그 이후에도 지속되었다. EAE 유도 후 8일째부터 30일째까지의 스코어의 합은 60.4 ± 2.7이었다. 그러나 EAE + RVS군에서의 행동학적인 증상은 EAE군에 비하여 감소하는 경향이었으며, EAE + RVS 100과 200 mg/kg군에서는 유의한 감소를 보였다. EAE 유도 후 8일째부터 30일째까지의 스코어의 합은 RVS 100 mg/kg 투여군에서 34.6 ± 3.1 이었고, 200 mg/kg 투여군에서 39.0 ± 8.4으로 100 mg/kg 투여군에서 가장 낮았다. The behavioral symptoms of the EAE group began to appear between
실시예 5. 조직병리학적 분석Example 5. Histopathological analysis
(1) 탈수초화(demyelination) 및 면역세포의 침윤(infiltration) 억제 확인(1) confirmation of inhibition of demyelination and immune cell infiltration
다발성 경화증(Multiple sclerosis) 환자의 전형적인 조직 병리학적 특징은 척수 내 병변의 탈수초이므로, 본 발명자들은 EAE의 임상 점수와 탈수초화의 수준 사이의 상관관계를 확인하였다.Because typical histopathological features of patients with multiple sclerosis are dehydration of spinal cord lesions, the present inventors have confirmed the correlation between the clinical score of EAE and the level of dehydration.
RVS의 EAE 동물모델에서의 탈수초 현상의 억제 효과를 확인하기 위하여 미엘린 수초의 주성분인 인지질을 염색하는 룩솔 페스트 블루(Luxol fast blue, LFB) 염색을 통해 탈수초화 정도를, H&E염색을 통해 면역세포의 침윤 정도를 확인하기 위해 시행하였다. In order to confirm the inhibitory effect of dehydration on the EAE animal model of RVS, dehydration oligosaccharide was stained with Luxol fast blue (LFB) stain, which is a main component of myelin aphid, Of the patients.
구체적으로, EAE 유도 후 임상증상이 가장 심한 시기인 18일 내지 22일 사이에 생쥐를 마취시키고, 4 % 파라 포름 알데히드 (PFA)액을 심장으로 관류하여 고정하고 허리부위의 척수(요추 4-5번; L4-5)를 적출하였고 적출한 척수는 4% PFA에 담가 4℃에서 하루 정도 고정시킨 다음 30% 수크로스(sucrose)로 바꾸어 4℃에서 3일 이상 기간 동안 보관하여 동결손상을 방지하였다. 동결 절편을 제작하여 10-μm 두께로 절단하였다. 상기 절편은 탈수초 및 면역 세포 침윤을 각각 평가 하기 위해 luxol fast blue (LFB) 및 H&E(Hematoxylin and eosin)로 염색되었다. 상기 절편은 탈수되고 커버슬립(coverslipped)되었다.Specifically, the mice were anesthetized between 18 and 22 days after the induction of EAE, and the 4% paraformaldehyde (PFA) solution was perfused through the heart, and the spinal cord at the waist region (lumbar spine 4-5 L4-5) and the spinal cord was immersed in 4% PFA for 4 days at 4 ℃ for 30 min. The supernatant was replaced with 30% sucrose and stored at 4 ℃ for more than 3 days to prevent freezing injury . Frozen sections were prepared and cut to a thickness of 10-μm. The sections were stained with luxol fast blue (LFB) and H & E (Hematoxylin and eosin) to assess dehydration and immune cell infiltration, respectively. The sections were dehydrated and coverslipped.
LFB 염색 후 탈수초의 수준을 평가하였다. The level of dehydrated chicks after LFB staining was evaluated.
0, 탈수초화 없음0, No dehydration
1, 주변 침윤 및 백질의 25 % 미만을 수반하는 약간의 탈수초화1, slight dehydration with accompanying infiltration and less than 25% of white matter
2, 백질의 50 % 미만을 수반하는 탈수초화2, Dehydrated chrysanthemum with less than 50% of white matter
3, 백질의 50 % 이상을 수반하는 확산과 광범위한 탈수초화.3, the spread accompanied by more than 50% of white matter and extensive dehydration.
H&E 염색 후 면역 세포의 이동(recruitment) 및 침윤(infiltration)정도는 다음 기준에 따라 점수를 매겼다. The degree of recruitment and infiltration of immune cells after H & E staining was scored according to the following criteria.
0, 병변 없음0, no lesion
1, 뇌척수(meninges)에서만 세포 이동/침윤1, cell migration / invasion only in meninges
2, 실질(parenchyma)에서 매우 이산(discrete)되고 표재성 침윤 (superficial infiltrates)2, highly discrete and superficial infiltrates in parenchyma,
3, 백질에 중간 정도의 침윤 (25 % 미만)3, moderate infiltration (<25%) in white matter,
4, 백질에 심각한 침윤 (50 % 미만)4, severe infiltration of white matter (less than 50%)
5, 백질에 더 심각한 침윤 (50 % 이상)5, more severe infiltration (over 50%) in white matter
도 2는 본 발명의 옻나무 추출물(RVS)을 처리를 통한 EAE가 유도된 동물 모델의 허리 척수 절편에서 탈수초화 및 면역세포 침윤 정도를 확인한 도이다.FIG. 2 is a graph showing the degree of dehydration and immune cell infiltration in the spinal cord section of an EAE-induced animal model through treatment with RVS extract (RVS) of the present invention.
구체적으로 도 2A 내지 도 2D는 탈수초화(demyelination) 현상의 억제 효과를 확인하기 위하여 수초(myelin)의 주성분인 인지질을 염색하는 룩솔 페스트 블루(luxol fast blue; LFB) 염색을 통해 탈수초화 정도를 비교한 도이고, 도 2E 내지 도 2H는 H&E (hematoxyline and eosin) 염색을 통해 면역세포의 침윤 정도를 비교한 도이다. 또한 도 2I는 도 2A 내지 도 2D의 LFB 염색을 정량적으로 나타낸 도이고, 도 2J는 도 2E 내지 도 2H의 H&E 염색을 정량적으로 나타낸 도이다. Specifically, FIGS. 2A to 2D show the results of comparing the degree of dehydration reduction by staining luxol fast blue (LFB) staining phospholipid, which is a main component of myelin, in order to confirm the inhibitory effect of demyelination phenomenon And FIGS. 2E to 2H compare the degree of invasion of immune cells through H & E (hematoxyline and eosin) staining. FIG. 2I is a quantitative view showing LFB staining of FIGS. 2A to 2D, and FIG. 2J is a diagram quantitatively showing H & E staining of FIGS. 2E to 2H.
상기 도 2의 결과로 보아 EAE군은 정상군에 비하여 탈수초화와 면역세포의 침윤 정도가 증가되었으며, RVS 투여군은 EAE군에 비하여 탈수초화 정도와 염증세포의 침윤 정도가 유의하게 감소된 것을 확인할 수 있다.2, the degree of dehydration and immune cell infiltration was increased in the EAE group, and the degree of dehydration and inflammatory cell infiltration was significantly decreased in the RVS group compared to the EAE group have.
(2) 면역조직화학염색법(2) Immunohistochemical staining
EAE 유도 후 임상증상이 가장 심한 시기인 18일 내지 22일 사이에 생쥐를 마취시키고, 4 % 파라 포름 알데히드 (PFA)액을 심장으로 관류하여 고정하고 허리부위의 척수(요추 4-5번; L4-5)를 적출하였고 적출한 척수는 4% PFA에 담가 4℃에서 하루 정도 고정시킨 다음 30% 수크로스(sucrose)로 바꾸어 4℃에서 3일 이상 기간 동안 보관하여 동결손상을 방지하였다. 동결 절편을 제작하여 10-μm 두께로 절단하였다(Choi et al., 2015; Lee et al., 2016a; Lee et al., 2016b, Lee et al., 2016c). 상기 절편은 척수 내 미세아교세포 및 별아교세포의 활성 정도를 확인하기 위해 면역조직화학 염색을 실시하였다.The mice were anesthetized between 18 and 22 days after the induction of EAE, and the 4% paraformaldehyde (PFA) solution was perfused through the heart and the spinal cord at the waist (lumbar spine 4-5; L4 -5). The spinal cord was immersed in 4% PFA and fixed at 4 ℃ for one day. The spinal cord was replaced with 30% sucrose and stored at 4 ℃ for more than 3 days to prevent freezing injury. (Lee et al., 2016a; Lee et al., 2016b, Lee et al., 2016c). The sections were immunohistochemically stained to confirm the degree of microglial cells and astrocytes in the spinal cord.
척수 절편은 1차 면역혈청(antiserum)으로 미세아교세포의 마커인 토끼 항-이온화 칼슘 결합 어뎁터 분자-1(Iba-1; ionized calcium-binding adapter molecule 1) (1:2,000; WAKO, Osaka, Japan) 또는 별아교세포의 마커인 토끼 항-아교 섬유 산성 단백질(GFAP; Glial fibrillary acidic protein) (1:2,000; DACO, USA)을 하루 동안 반응시켰다. 2차 면역혈청으로 바이오틴화 토끼 IgG 항체 (1:200; Vector Laboratories, USA), 그리고 아비딘-바이오틴화 겨자무과산화효소 (horseradish peroxidase)-복합체 (1:200; Vector Laboratories), 및 3,3'-diamino-benzidine를 이용하여 면역염색을 하였다.The spinal cord slices were incubated with a primary immunoreactive serum (IAB-1) (1: 2,000; WAKO, Osaka, Japan) as a marker of microglial cells ) Or glial fibrillary acidic protein (GFAP) (1: 2,000; DACO, USA), a marker of astrocytic cells, was reacted for one day. Biotinylated rabbit IgG antibody (1: 200; Vector Laboratories, USA) and avidin-biotinylated horseradish peroxidase-complex (1: 200; Vector Laboratories) -diamino-benzidine. < / RTI >
(3) 웨스턴블롯 분석(3) Western blot analysis
EAE 유도 후 임상증상이 가장 심한 시기인 18일 내지 22일 사이에 생쥐를 마취시키고 안락사 한 뒤 허리부위의 척수(요추 4-5번; L4-5)를 적출하였고, 이를 단백질 용해 완충액(protein lysis buffer; 10 mM 트리스, 0.5 mM EDTA, 0.25 M 수크로스) 200 ul를 넣고 분쇄기를 이용하여 단백질을 분리하였다. 분리된 단백질은 소혈청알부민(BSA, bovine serum albumin, sigma, 미국)를 사용한 Bradford(Bio-rad, 미국) 방법을 이용하여 각 개체의 단백질을 정량한 뒤 단백질 30 ㎍은 10% SDS-PAGE(Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis)에 의해 분리하여 PVDF(polyvinylidene difluoride)멤브레인(membrane, Gendepot, 영국)으로 이동시켰다. PVDF 멤브레인은 5% 탈지유(skimmed milk, BD, 미국)에서 1시간 동안 블로킹시켰다. TBST로 씻은 후 1차 항체[CD11b(단핵구 마커), glial fibrillary acidic protein (GFAP; 별아교세포 마커), platelet and endothelial cell adhesion molecule (PECAM)-1과 fibronectin (혈액뇌장벽의 마커), nuclear factor kappa B (NF-kB), phospho-I-kappa-B (p-IkB), phospho-extracellular signal-regulated kinase 1/2(p-ERK), phospho-c-Jun N-terminal kinase (p-JNK) 및 phospho-p38 (p-p38) 및 GAPDH]를 3% 탈지유에 1:1,000으로 희석시켜 4℃에서 하루 동안 반응시킨 후 TBST로 10분씩 3회 씻고 2차 항체와 함께 상온에서 1시간 동안 반응시켰다. 2차 항체 반응 후 TBST에 씻은 뒤 ECL system(Santacruz, 미국)로 밴드를 확인하였다. 단백질의 확인 및 정량 분석은 이미지 장비 ChemiDoc XRS+(Bio-Rad)를 이용하였다.The mice were anesthetized and euthanized between 18 and 22 days after the induction of EAE, and the spinal cord (lumbar spine 4-5; L4-5) was excised and lysed in protein lysis buffer buffer; 10 mM Tris, 0.5 mM EDTA, 0.25 M sucrose) was added to the wells and the proteins were separated using a pulverizer. Proteins of each individual were quantitated using Bradford (Bio-Rad, USA) method using bovine serum albumin (Sigma, USA) and 30 μg of protein was separated on a 10% SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene difluoride) membrane (Gendepot, UK). The PVDF membrane was blocked in 5% skimmed milk (BD, USA) for 1 hour. After washing with TBST, the primary antibody [CD11b (mononuclear marker), glial fibrillary acidic protein (GFAP), platelet and endothelial cell adhesion molecule (PECAM) -1 and fibronectin (marker of blood brain barrier) (P-ERK), phospho-c-Jun N-terminal kinase (p-JNK), phospho-I-kappa-B And phospho-p38 (p-p38) and GAPDH] were diluted 1: 1,000 in 3% skim milk, reacted at 4 ° C for one day, washed three times for 10 minutes each with TBST and reacted for 1 hour at room temperature with secondary antibody . After the second antibody reaction, the cells were washed with TBST and the band was confirmed by ECL system (Santacruz, USA). Identification and quantitative analysis of proteins were performed using the imaging equipment ChemiDoc XRS + (Bio-Rad).
(4) 역전사 중합효소 연쇄반응(RT-PCR, reverse transcription-polymerase chain reaction)(4) reverse transcription-polymerase chain reaction (RT-PCR)
mRNA 발현 확인을 위해 EAE 유도 후 임상 증상이 피크 단계 (18-22일)에서, 허리부위의 척수를 적출한 총 RNA를 TRIsure 시약을 이용하여 공지된 메뉴엘에 따라 추출하였다(Bioline, UK). 척수조직에 트리졸(Trizol, Invitrogen, 미국) 1 ml을 넣고 분쇄기로 갈고, 원심분리하여 RNA를 분리하였다. cDNA를 합성하기 위해 0.5 μg의 Oligo dT, 0.5 mM dNTP mix, 5x first-strand buffer, RNase out, 5 mM dithiothreitol (DTT), 및 M-MLV 역전사효소를 포함하는 반응혼합물(reaction mixture) 중에서 1 μg의 총 RNA를 37℃에서 1시간 동안 반응시켰다. RT-PCR 분석은 RT-PCR kit의 제조사의 메뉴얼에 따라 수행되었다(Roche, Germany). PCR 증폭을 위해, 특정 올리고뉴클레오티드 프라이머 쌍은 6 μl of PCR Master Mix (Lucigen, WI, USA) 중에서 1 μl의 cDNA 및 0.6 U의 Econo TaqDNA 중합효소를 반응시켰다. 결과물의 5-7 μl를 2% 아가로스 젤에서 전기영동하고 트랜드일루미네이터에서 브롬화 에티듐(ethidium bromide)으로 염색한 후에 확인하였다. 각 유전자의 발현 수준은 GAPDH (glyceraldehyde 3-phosphate dehydrogenase)로 표준화하였다.For confirmation of mRNA expression, the total RNA extracted from the spinal cord at the lumbar region at the peak stage (18-22 days) after EAE induction was extracted according to a known menu ELI using TRIsure reagent (Bioline, UK). One milliliter of trizol (Trizol, Invitrogen, USA) was added to the spinal cord tissue, ground with a grinder, and centrifuged to separate the RNA. To synthesize the cDNA, 1 μg / ml of a reaction mixture containing 0.5 μg of Oligo dT, 0.5 mM dNTP mix, 5 × first-strand buffer, RNase out, 5 mM dithiothreitol (DTT), and M-MLV reverse transcriptase Of total RNA was reacted at 37 DEG C for 1 hour. RT-PCR analysis was performed according to the manufacturer's manual of the RT-PCR kit (Roche, Germany). For PCR amplification, specific oligonucleotide primer pairs were reacted with 1 μl of cDNA and 0.6 U of Econo Taq DNA polymerase in 6 μl of PCR Master Mix (Lucigen, WI, USA). 5-7 μl of the resultant was confirmed by electrophoresis on 2% agarose gel and staining with ethidium bromide in a trend illuminator. Expression levels of each gene were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
또한, 실시간(Real-time) PCR의 경우 SYBR Green PCR Master Mix (Applied Biosystems, Franklin Lakes, NJ, USA)을 이용하여 수행하였으며, Foldinduction은 2-ΔΔCT방법을 이용하여 계산되었다. PCR 증폭은 적어도 세 번 수행하였다.Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Franklin Lakes, NJ, USA). Foldinduction was calculated using the 2-ΔΔCT method. PCR amplification was performed at least three times.
도 3은 다발성경화증 동물모델(EAE)의 척수 내에 분포하는 미세아교세포의 활성도를 확인한 도이다. FIG. 3 is a graph showing the activity of microglial cells distributed in the spinal cord of an animal model of multiple sclerosis (EAE).
구체적으로, 도 3A는 다발성경화증 동물모델(EAE)의 척수에서 CD11b의 RT-PCR 분석 결과 및 이를 정량화한 도이고, 도 3B는 CD11b의 웨스턴블롯 분석 결과 및 이를 정량화 한 도이며, 도 3C는 Iba-1 단백질의 면역 조직 화학적 염색 결과를 나타낸 도이다. 3A is a result of quantitative analysis of CD11b in the spinal cord of an MS model of multiple sclerosis (EAE), and FIG. 3B is a result of Western blot analysis of CD11b and quantification thereof. -1 < / RTI > protein.
상기 도 3의 결과로 척수에서 CD11b의 mRNA와 단백질의 발현은 EAE군에서 증가되었나, EAE + RVS군에서는 감소됨을 확인할 수 있었다. 또한, 활성화된 Iba-1(+) 미세아교세포와 말초에서 중추로 침윤된 대식세포가 EAE군에서 증가되었으나, EAE + RVS 군에서는 감소된 것을 확인할 수 있었다. As shown in FIG. 3, the expression of CD11b mRNA and protein in the spinal cord was increased in the EAE group but decreased in the EAE + RVS group. In addition, it was confirmed that the activated Iba-1 (+) microglia and peripheral macrophages infiltrated into the EAE group but decreased in the EAE + RVS group.
도 4는 다발성경화증 동물모델(EAE)의 척수에서 RT-PCR 분석을 시행하여 염증매개인자의 활성도를 검사하고 정량화를 한 결과를 나타낸 도이다. FIG. 4 is a graph showing the results of quantitative analysis of the activity of inflammatory mediators by RT-PCR analysis of the spinal cord of an animal model of multiple sclerosis (EAE).
구체적으로, 도 4A는 MCP-1, 도 4B는 MIP-1α, 도 4C는 RANTES, 도 4D는 IL-1β, 도 4E는 IL-6, 도 4F는 TNF-α, 도 4G는 COX-2, 도 4H는 iNOS의 염증매개인자의 활성도에 대한 정량화를 나타낸 도이다. 4F, IL-1, IL-4, IL-6, IL-4, IL-4, IL-4, IL-6 and IL-6 are shown in FIG. 4A, MIP- Figure 4H is a graphical representation of the activity of inflammatory mediators of iNOS.
도 4의 결과로 보아, 대표적인 케모카인(MCP-1, MCP-1α 및 RANTES), 전염증성 사이토카인(IL-1β, IL-6 및 TNF-α), COX-2 및 iNOS에 대한 mRNA 발현이 EAE군에서 증가되었나, EAE + RVS군에서는 감소된 것을 확인할 수 있었다. 4, mRNA expression for representative chemokines (MCP-1, MCP-1α and RANTES), proinflammatory cytokines (IL-1β, IL-6 and TNF-α), COX-2 and iNOS, , But decreased in the EAE + RVS group.
도 5는 다발성경화증 동물모델(EAE)의 척수에서 혈액뇌장벽의 변화를 조사하고 정량화한 결과를 나타낸 도이다. FIG. 5 is a graph showing the results of investigation and quantification of changes in the blood brain barrier in the spinal cord of the multiple sclerosis animal model (EAE).
도 5A는 혈관뇌장벽의 마커인 PECAM-1(platelet-endothelial celladhesion molecule-1; 혈소판-내피세포 결합 단백질-1)에 대한 웨스턴블롯의 분석 결과를 나타낸 도이고, 도 5B는 혈관뇌장벽의 마커인 피브로넥틴(Fibronectin)에 대한 웨스턴블롯의 분석 결과를 나타낸 도이다. FIG. 5A is a graph showing the results of Western blot analysis on PECAM-1 (platelet-endothelial celladhesion molecule-1; platelet-endothelial cell binding protein-1), which is a marker of blood vessel brain barrier, Lt; / RTI > showing the results of Western blot analysis for Fibronectin.
도 5C는 GFAP (glial fibrillary acidic protein; 신경교섬유질산성단백질)에 대한 웨스턴블롯 분석과 이를 정량적으로 나타낸 도이며, 도 5D 내지 도 5G는 GFAP의 면역 조직화학적 염색 결과를 나타낸 도이다. FIG. 5C is a Western blot analysis and quantitative analysis of GFAP (glial fibrillary acidic protein). FIGS. 5D to 5G are immunohistochemical staining results of GFAP. FIG.
도 5H 내지 도 5J는 결합 분자(junctional molecules; Claudin3, Claudin5 및 ZO-1)의 실시간 PCR 분석을 나타낸 도이다. Figures 5H through 5J show real time PCR analysis of junctional molecules (
도 5의 결과를 보면, 혈액뇌장벽 마커인 두 단백질의 발현(PECAM-1, fibronectin)은 EAE군에서 증가되었으나, EAE + RVS군에서는 감소되었다. 혈액뇌장벽의 주요 구성성분의 하나인 GFAP의 발현을 확인한 결과, GFAP의 단백질 발현 정도와 GFAP 면역양성 별아교세포의 활성화의 정도는 EAE군에서 증가하였으나, EAE + RVS군에서 감소하였다. 또한, 세포 사이의 결합 분자 (Claudin3, Claudin5 및 ZO-1)에 대한 mRNA의 발현 정도도 EAE군에서 증가하였으나, EAE + RVS군에서 감소하였다. In FIG. 5, expression of two proteins (PECAM-1, fibronectin) as a blood brain barrier marker was increased in the EAE group but decreased in the EAE + RVS group. GFAP protein expression and GFAP immunoreactivity were increased in the EAE group but decreased in the EAE + RVS group, which is one of the major components of the blood brain barrier. In addition, the expression level of mRNA in intercellular binding molecules (Claudin3, Claudin5 and ZO-1) was increased in the EAE group but decreased in the EAE + RVS group.
도 6은 다발성경화증 동물모델(EAE)의 척수에서 CD4+ T 세포(T helper 세포; Th)의 활성도를 조사하고 정량화한 결과를 나타낸 도이다. 6 is a graph showing the results of quantitative analysis of the activity of CD4 + T cells (T helper cells; Th) in the spinal cord of an animal model of multiple sclerosis (EAE).
도 6A는 IFN-r (Th1 세포의 분비물)에 대한 실시간 PCR 분석을 나타낸 도이고, 도 6B는 IL-17 (Th17 세포의 분비물)에 대한 실시간 PCR 분석을 나타낸 도이며, 도 6C는 IL-4 (Th2 세포의 분비물)에 대한 실시간 PCR 분석을 나타낸 도이고, 도 6D는 Foxp3 (Treg 세포의 마커)에 대한 실시간 PCR 분석을 나타낸 도이다. FIG. 6A is a real time PCR analysis of IFN-r (secretion of Th1 cells), FIG. 6B is a real time PCR analysis of IL-17 (secretion of Th17 cells) (Secretion of Th2 cells), and FIG. 6D shows a real time PCR analysis of Foxp3 (a marker of Treg cells).
도 6의 결과로 보아, CD4+/IFN-r+ (Th1 세포)와 CD4+/IL-17+(Th17 세포)의 mRNA 발현은 EAE군의 척수에서 증가하였으나, EAE + RVS군의 척수에서는 감소하였다. 억제(suppressor) T 세포로 알려진 CD4+/Foxp3+ T 세포와 CD4+/IL-4+(Th2 세포)의 mRNA 발현은 EAE 유도와 RVS투여에 의하여 유의한 영향을 받지 않음을 확인할 수 있었다6, mRNA expression of CD4 + / IFN-r + (Th1 cells) and CD4 + / IL-17 + (Th17 cells) was increased in the spinal cord of the EAE group but decreased in the spinal cord of the EAE + RVS group Respectively. The mRNA expression of CD4 + / Foxp3 + T cells and CD4 + / IL-4 + (Th2 cells), known as suppressor T cells, was not significantly affected by EAE induction and RVS administration
도 7은 다발성경화증 동물모델(EAE)의 척수에서 웨스턴 블롯 분석을 통해 MAPKs와 NF-kB 신호기전의 활성화 정도를 나타낸 도이다. FIG. 7 is a graph showing the degree of activation of MAPKs and NF-kB signaling via Western blot analysis in spinal cord of an animal model of multiple sclerosis (EAE).
도 7A 내지 도 7C는 각각 MAPKs 신호기전인 p-ERK, p-JNK 및 p-p38에 대한 웨스턴블롯의 분석 결과를 나타낸 도이고, 도 7D 내지 도 7E는 각각 NF-kB 신호기전인 NF-kB와 p-IkB에 대한 웨스턴블롯의 분석 결과를 나타낸 도이다. 7A to 7C are graphs showing results of Western blot analysis for p-ERK, p-JNK and p-p38, respectively, before MAPKs signaling, and Figs. 7D to 7E are graphs showing results of analysis of NF- -IkB. ≪ / RTI >
도 7의 결과로 p-NF-kB, p-IkB, p-ERK, p-JNK 및 p-p38의 단백질 발현은 EAE군의 척수에서 증가하였으나, EAE + RVS군의 척수에서는 감소하였음을 확인할 수 있었다.7, protein expression of p-NF-kB, p-IkB, p-ERK, p-JNK and p-p38 was increased in the spinal cord of EAE group but decreased in spinal cord of EAE + RVS group there was.
상기 실시예들의 결과로 보아, 본 발명에 따른 옻나무 추출물은 다발성 경화증의 신경학적인 임상증상 척도를 개선하고, 척수의 탈수초화를 억제하며, 미세아교세포의 활성화를 억제하고, 혈액뇌장벽 투과성을 억제하며, 케모카인 및 염증성 사이토카인의 생성을 억제하고, COX-2 및 iNOS의 유전자 발현을 억제하고, MAPK 및 NF-KB의 신호 기전을 억제한다는 것을 확인할 수 있다. 따라서, 본 발명에 따른 옻나무 추출물을 유효성분으로 포함하는 조성물은 다발성 경화증의 발생을 예방하거나, 치료 또는 개선용의 약제학적 조성물 또는 건강기능식품으로 유용하다는 것을 확인할 수 있다. As a result of the above examples, the Rhus verniciflua extract according to the present invention improves the neurological symptom scale of multiple sclerosis, inhibits dehydration of spinal cord, inhibits activation of microglial cells, inhibits blood brain barrier permeability , Inhibits the production of chemokines and inflammatory cytokines, inhibits gene expression of COX-2 and iNOS, and suppresses the signaling pathway of MAPK and NF-KB. Accordingly, it can be confirmed that the composition comprising the extract of Rhus verniciflua according to the present invention as an active ingredient is useful as a pharmaceutical composition for treating or ameliorating the occurrence of multiple sclerosis, or as a health functional food.
Claims (10)
상기 옻나무 분획물은 옻나무의 나무껍질을 추출한 것인 다발성 경화증의 예방 또는 치료용 약제학적 조성물. The method according to claim 1,
Wherein the fraction of Rhus verniciflua is extracted from the bark of Rhus verniciflua.
상기 옻나무 분획물은 열수 추출물을 n-헥산으로 분획한 것인 다발성 경화증의 예방 또는 치료용 약제학적 조성물.The method according to claim 1,
Wherein said Rhus verniciflua fraction is obtained by fractionating a hydrothermal extract with n-hexane to prevent or treat multiple sclerosis.
상기 열수 추출 시 추출 온도는 90℃ 내지 100℃인 것인 다발성 경화증의 예방 또는 치료용 약제학적 조성물. The method of claim 3,
Wherein the extraction temperature during the hot-water extraction is 90 ° C to 100 ° C.
상기 옻나무 분획물은 옻나무의 나무껍질을 추출한 것인 다발성 경화증의 예방 또는 개선용 건강기능식품. The method according to claim 6,
Wherein the fraction of Rhus verniciflua is extracted from the bark of Rhus verniciflua, which is a health functional food for preventing or ameliorating multiple sclerosis.
상기 옻나무 분획물은 열수 추출물을 n-헥산으로 분획한 것인 다발성 경화증의 예방 또는 개선용 건강기능식품. The method according to claim 6,
Wherein said Rhus verniciflua fraction is obtained by fractionating a hydrothermal extract with n-hexane to prevent or ameliorate multiple sclerosis.
상기 열수 추출 시 추출 온도는 90℃ 내지 100℃인 것인 다발성 경화증의 예방 또는 개선용 건강기능식품. 9. The method of claim 8,
Wherein the extraction temperature during the hot water extraction is 90 ° C to 100 ° C.
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