KR20210131231A - Composition Comprising Puerarin for Wound Healing - Google Patents
Composition Comprising Puerarin for Wound Healing Download PDFInfo
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- KR20210131231A KR20210131231A KR1020210041951A KR20210041951A KR20210131231A KR 20210131231 A KR20210131231 A KR 20210131231A KR 1020210041951 A KR1020210041951 A KR 1020210041951A KR 20210041951 A KR20210041951 A KR 20210041951A KR 20210131231 A KR20210131231 A KR 20210131231A
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- puerarin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
- A61K8/602—Glycosides, e.g. rutin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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Abstract
Description
본 발명은 푸에라린을 포함하는 상처 치료용 조성물에 관한 것이다.The present invention relates to a composition for treating wounds comprising puerarine.
우리 몸에서 피부는 여러 가지 기능을 담당하고 있으며, 그 중에서도 가장 중요한 기능은 외부로부터 우리의 몸을 보호하는 장벽 역할이라 할 수 있다. 피부의 정상적인 구조가 파괴된 상태를 창상 혹은 상처라고 한다. 이와 같이 피부가 상처를 입게 되면 상처 부위가 노출되어 외부 균의 침입을 받을 수 있다. 따라서, 신속한 상처치료가 되어야 추가 감염(infection)을 막을 수 있고 또한, 그로 인한 과도한 염증반응(inflammation)을 막을 수 있다. 과도한 염증반응은 상처치유 기간을 연장시킬 뿐 아니라 흉터의 원인이 되기도 한다.In our body, the skin is responsible for various functions, and the most important function among them is a barrier that protects our body from the outside. A condition in which the normal structure of the skin is destroyed is called a wound or wound. When the skin is injured in this way, the wound site is exposed and can be invaded by external bacteria. Therefore, it is possible to prevent further infection and also to prevent excessive inflammatory reaction (inflammation) resulting therefrom only when the wound is treated promptly. Excessive inflammatory response not only prolongs the healing period but also causes scarring.
상처치유(wound healing)란 임상적으로 피부가 완전하게 봉합되는 것을 의미하며, 만성상처(chronic wound)가 아닌 일반적인 상처는 대략 3-14일 안에 완전히 치유된다. 상처치유는 각질세포(keratinocyte), 섬유아세포(fibroblast), 혈관내피세포(endothelial cell), 대식세포(macrophage), 혈소판(platelet) 등 여러 종류의 세포가 상호작용하면서 조절하는 복잡한 과정으로 이루어진다.Wound healing clinically means that the skin is completely sutured, and normal wounds that are not chronic wounds heal completely within about 3-14 days. Wound healing consists of a complex process controlled by interaction of several types of cells, such as keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets.
스테로이드제제는 면역억제를 통하여 피부염증을 조절해주지만, 각질형성세포의 증식을 억제하거나, 세포증식을 억제함으로써 상처치유를 지연시킨다. 이와 같이, 스테로이드제 등으로 인하여 상처 치료가 지연되면 2차 감염 등 다른 위험이 증가하게 되므로, 상처치료제의 개발은 여전히 필요한 실정이다. Steroids control skin inflammation through immunosuppression, but delay wound healing by inhibiting the proliferation of keratinocytes or inhibiting cell proliferation. As such, when wound treatment is delayed due to steroids, etc., other risks such as secondary infection increase, so the development of a wound treatment agent is still necessary.
일 양상은 푸에라린(Puerarin)을 포함하는 상처 치료용 약학적 조성물을 제공하는 것이다. One aspect is to provide a pharmaceutical composition for the treatment of wounds comprising Puerarin (Puerarin).
다른 양상은 푸에라린(Puerarin)을 포함하는 상처 치료 촉진용 의약외품 조성물을 제공하는 것이다. Another aspect is to provide a quasi-drug composition for promoting wound healing comprising Puerarin.
일 양상은 푸에라린(Puerarin)을 포함하는 상처 치료용 약학적 조성물을 제공하는 것이다. One aspect is to provide a pharmaceutical composition for the treatment of wounds comprising Puerarin (Puerarin).
상기 푸에라린은 콩과에 속한 칡의 뿌리에 함유된 이소플라보노이드 성분일 수 있으며, 하기 화학식 1과 같은 물질일 수 있다.The puerarin may be an isoflavonoid component contained in the root of arrowroot belonging to the legume family, and may be a substance represented by Chemical Formula 1 below.
상기 상처는 외부의 압력 등에 의한 피부 상피조직의 온전함이 파괴되는 것을 나타내는 의미로서, 창상(創傷)이라고도 할 수 있다. 상기 상처의 종류로는 찰과상, 타박상, 열상, 칼날에 의한 절창, 자창(刺創), 할창(割創), 총창(銃創), 폭창, 교창(咬創) 등이 있으나, 이에 제한되지 않는다.The wound means that the integrity of the skin epithelial tissue is destroyed by external pressure, etc., and may be referred to as a wound (創傷). The types of wounds include, but are not limited to, abrasions, bruises, lacerations, cuts by a knife, spears, spears, spears, spears, and spears.
일 구체예에 따르면, 상기 상처는 스테로이드제로 인하여 상처치유가 지연된 상처일 수 있다. 상기 스테로이드제제로 인하여 지연된 상처는 스테로이드제제가 각질형성세포의 증식을 억제하거나, 세포증식을 억제함으로써 지연되는 것일 수 있다. 상기 상처치유가 지연된 상처는 스테로이드제를 사용하여 상처가 지연된 것일 수 있고, 스테로이드제를 지속하여 사용하고 있어 상처 치유의 지연이 진행되고 있는 상처일 수 있다. According to one embodiment, the wound may be a wound in which wound healing is delayed due to a steroid agent. The wound delayed due to the steroid agent may be delayed by the steroid agent inhibiting the proliferation of keratinocytes or inhibiting cell proliferation. The wound in which the wound healing is delayed may be a wound in which the wound is delayed using a steroid, or a wound in which the wound healing is delayed due to the continuous use of the steroid.
일 구체예에 따르면, 상기 스테로이드제는 부신피질호르몬제, 남성호르몬제, 여성호르몬제일 수 있으나, 예를 들면 부신피질호르몬제 일 수 있다. 상기 부신피질호르몬제는 항염증 효과 및 면역억제효과를 가지는 약물로, 발적, 부종, 열감, 압통 등의 증상을 현저히 감소시킬 수 있는 제제일 수 있으며, 신체의 면역기능이 저하되어 세균 감염이 잘 유발되거나 악화되는 부작용이 나타날 수 있다. 상기 부신피질호르몬제는 덱타메타손, 히드로코르티손, 프레드니솔론, 베타메타손일 수 있으나, 이에 제한되지 않는다.According to one embodiment, the steroid agent may be a corticosteroid agent, a male hormone agent, or a female hormone agent, but may be, for example, a corticosteroid agent. The corticosteroid agent is a drug having an anti-inflammatory effect and an immunosuppressive effect, and may be a drug that can significantly reduce symptoms such as redness, edema, heat, tenderness, etc. Side effects may be induced or exacerbated. The corticosteroid agent may be dextamthasone, hydrocortisone, prednisolone, or betamethasone, but is not limited thereto.
일 실시예에 따르면, 스테로이드제를 처리한 상처의 경우, 상처치유가 되지 않았으며, 스테로이드제를 처리한 상처에 푸에라린을 사용한 경우 스테로이드제만 처리한 상처 대비 현저히 상처치유가 진행되는 것을 확인하였다. According to one embodiment, in the case of a wound treated with a steroid, the wound was not healed, and when puerarine was used for a wound treated with a steroid, it was confirmed that the wound healing progressed significantly compared to a wound treated with only a steroid. did.
상기 '상처 치료'는 상처치유와 동등한 의미일 수 있으며, 파괴된 피부의 상피조직이 온전해지는 모든 과정을 포함하는 의미이다.The 'wound treatment' may have the same meaning as wound healing, and includes all processes in which the epithelial tissue of the destroyed skin is intact.
일 구체예에 따르면, 상기 조성물은 상처에서 각질형성세포를 형성하거나 증식시키는 것일 수 있다. According to one embodiment, the composition may form or proliferate keratinocytes in a wound.
일 실시예에 따르면, 스테로이드제를 처리한 상처의 경우, 각질 세포의 형성이 지연되었으나, 스테로이드제 및 푸에라린을 처리한 경우 각질 세포가 형성되는 것을 확인하였다. According to one embodiment, in the case of a wound treated with a steroid, the formation of keratinocytes was delayed, but it was confirmed that keratinocytes were formed when the wound was treated with a steroid and puerarin.
일 실시예에 따르면, 스테로이드제를 처리한 상처의 경우, 세포 증식 지수인 Ki67(+)의 비율이 억제되었으나, 스테로이드제 및 푸에라린을 처리한 경우 Ki67(+)의 비율이 대조군과 유사한 정도로 증가되는 것을 확인하였다. According to one embodiment, in the case of a wound treated with a steroid, the ratio of Ki67(+), which is the cell proliferation index, was suppressed, but when treated with a steroid and puerarin, the ratio of Ki67(+) was similar to the control. was confirmed to increase.
한편, 본 발명의 약학적 조성물은 각각 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있다. 예컨대, 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽 등의 경구형 제형으로 제형화할 수 있고, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 다만, 본 발명의 조성물은 상처 치료라는 목적상 피부 외용제의 형태로 제공되는 것일 수 있다. 예를 들면, 액제, 연고제, 크림제, 로션제, 스프레이제, 패취제, 겔제 또는 에어로졸제 등의 피부 외용제의 형태로 사용될 수 있다.On the other hand, the pharmaceutical composition of the present invention can be formulated in various forms according to a conventional method, respectively. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated in the form of external preparations, suppositories, and sterile injection solutions. However, the composition of the present invention may be provided in the form of a skin external preparation for the purpose of wound treatment. For example, it may be used in the form of external preparations for skin, such as liquids, ointments, creams, lotions, sprays, patches, gels, or aerosols.
또한, 각각의 제형에 따라 약학적으로 허용 가능한 담체, 예컨대 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제, 기제, 부형제, 윤활제 등 당업계에 공지된 담체를 추가로 포함하여 제조할 수 있다. 예컨대, 상처 부위에 국부적으로 사용되는 피부 외용제인 경우에는 통상적인 첨가제, 예를 들어 보존제, 의약 침투를 보조하는 용매, 연고 및 크림의 경우 연화제 등을 포함할 수 있으며, 에탄올 또는 올레일 알코올과 같은 통상적 담체를 함유할 수 있다. In addition, according to each dosage form, a pharmaceutically acceptable carrier, such as a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc., may be prepared by further including a carrier known in the art. can For example, in the case of an external preparation for skin used locally on the wound site, conventional additives, for example, a preservative, a solvent to aid drug penetration, and an emollient in the case of ointments and creams, such as ethanol or oleyl alcohol, may be included. It may contain conventional carriers.
또한, 본 발명의 약학적 조성물에는, 상처 치료에 일반적으로 사용되는 화합물, 예컨대 항생제, 피부 치료용 조제, 마취제 및 진통제 등을 추가로 포함할 수 있다. 이는, 각각의 제형에 따라 약간씩 변화될 수 있으며, 바람직하게는 피부 외용제의 형태로 제공되는 경우에 포함될 수 있으나, 이에 제한되는 것은 아니다. 일반적으로, 상처부위의 감염을 방지하거나 감염으로 인한 상처의 악화를 방지하기 위하여 상처부위에 항생제, 항균제 등의 약제를 공급할 수 있고, 또한 통증의 완화를 위하여 국소 마취제 및 진통제 등을 공급할 수 있다. 다양한 기능성 약제들을 함유시켜 공급함으로써, 사용상의 편리성을 도모하고, 효율적인 상처치유에 도움이 되는 환경을 조성함으로써 상처치유 효과를 더욱 향상시킬 수 있다.In addition, the pharmaceutical composition of the present invention may further include a compound commonly used for wound treatment, such as antibiotics, skin treatment preparations, anesthetics and analgesics. This may be slightly changed according to each formulation, and may preferably be included when provided in the form of an external preparation for skin, but is not limited thereto. In general, in order to prevent infection of the wound site or to prevent deterioration of the wound due to infection, drugs such as antibiotics and antibacterial agents may be supplied to the wound site, and local anesthetics and analgesics may be supplied to relieve pain. By containing and supplying various functional drugs, it is possible to promote convenience in use and to further improve the wound healing effect by creating an environment conducive to effective wound healing.
한편, 본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명의 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 상처의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.On the other hand, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level depends on the patient's health condition, The type of wound, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, duration of treatment, factors including drugs used in combination or concomitantly, and other factors well known in the medical field. can
다른 양상은 푸에라린(Puerarin)을 포함하는 상처 치료 촉진용 의약외품 조성물을 제공하는 것이다. Another aspect is to provide a quasi-drug composition for promoting wound healing comprising Puerarin.
상기 푸에라린, 상처, 및 푸에라린의 상처 치료 효능에 대해서는 상기 설명한 바와 같으며, 본 발명의 조성물은 상처의 치료 또는 개선 목적으로 의약외품에 첨가될 수 있다. The pueralin, the wound, and the wound healing efficacy of pueralin are the same as described above, and the composition of the present invention may be added to a quasi-drug for the purpose of treating or improving a wound.
상기 '상처 치료 촉진'은 상처치유 촉진과 동일한 의미일수 있으며, 파괴된 피부의 상피조직이 온전해지는 모든 과정이 촉진되는 것을 포함하는 의미이다.The 'promoting wound healing' may have the same meaning as promoting wound healing, and includes promoting all processes in which the epithelial tissue of the destroyed skin is intact.
일 구체예에 따르면, 상기 조성물은 상처에서 각질형성세포의 형성 또는 증식을 촉진시키는 것일 수 있다. According to one embodiment, the composition may promote the formation or proliferation of keratinocytes in the wound.
일 실시예에 따르면, 스테로이드제를 처리한 상처의 경우, 각질 세포의 형성이 지연되었으나, 스테로이드제 및 푸에라린을 처리한 경우 각질 세포가 형성되는 것을 확인하였다. According to one embodiment, in the case of a wound treated with a steroid, the formation of keratinocytes was delayed, but it was confirmed that keratinocytes were formed when the wound was treated with a steroid and puerarin.
일 실시예에 따르면, 스테로이드제를 처리한 상처의 경우, 세포 증식 지수인 Ki67(+)의 비율이 억제되었으나, 스테로이드제 및 푸에라린을 처리한 경우 Ki67(+)의 비율이 대조군과 유사한 정도로 증가되는 것을 확인하였다. According to one embodiment, in the case of wounds treated with steroids, the ratio of Ki67(+), which is the cell proliferation index, was inhibited, but when treated with steroids and puerarin, the ratio of Ki67(+) was similar to that of the control group. was confirmed to increase.
상기 '의약외품'은 사람이나 동물의 질병을 치료, 경감, 처치 또는 예방할 목적으로 사용되는 섬유, 고무제품 또는 이와 유사한 것, 인체에 대한 작용이 약하거나 인체에 직접 작용하지 아니하며, 기구 또는 기계가 아닌 것과 이와 유사한 것, 감염병 예방을 위하여 살균, 살충 및 이와 유사한 용도로 사용되는 제제 중 하나에 해당하는 물품으로서, 사람이나 동물의 질병을 진단, 치료, 경감, 처치 또는 예방할 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것 및 사람이나 동물의 구조와 기능에 약리학적 영향을 줄 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것을 제외한 물품을 의미한다.The above 'quasi-drugs' refer to fibers, rubber products, or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, have weak effects on the human body or do not directly act on the human body, and are not instruments or machines. Items similar to those used for sterilization, insecticide and similar purposes for the prevention of infectious diseases , refers to items other than instruments, machines, or devices, which are not machines or devices, and items used for the purpose of pharmacologically affecting the structure and function of humans or animals.
본 발명의 조성물을 의약외품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 의약외품 또는 의약외품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적에 따라 적합하게 결정될 수 있다.When the composition of the present invention is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use.
본 발명의 의약외품 조성물은 이에 제한되지는 않으나, 바람직하게는 소독청결제, 샤워폼, 구강 청정제(가그린), 물티슈, 세제비누, 핸드워시, 가습기 충진제, 마스크, 연고제 또는 필터충진제일 수 있다.The quasi-drug composition of the present invention is not limited thereto, but preferably disinfectant cleaner, shower foam, mouth freshener (Gagreen), wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.
본 발명에 따른 푸에라린을 포함하는 조성물을 사용할 경우, 각질세포의 형성 및 증식을 증가시키고 상처 치료 효능이 뛰어나므로, 상처 치료를 위한 피부 외용제를 비롯한 의약품 등에 적용되어 사용될 수 있을 것이다.When the composition containing puerarine according to the present invention is used, it increases the formation and proliferation of keratinocytes and has excellent wound healing efficacy, so it may be applied to and used in pharmaceuticals including external preparations for wound treatment.
도 1은 스테로이드제 및 푸에라린 처리 후 시간에 따라 HaCaT 세포의 형성 정도를 확인한 데이터이다.
도 2의 A는 각 처리군에 따라 Ki67(+) 세포의 비율을 본 데이터이고, B는 A의 비율을 그래프로 나타낸 데이터이다.
도 3은 각 처리군에 따라 K16의 발현을 확인한 데이터이다.
도 4의 A는 각 처리군의 상처를 사진으로 확인한 데이터이고, B는 처리군의 상처 크기를 그래프로 나타낸 데이터이다.1 is data confirming the degree of formation of HaCaT cells according to time after treatment with steroids and pueralin.
2A is data showing the ratio of Ki67(+) cells according to each treatment group, and B is data showing the ratio of A as a graph.
3 is data confirming the expression of K16 according to each treatment group.
4A is data confirming the wounds of each treatment group with pictures, and B is data showing the size of the wounds in the treatment group as a graph.
이하 하나 이상의 구체예를 실시예를 통해 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.
실시예 1. 상처 회복 분석(Wound scratch assay) Example 1. Wound scratch assay
상처 치료 효과를 확인하기 위하여, HaCaT 세포로 상처 회복 분석을 실시하였다. HaCaT 세포를 12 웰-플레이트에 4×105 세포/웰로 접종한 후, 10% FBS를 포함하는 EMEM 배지에서 100% confluency로 배양하였다. 배양한 세포에 스크래치를 낸 후, 덱사메타손(Dexamethasone, (DEX), Sigma Aldrich, Korea) 100 nM과 푸에라린(Puerarin, ChemFaces) 1μM, 10μM을 처리하였다. 처리 직후, 12시간 후, 24시간 후 Lionheart FX Automated를 사용하여 스크래치가 난 부분의 너비를 캡쳐하고 live-imaging software Ven5 Imager software(Biotek Instruments Inc, Winooski, VT, USA)를 사용하여 측정하였다.In order to confirm the wound healing effect, a wound healing assay was performed with HaCaT cells. HaCaT cells were seeded in 12 well-plates at 4×10 5 cells/well, and then cultured at 100% confluency in EMEM medium containing 10% FBS. After scratching the cultured cells, 100 nM of dexamethasone (DEX), Sigma Aldrich, Korea) and 1 μM and 10 μM of puerarin (Puerarin, ChemFaces) were treated. Immediately after treatment, 12 hours, and 24 hours later, the width of the scratched area was captured using Lionheart FX Automated and measured using live-imaging software Ven5 Imager software (Biotek Instruments Inc, Winooski, VT, USA).
도 1에서 보이는 바와 같이, 스테로이드제제인 덱사메타손(DEX)을 처리한 경우 정상군 세포(NC)에 비해 각질 세포인 HaCaT의 형성이 지연되는 것을 확인하였다. 반면, 스테로이드제 및 푸에라린을 함께 처리한 경우, HaCaT의 형성이 촉진되는 것을 확인하였다. As shown in FIG. 1 , it was confirmed that the formation of keratinocytes, HaCaT, was delayed compared to normal group cells (NC) when dexamethasone (DEX), a steroid, was treated. On the other hand, it was confirmed that the formation of HaCaT was accelerated when steroids and puerarin were treated together.
실시예 2. Ki67 증식반응 측정(Proliferation assay)Example 2. Ki67 Proliferation Assay
Ki67 증식 분석은 Muse Ki67 Proliferation kit(Luminex Corporaiton, Austin, Tx, USA)를 사용하였으며, 제조사의 프로토콜에 따라 진행하였다. HaCaT 세포를 6웰 플레이트에 5×105 세포/웰로 접종하고 난 24 시간 뒤, 덱사메타손 100nM과 푸에라린 1μM, 10μM을 처리하고 48시간 동안 배양하였다. 그 후 세포를 고정시키고, 투과성을 높여 Muse human Ki67-PE와 IgG1-PE 항체와 배양하였다. 그 다음 세포들을 분석하였고, Muse Cell Analyzer(merck KGaA, Darmstadt, Germany)로 Ki67(+) 비율을 측정하였다.Ki67 proliferation analysis was performed using the Muse Ki67 Proliferation kit (Luminex Corporaiton, Austin, Tx, USA), and was performed according to the manufacturer's protocol. 24 hours after HaCaT cells were inoculated into a 6-well plate at 5×10 5 cells/well, 100 nM of dexamethasone, 1 μM of puerarine, and 10 μM were treated and cultured for 48 hours. Thereafter, the cells were fixed and permeability was increased and incubated with Muse human Ki67-PE and IgG1-PE antibodies. Then, the cells were analyzed, and the Ki67(+) ratio was measured with Muse Cell Analyzer (merck KGaA, Darmstadt, Germany).
도 2에서 보이는 바와 같이, 대조군의 Ki67(+) 비율 90.85% 대비 스테로이드제제 처리군의 Ki67(+) 비율은 73.20%로 약 18% 감소하였다. 반면 스테로이드제제와 푸에라린 1μM을 처리한 경우는 86.45%, 스테로이드제제와 푸에라린 10μM을 처리한 경우는 86.30%로 Ki67(+)의 비율이 증가하여 대조군의 비율과 유사할 정도로 세포가 증식된다는 것을 확인하였다. As shown in FIG. 2 , the Ki67(+) ratio of the control group was reduced by about 18% to 73.20%, compared to 90.85% of the Ki67(+) ratio of the control group. On the other hand, the ratio of Ki67(+) increased to 86.45% when steroids and 1 μM of puerarin were treated, and 86.30% when steroids and 10 μM of puerarin were treated. confirmed that it will be
실시예 3. K16 발현 유도 효과 확인Example 3. Confirmation of K16 expression induction effect
K16(Keratin 16)의 발현을 확인하기 위하여, 웨스턴 블롯 분석을 하였다. HaCaT 세포를 6 웰-플레이트에 5×105 세포/웰로 접종한 후, 덱사메타손(Dexamethasone, (DEX), Sigma Aldrich, Korea) 100 nM과 푸에라린(Puerarin, Chemfaces) 1μM, 10μM을 처리하였다. 덱사메타손 및 푸에라린을 처리한 HaCaT 세포를 각각 조직 추출 시약(Thermo Fisher Scientific, IL, USA)로 용해시켰다. 단백질을 15분 동안 8,000 Х g 의 4℃에서 원심 분리하여 추출한 후, 상등액을 회수하였다. 그 후, 50μg의 단백질을 10% SDS-PAGE 전기영동으로 분석한 다음, 폴리비닐리덴 디플루오라이드(PVDF) 멤브레인(Merck Millipore, Carrigtwohill, Ireland)으로 옮겼다. 멤브레인을 실온에서 2시간 동안 1ХPBS의 5% 탈지유로 블로킹하고, 1차 항체와 배양한 다음, 겨자무과산화효소(horseradish peroxidase)가 컨쥬게이트된 anti IgG 2차 항체와 배양하였다. 그 후, 강화 화학발광(enhanced chemiluminescence, BioRad, CA, USA)으로 모든 멤브레인을 검출하였다. 단백질의 밴드 강도는 GelPro V3.1 소프트웨어(Media Cybernetics, MD, USA)를 사용하여 정량화하였다.To confirm the expression of K16 (Keratin 16), Western blot analysis was performed. HaCaT cells were inoculated into a 6-well-plate at 5×10 5 cells/well, followed by treatment with 100 nM of dexamethasone (DEX), Sigma Aldrich, Korea and 1 μM, 10 μM of puerarin (Puerarin, Chemfaces). HaCaT cells treated with dexamethasone and puerarin were each lysed with tissue extraction reagent (Thermo Fisher Scientific, IL, USA). After the protein was extracted by centrifugation at 8,000 Х g at 4°C for 15 minutes, the supernatant was recovered. Then, 50 μg of the protein was analyzed by 10% SDS-PAGE electrophoresis, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Carrigtwohill, Ireland). The membrane was blocked with 5% skim milk in 1ХPBS for 2 hours at room temperature, incubated with a primary antibody, and then incubated with an anti-IgG secondary antibody conjugated with horseradish peroxidase. All membranes were then detected by enhanced chemiluminescence (BioRad, CA, USA). The band intensities of proteins were quantified using GelPro V3.1 software (Media Cybernetics, MD, USA).
그 결과, 도 3에서 보이는 바와 같이, 스테로이드제제인 덱사메타손만 처리한 세포에서는 대조군 대비 K16 발현이 감소하였으나, 덱사메타손 및 푸에라린을 처리한 세포에서는 K16의 발현이 현저히 증가된 것을 확인하였다. As a result, as shown in FIG. 3 , it was confirmed that K16 expression was decreased in cells treated with only dexamethasone, a steroid, compared to the control, but K16 expression was significantly increased in cells treated with dexamethasone and puerarin.
실시예 4. 상처 치료 효과 확인Example 4. Confirmation of wound healing effect
실험동물은 5주령 수컷 BALB/c 마우스(코아텍, 서울)를 사용하였다. 마우스는 6 마리씩 4 그룹(대조군, 스테로이드 처리군, 스테로이드 및 푸에라린 0.1% 처리군, 스테로이드 및 푸에라린 1% 처리군)으로 무작위로 나누어 실험 하였다. 스테로이드는 아세톤에 덱사메타손(Dexamethasone, DEX)을 0.1% 로 희석하여 준비하였고, 푸에라린(Puerarin)은 바세린 50μg에 0.1% 또는 1%로 희석하여 준비하였다. As experimental animals, 5-week-old male BALB/c mice (Coatec, Seoul) were used. Mice were randomly divided into 4 groups of 6 mice each (control group, steroid treatment group, steroid and puerarin 0.1% treatment group, steroid and
상처 재생 실험을 위하여, 각 그룹의 마우스 등을 면도한 후 직경 5mm 생검펀치(Kai Group, Seki, Japan)로 피부 결손을 만들었다. 덱사메타손을 대조군을 제외한 모든 그룹의 상처에 같은 양으로 14일간 매일 도포하였다. 또한, 스테로이드 및 푸에라린 0.1% 또는 1% 처리군의 상처에는 희석한 푸에라린을 각 농도에 맞게 같은 양으로 14일간 매일 도포하였다. 상처의 크기는 피부를 결손 시킨 후 당일, 7일째, 14일째에 측정하여 평균을 계산하였다. For the wound regeneration experiment, after shaving the back of the mice of each group, a skin defect was made with a 5 mm diameter biopsy punch (Kai Group, Seki, Japan). Dexamethasone was applied daily for 14 days in the same amount to the wounds of all groups except the control group. In addition, the same amount of diluted puerarine was applied to the wounds of the steroid and puerarin 0.1% or 1% treatment groups every day for 14 days in accordance with each concentration. The size of the wound was measured on the same day, the 7th day, and the 14th day after the skin defect was calculated and the average was calculated.
도 4에서 보이는 바와 같이, 처음 상처 크기가 모두 똑같았지만, 7일째 측정부터는 스테로이드제제를 처리하지 않은 대조군 마우스의 상처가 가장 빠르게 아물었다. 스테로이드제제를 사용한 경우, 14일째에 스테로이드제제를 사용한 마우스 대비 푸에라린을 사용한 마우스의 상처 크기가 현저히 작아진 것을 확인하였고, 이는 대조군 마우스와도 유사한 크기임을 확인하였다. As shown in FIG. 4 , the initial wound sizes were all the same, but from the 7th day, the wounds of the control mice not treated with the steroids healed the fastest. In the case of using a steroid formulation, it was confirmed that the wound size of the mice using the puerarine was significantly smaller on the 14th day compared to the mice using the steroid formulation, and it was confirmed that the size was similar to that of the control mice.
Claims (5)
A pharmaceutical composition for wound treatment comprising Puerarin.
상기 상처는 스테로이드제로 인하여 상처치유가 지연된 상처인,
상처 치료용 약학적 조성물.
The method of claim 1,
The wound is a wound in which wound healing is delayed due to steroids,
A pharmaceutical composition for the treatment of wounds.
상기 스테로이드제는 부신피질호르몬제, 남성호르몬제, 여성호르몬제인,
상처 치료용 약학적 조성물.
3. The method of claim 2,
The steroid is a corticosteroid, male hormone, female hormone,
A pharmaceutical composition for the treatment of wounds.
상기 조성물은 액제, 연고제, 크림제, 로션제, 스프레이제, 패취제, 겔제 또는 에어로졸제인 것을 특징으로 하는,
상처 치료용 약학적 조성물.
The method of claim 1,
The composition is characterized in that it is a solution, an ointment, a cream, a lotion, a spray, a patch, a gel or an aerosol,
A pharmaceutical composition for the treatment of wounds.
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| KR102222919B1 (en) | 2018-07-06 | 2021-03-04 | 주식회사 씨케이바이오텍 | composition for preventing or treating of wound comprising Indirubin derivative compound |
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