KR100515066B1 - Novel compounds showing tyrosinase-inhibitive effect purified from Veratrum grandiflorum, and whitening compositions the same - Google Patents
Novel compounds showing tyrosinase-inhibitive effect purified from Veratrum grandiflorum, and whitening compositions the same Download PDFInfo
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Abstract
본 발명은 박새로부터 분리한 타이로시네이즈 저해활성을 가지는 화합물 및 이를 포함하는 피부미백제에 관한 것으로서, 더욱 상세하게는 박새(Veratrum grandiflorum) 지하부를 메탄올로 추출한 추출액 중 부탄올 가용부를 분리하여 젤여과크로마토그래피에 의하여 단리된 타이로시네이즈 저해활성을 가지는 신규 화합물과, 이러한 신규 화합물이 함유된 피부미백제에 관한 것이다.The present invention relates to a compound having a tyrosinase inhibitory activity isolated from the larvae, and a skin whitening agent comprising the same, and more particularly, gel filtration chromatography by separating the soluble part of butanol from the extract extracted from the underground of Veratrum grandiflorum with methanol. The present invention relates to novel compounds having tyrosinase inhibitory activity isolated by graphics, and skin lightening agents containing such novel compounds.
Description
본 발명은 박새로부터 분리한 타이로시네이즈 저해활성을 가지는 화합물 및 이를 포함하는 피부미백제에 관한 것으로서, 더욱 상세하게는 박새(Veratrum grandiflorum) 지하부를 메탄올로 추출한 추출액 중 부탄올 가용부를 분리하여 젤여과크로마토그래피에 의하여 단리된 타이로시네이즈 저해활성을 가지는 신규 화합물과, 이러한 신규 화합물이 함유된 피부미백제에 관한 것이다.The present invention relates to a compound having a tyrosinase inhibitory activity isolated from the larvae, and a skin whitening agent comprising the same, and more particularly, gel filtration chromatography by separating the soluble part of butanol from the extract extracted from the underground of Veratrum grandiflorum with methanol. The present invention relates to novel compounds having tyrosinase inhibitory activity isolated by graphics, and skin lightening agents containing such novel compounds.
멜라닌은 색소 세포 내에 존재하는 타이로시네이즈 작용에 의해 타이로신으로부터 도파(dopa), 도파퀴논(dopaquinone)으로 변환되어 도파크롬(dopachrome) 등을 거처 생성되어 진다. 이 멜라닌은 피부에 존재하여 자외선 등으로부터 신체를 보호하는 중요한 기능을 가지고 있다. 그러나 멜라닌이 과잉생산됨으로써 기미, 주근깨 등을 형성하고, 피부노화를 촉진하며, 피부암 유발에 중요한 작용을 하는 것으로 알려져, 최근에는 멜라닌 과잉생성을 예방하는 약제 개발이 활발히 진행되고 있다. 이미 미백효과제로서는 파라-메톡시페놀(p-metoxyphenol), 하이드로퀴논(hydroquinone), 코지산(kojic acid), 알부틴(arbutin) 등이 사용되고 있으나, 이들은 활성이 약하거나 색소세포의 변성 또는 치사를 일으키거나 세포 본래의 기능을 손상시키는 등 부작용이 있는 경우도 있다. 한편, 멜라닌 생성 억제를 목적으로 비타민 C 및 그 유도체 등이 사용되고 있으나, 이들 또한 저해활성이 낮다는 단점을 가지고 있다. 따라서 소량으로도 멜라닌 생합성에 관여하는 타이로시네이즈 효소활성을 나타내는 물질의 개발이 요구된다.Melanin is converted from tyrosine to dopa and dopaquinone by tyrosinase action present in pigmented cells, and is produced via dopachrome and the like. This melanin is present in the skin and has an important function of protecting the body from ultraviolet rays. However, over-production of melanin is known to form blemishes, freckles, etc., promote skin aging, and play an important role in inducing skin cancer. Recently, development of drugs for preventing melanin overproduction is being actively conducted. P- metoxyphenol, hydroquinone, kojic acid, arbutin, and the like are already used as whitening agents, but they are weak in activity or denature or kill pigment cells. There are also side effects, such as causing or impairing the cell's original function. On the other hand, vitamin C and derivatives thereof are used for the purpose of inhibiting melanin production, but they also have the disadvantage of low inhibitory activity. Therefore, there is a need for development of a substance showing tyrosinase enzyme activity involved in melanin biosynthesis even in small amounts.
이에, 본 발명자들은 타이로시네이즈 저해 활성을 가지는 물질을 천연물로부터 분리하는 연구를 하였고, 그 결과 백합과 식물인 박새로부터 유효 활성물질을 추출 정제함으로써 본 발명을 완성하게 되었다. Therefore, the present inventors have conducted a study for separating a substance having tyrosinase inhibitory activity from natural products, and as a result, the present invention has been completed by extracting and purifying an active substance from the lily of the herbaceous locust.
따라서, 본 발명은 박새(Veratrum grandiflorum)로부터 분리된 타이로시네이즈 저해활성을 나타내는 신규 화합물을 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide a novel compound exhibiting tyrosinase inhibitory activity isolated from Veratrum grandiflorum .
또한, 본 발명은 박새(Veratrum grandiflorum)를 알콜 추출 및 젤여과크로마토그래피하여 타이로시네이즈 저해활성을 가지는 화합물을 분리하는 방법을 제공하는데 다른 목적이 있다.Another object of the present invention is to provide a method for separating compounds having tyrosinase inhibitory activity by alcohol extraction and gel filtration chromatography of Vertrum grandiflorum .
또한, 본 발명은 박새(Veratrum grandiflorum) 추출물, 또는 박새로부터 분리된 신규 화합물 또는 약제학적으로 허용 가능한 이의 염이 유효활성 성분으로 포함되어 있어 타이로시네이즈 저해하는 피부미백제를 제공하는데 또다른 목적이 있다.In another aspect, the present invention is to provide a skin whitening agent that inhibits tyrosinase, containing Veratrum grandiflorum extract, or a novel compound or a pharmaceutically acceptable salt thereof isolated from the larvae as an active ingredient. have.
본 발명은 우수한 타이로시네이즈 저해활성을 가지는 다음 화학식 1로 표시되는 신규 화합물 및 약제학적으로 허용 가능한 이의 염을 그 특징으로 한다.The present invention is characterized by a novel compound represented by the following formula (1) and a pharmaceutically acceptable salt thereof having excellent tyrosinase inhibitory activity.
또한, 본 발명은 박새(Veratrum grandiflorum) 추출물 또는 상기 화학식 1로 표시되는 신규 화합물또는 이의 염이 유효성분으로 함유되어 있어 타이로시네이즈의 활성을 저해하는 피부미백제를 또다른 특징으로 한다.In another aspect, the present invention is characterized by a skin whitening agent that inhibits the activity of tyrosinase by containing Veratrum grandiflorum extract or a novel compound represented by Formula 1 or a salt thereof as an active ingredient.
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.
본 발명은 우수한 타이로시네이즈 저해 활성성분으로서의 상기 화학식 1로 표시되는 화합물과, 박새 식물로부터 이러한 활성물질을 추출 분리하는 방법에 관한 것이다. 상기한 바대로, 본 발명에 따른 상기 화학식 1로 표시되는 화합물은 신규 화합물로서 유기 합성방법으로 합성할 수 있고, 또는 본 발명의 방법에 따라 박새로부터 추출 분리할 수도 있다.The present invention relates to a compound represented by the formula (1) as an excellent tyrosinase inhibitory active ingredient, and to a method for extracting and separating such an active substance from the shoots. As described above, the compound represented by Chemical Formula 1 according to the present invention may be synthesized by an organic synthesis method as a novel compound, or may be extracted and separated from the fly according to the method of the present invention.
박새로부터 상기 화학식 1로 표시되는 화합물을 추출 분리하는 방법은 다음 단계를 포함한다: 1) 박새(Veratrum grandiflorum)를 메탄올로 추출하는 단계, 2) 상기 메탄올 추출액을 부탄올으로 진탕추출하여 부탄올 가용부를 회수하는 단계, 3) 상기 부탄올 가용부를 젤여과크로마토그래피하여 활성분획을 얻는 단계, 및 4) 상기 활성분획을 동결 건조하는 단계.The method of extracting and separating the compound represented by Chemical Formula 1 from the larvae includes the following steps: 1) extracting Veratrum grandiflorum with methanol, 2) recovering the butanol soluble part by shaking the methanol extract with butanol 3) gel filtration of the butanol soluble part to obtain an active fraction, and 4) freeze drying the active fraction.
박새 식물로부터 박새 추출물 또는 활성물질의 분리과정을 그 단계별로 상세히 설명하면 다음과 같다. 먼저, 자생 또는 재배한 박새를 채취하여 쇄절한다. 그리고, 100% 메탄올으로 24 시간 환류추출하고 감압여과한 후, 그 여액을 20 ∼ 50 ℃ 온도에서 감압농축한다. 그런 다음, 농축된 메탄올 추출액을 과량의 물에 현탁하여 분액여두에 넣은 후 과량의 부탄올을 넣고 진탕추출한다. 상등액을 회수하여 40 ∼ 60 ℃ 온도에서 감압농축하여 부탄올 가용부를 얻는다. 농축된 부탄올 가용부를 소량의 메탄올에 용해한 후 젤여과크로마토그래피하여 활성 분액을 모은다. 이때, 크로마토그래피로는 세파덱스 LH-20 젤 칼럼 크로마토그래피(sephadex LH-20 gel column chromatography), YMC 젤 ODS-A 칼럼 크로마토그래피(YMC gel column chromatography)를 수행하여 활성분액을 모은다. 그리고, 모아진 활성분액은 40 ∼ 60 ℃ 온도에서 감압농축하여 분말로 얻은 후 소량의 증류수에 용해한 후 동결건조한다.The separation process of the extracts or active substances from the leaves of the plant is described in detail step by step as follows. First, the native or cultivated chickadee is harvested and broken up. After refluxing with 100% methanol for 24 hours and filtration under reduced pressure, the filtrate was concentrated under reduced pressure at a temperature of 20 to 50 ° C. Then, the concentrated methanol extract is suspended in excess water, placed in a separatory filter, and the excess butanol is added and shaken. The supernatant is recovered and concentrated under reduced pressure at a temperature of 40 to 60 ° C. to obtain a butanol soluble part. The concentrated butanol soluble part is dissolved in a small amount of methanol, and then gelled chromatography to collect the active aliquots. At this time, as an chromatography, Sepadex LH-20 gel column chromatography and YMC gel ODS-A column chromatography are performed to collect active liquids. The collected active fractions are concentrated under reduced pressure at a temperature of 40 to 60 ° C. to obtain a powder, dissolved in a small amount of distilled water, and then lyophilized.
상기한 추출 분리과정 중의 활성물질의 추적은 항 타이로시네이즈 (tyrosinase) 활성의 측정에 기준을 두어 다음과 같이 행하였다. 먼저, 검체 15 ㎕를 마이크로플레이트(96 well microplate)에 넣고, 0.1 M 인산완충액(pH 6.5) 150 ㎕와 1.5 mM L-타이로신 용액 25 ㎕를 넣은 후, 효소용액(타이로시네이즈, 시그마제) 7 ㎕를 첨가하여 마이크로플레이트 리이더(Microplate reader, 바이오라드제)를 이용하여 490 nm에서 흡광도를 측정하였다. 이 플레이트를 37 ℃에서 10 분간 반응시킨 후 다시 490 nm에서 흡광도를 측정한 후 타이로시네이즈 저해율(%)을 계산하였다.Tracking of the active material during the extraction separation process was performed as follows based on the measurement of anti-tyrosinase activity. First, 15 μl of the sample was put into a microplate (96 well microplate), 150 μl of 0.1 M phosphate buffer (pH 6.5) and 25 μl of 1.5 mM L-tyrosine solution were added, followed by enzyme solution (tyrosinase, sigma). 7 μl was added and the absorbance was measured at 490 nm using a Microplate reader (manufactured by Biorad). The plate was reacted at 37 ° C. for 10 minutes, and then absorbance was measured at 490 nm, and then the tyrosinase inhibition rate (%) was calculated.
이상의 추출 분리공정에 의해 최종적으로 분리된 활성화합물은 타이로시네이즈 저해 효과가 있는 바, 따라서 본 발명은 박새 식물의 추출물 또는 상기 화학식 1로 표시되는 화합물이 유효성분으로 함유된 피부미백제를 포함한다. 본 발명에서의 피부미백제는 의약품으로서 직접 사용하거나 또는 화장품, 식품 등에 함유되어 피부질환치료 및 피부 미백효과를 발현 또는 식품의 갈변을 방지하게 된다.The active compound finally separated by the above extraction and separation process has a tyrosinase inhibitory effect, and thus the present invention includes an extract of the chickpea plant or a skin lightening agent containing the compound represented by the formula 1 as an active ingredient. . Skin whitening agent in the present invention can be used directly as a medicine or contained in cosmetics, foods, etc. to treat skin diseases and skin whitening effect or to prevent browning of food.
상기 화학식 1로 표시되는 화합물은 약학적으로 허용 가능한 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 상기 화학식 1로 표시되는 화합물은 당해 기술 분야에서 통상적인 방법에 따라 약학적으로 형용되는 산 부가염을 형성할 수 있다. 유리산으로는 유리산과 무기산을 사용할 수 있으며, 무기산으로는 염산, 브롬산, 황산, 인산 등을 사용할 수 있고 유기산으로는 구연산(citric acid), 초산, 젖산, 주석산(tartaric acid), 말레인산, 푸마르산(fumaric acid), 포름산, 프로피온산(propionic acid), 옥살산, 트리플루오로아세트산, 벤조산, 글루콘산, 메탄술폰산, 글리콜산, 숙신산, 4-톨루엔술폰산, 갈룩투론산, 엠본산, 글루탐산 또는 아스파르트산 등을 사용할 수 있다.The compound represented by Chemical Formula 1 may be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. The compound represented by Chemical Formula 1 may form an acid addition salt which is pharmaceutically acceptable according to a conventional method in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.
본 발명의 피부미백제를 의약품으로 사용하는 경우, 박새 식물의 추출물 또는 상기 화학식 1로 표시되는 화합물 또는 약학적으로 허용 가능한 염을 임상적으로 이용시에는 약학적 분야에서 통상적인 담체와 함께 배합하여 약학적 분야에서 통상적인 제제, 예를들면 정제, 캅셀제, 트로키제, 액제, 현탁제 등의 경구투여용 제제; 주사용 용액 또는 현탁액, 또는 주사시에 주사용 증류수로 제조하여 사용할 수 있는 즉시 사용형 주사용 건조분말 등의 형태인 주사용 제제; 또는 연고제 등의 다양한 제제로 제형화할 수 있다. 통상적인 담체를 상용하여 제조된 약학적 제제는 경구적으로 투여하거나, 비경구적으로 예를들면 정맥내, 피하, 복강내 또는 국소적용할 수 있다. 본 발명의 박새 추출물 또는 상기 화학식 1로 표시되는 화합물의 투여량은 환자의 나이, 상태 등에 따라 차이가 있으나, 일반적으로 성인에게 1일에 10 ∼ 500 ㎎, 바람직하게는 50 ∼ 300 ㎎의 양이 투여되도록 하며, 의사 또는 약사의 판단에 따라 일정시간 간격으로 1일 수회, 바람직하기로는 1회 내지는 6회 분할투여할 수 있다.In the case of using the skin lightening agent of the present invention as a medicine, the extract of the creeper plant or the compound represented by the formula (1) or a pharmaceutically acceptable salt when used clinically in combination with a conventional carrier in the pharmaceutical field Preparations orally administered such as tablets, capsules, troches, solutions, suspensions, etc., which are customary in the field; Injectable preparations in the form of injectable solutions or suspensions, or ready-to-use injectable dry powders which can be prepared and used as injectable distilled water at the time of injection; Or in various preparations such as ointments. Pharmaceutical formulations prepared using conventional carriers can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically. Dose extract of the present invention or the compound represented by the formula (1) varies depending on the age, condition, etc. of the patient, but in general, the amount of 10 to 500 mg, preferably 50 to 300 mg per day in adults To be administered, it can be divided into several times a day, preferably once to six times divided by a predetermined time interval, depending on the judgment of the doctor or pharmacist.
또한, 본 발명의 피부미백제를 화장품으로 사용하는 경우, 박새 추출물 또는 상기 화학식 1로 표시되는 화합물을 기초제품 화장료(화장수, 크림, 에센스, 클렌징 폼, 클렌징 워터, 팩), 바디제품 화장료(바디 로션, 바디 오일, 바디 젤), 색조제품 화장료(화운데이션, 립스틱, 마스카라, 메이크업 베이스), 두발제품 화장료(샴푸, 린스, 헤어 콘디셔너, 헤어 젤) 등에 화장료의 건조중량에 대하여 0.05 ∼ 10.0 중량% 함량으로 배합하여 사용할 수 있다.In addition, in the case of using the skin whitening agent of the present invention as a cosmetic, using the extract of the compound or the compound represented by the formula (1) basic product cosmetics (cosmetics, creams, essences, cleansing foam, cleansing water, packs), body products cosmetics (body lotion , Body oil, body gel), cosmetic products (foundation, lipstick, mascara, makeup base), hair products cosmetics (shampoo, rinse, hair conditioner, hair gel), etc. It can mix and use.
또한, 본 발명의 박새 추출물 또는 상기 화학식 1로 표시되는 화합물을 각종 식료품의 건조중량에 대하여 0.05 ∼ 10.0 중량% 함량으로 배합하여 미백효과를 가지는 식품을 제조할 수도 있다.In addition, it is also possible to prepare a food having a whitening effect by blending the extract according to the present invention or the compound represented by the formula (1) in a content of 0.05 to 10.0% by weight based on the dry weight of various food products.
이와 같은 본 발명을 실시예에 의거하여 상세히 설명하면 다음과 같은 바, 본 발명이 실시예에 한정되는 것은 아니다.If the present invention will be described in detail based on the embodiment as follows, the present invention is not limited to the embodiment.
실시예 1 : 박새 추출물의 제조Example 1 Preparation of Aqueous Extract
전국 각처의 산야지에서 채취한 박새 3 kg를 쇄절하고 100% 메탄올로 24 시간 동안 환류추출하여 감압여과한 후 그 여액을 40 ℃의 수조에서 감압농축하여 농축된 메탄올 농축액을 2 L의 증류수에 현탁하였다. 증류수에 현탁시킨 메탄올 추출물은 분액여두에서 2 L의 부탄올으로 진탕추출하여 부탄올 가용부를 추출하여 얻은 다음 다시 추출액을 50 ℃의 수조에서 감압농축하였다. 농축후 얻어진 엑기스는 엑기스 총량의 10배의 증류수로 3회 공비 농축하고 동량의 증류수를 가하여 균질하게 현탁시킨 뒤, 동결 건조시켜 분말 상태의 박새 추출물을 얻었다.3 kg of vultures collected from wild fields from all over the country were broken down, and refluxed with 100% methanol for 24 hours, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure in a 40 ° C water bath, and the concentrated methanol concentrate was suspended in 2 L of distilled water. It was. Methanol extract suspended in distilled water was extracted with 2 L butanol in a separatory extract to extract the butanol soluble part, and the extract was concentrated under reduced pressure in a water bath at 50 ° C. The extract obtained after concentration was azeotropically concentrated three times with distilled water 10 times the total amount of the extract, and the same amount of distilled water was added to suspend homogeneously.
실시예 2 : 박새 추출물로부터 활성화합물의 단리Example 2 Isolation of the Active Compound from the Aqueous Extract
상기 실시예 1에서 얻은 박새 추출물 20 g을 메탄올 5 ㎖에 용해시킨 후 세파덱스 LH 젤 칼럼 크로마토그래피를 수행하여 활성분획을 모았다. 상기 활성분획을 80% 메탄올을 용출용매로 이용하는 YMC 젤 크로마토그래피를 수행하여 활성분획을 분리하고 감압건조기로 용매를 제거한 후 얻은 잔사(residue)를 냉동건조하여 활성물질(화학식 1로 표시되는 화합물)을 얻었다.After dissolving 20 g of the extract of the locusts obtained in Example 1 in 5 ml of methanol, an active fraction was collected by Sephadex LH gel column chromatography. The active fraction was subjected to YMC gel chromatography using 80% methanol as the eluting solvent. The active fraction was separated and the solvent was removed using a reduced pressure dryer. Got.
활성물질에 대해서는 ESI-MS(electron spray ionization mass spectrometer), 핵자기 공명 스펙트럼 등의 방법을 이용하여 이화학적 특성을 분석하였고, 그 결과 다음과 같은 이화학적 특성을 갖는 것으로 확인되었다.For the active material, physicochemical characteristics were analyzed by using an electron spray ionization mass spectrometer (ESI-MS) and nuclear magnetic resonance spectra. As a result, it was confirmed that the physicochemical characteristics were as follows.
[화학식 1][Formula 1]
① 물질 성상 : 분말① Material Property: Powder
② 분자량: 552② Molecular Weight: 552
③ 분자식: C26H32O13 ③ Molecular Formula: C 26 H 32 O 13
④ 질량분석치(M+Na): 575(m/z)④ Mass Spec. (M + Na): 575 ( m / z )
또한, 듀트로 메탄올(Methanol-d4) 용매로 녹여 5 ㎜ NMR 튜브에서 측정하였으며, 각 용매의 피크(peak)를 내부 표준물질로 하거나 테트라메틸실란(TMS)의 피크를 기준으로 하여 화학이동을 측정한 수소 핵자기공명 스펙트럼 및 탄소 핵자기공명 스펙트럼 분석결과, 현재까지 보고되지 않은 타이로시네이즈 저해 효과를 나타내는 신규한 상기 화학식 1로 표시되는 화합물로 판명되었다.In addition, the resultant was dissolved in methanol (Methanol- d4 ) solvent and measured in a 5 mm NMR tube. The chemical shift was measured based on the peak of each solvent or based on the peak of tetramethylsilane (TMS). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectrum analysis revealed a novel compound represented by the formula (1) exhibiting a tyrosinase inhibitory effect that has not been reported to date.
13C NMR(메탄올-d 6 ) δ 60.7, 69.7, 69.8, 73.2, 76.6, 76.7, 77.1, 77.1, 100.3, 100.7, 103.0, 104.9, 107.4, 116.4, 126.7, 127.6, 128.0, 130.7, 139.1, 157.1, 158.4, 158.9 13 C NMR (Methanol- d 6 ) δ 60.7, 69.7, 69.8, 73.2, 76.6, 76.7, 77.1, 77.1, 100.3, 100.7, 103.0, 104.9, 107.4, 116.4, 126.7, 127.6, 128.0, 130.7, 139.1, 157.1, 158.4, 158.9
1H NMR (메탄올-d 6 ) δ 3.4, 3.5, 3.7, 3.9, 6.4, 6.6, 6.8, 6.9, 7.0, 7.1, 7.5 1 H NMR (Methanol- d 6 ) δ 3.4, 3.5, 3.7, 3.9, 6.4, 6.6, 6.8, 6.9, 7.0, 7.1, 7.5
실시예 3 : 물질의 역가Example 3: Titer of Material
상기 실시예 1에서 얻은 박새 추출물 및 실시예 2에 따른 박새로부터 추출 분리된 활성 물질에 대한 타이로시네이즈 저해활성을 다음과 같은 방법으로 조사하였다. 검체 15 ㎕를 마이크로플레이트(96 well microplate)에 넣고, 0.1 M 인산완충액(pH 6.5) 150 ㎕와 1.5 mM L-타이로신 용액 25 ㎕를 넣은 후, 효소용액(타이로시네이즈, 시그마제) 7 ㎕를 첨가하여 마이크로플레이트 리이더(Microplate reader, 바이오라드제)를 이용하여 490 nm에서 흡광도를 측정하였다. 이 플레이트를 37 ℃에서 10 분간 반응시킨 후 다시 490 nm에서 흡광도를 측정한 후 다음 수학식 1에 의하여 타이로시네이즈 저해율(%)을 계산하였다.Tyrosinase inhibitory activity against the extract of the hibiscus obtained in Example 1 and the extract extracted from the larva according to Example 2 was investigated in the following manner. 15 μl of the sample was put into a microplate (96 well microplate), 150 μl of 0.1 M phosphate buffer (pH 6.5) and 25 μl of 1.5 mM L-tyrosine solution were added, followed by 7 μl of enzyme solution (tyrosinase, sigma). The absorbance was measured at 490 nm using a microplate reader (manufactured by Biorad). After reacting the plate at 37 ° C. for 10 minutes, absorbance was measured at 490 nm again, and then the tyrosinase inhibition rate (%) was calculated by the following equation (1).
상기 수학식 1에서, Sb는 저해제를 넣은 시료의 반응전 흡광도를 나타내고, Sa는 저해제를 넣은 시료의 반응후 흡광도를 나타내고, Bb는 저해제를 넣지 않은 시료의 반응전 흡광도를 나타내고, Ba는 저해제를 넣지 않은 시료의 반응후 흡광도를 나타낸다. 타이로시네이즈 저해활성 조사 결과는 다음 표 1에 나타내었다.In Equation 1, S b represents the absorbance before the reaction of the sample containing the inhibitor, S a represents the absorbance after the reaction of the sample containing the inhibitor, B b represents the absorbance before the reaction of the sample without the inhibitor, B a shows the absorbance after reaction of the sample without inhibitor. The tyrosinase inhibitory activity investigation results are shown in Table 1 below.
상기 표 1에 따르면, 박새 추출물 그리고 상기 화학식 1로 표시되는 화합물은 모두 타이로시네이즈 저해활성을 나타내는 것을 확인할 수 있었다.According to the Table 1, both the extract and the compound represented by the formula (1) was confirmed to exhibit tyrosinase inhibitory activity.
실시예 4 : 정제의 제조Example 4: Preparation of Tablets
본 발명의 박새 추출물을 이용하여 다음과 같은 조성으로 경구투여용 정제를 습식과립법 및 건식과립법을 이용하여 제조하였다.Tablets for oral administration were prepared using the wet granules method and the dry granules method with the composition as follows.
[조성][Furtherance]
박새 추출물 200 mg, 경질 무수규산 10 mg, 스테아린산 마그네슘 2 mg, 미세결정 셀룰로오즈 50 mg, 전분 글리콜산 나트륨 25 mg, 옥수수 전분 113 mg, 무수에탄올 적량.200 mg extract, hard silicic acid 10 mg, magnesium stearate 2 mg, microcrystalline cellulose 50 mg, starch glycolate 25 mg, corn starch 113 mg, ethanol anhydride.
실시예 5 : 연고제의 제조Example 5 Preparation of Ointment
본 발명의 박새 추출물을 이용하여 다음과 같은 조성으로 연고제를 제조하였다.Using oak extract of the present invention was prepared an ointment with the following composition.
[조성][Furtherance]
박새 추출물 5 g, 세틸팔미테이트 20 g, 세탄올 40 g, 스테아릴알콜 40 g, 미리스탄이소프로필 80 g, 모노스테아린산 소르비탄 20 g, 폴리솔베이트 60 g, 파라옥시안식향산 프로필 1 g, 파라옥시안식향산 메틸 1 g, 인산 및 정제수 적량Methanol extract 5 g, cetyl palmitate 20 g, cetanol 40 g, stearyl alcohol 40 g, myristan isopropyl 80 g, monostearic acid sorbitan 20 g, polysorbate 60 g, paraoxybenzoic acid propyl 1 g, para 1 g of methyl oxyanate, phosphoric acid and purified water
실시예 6 : 주사제의 제조Example 6: Preparation of Injection
본 발명의 박새 추출물을 이용하여 다음과 같은 조성으로 주사제를 제조하였다.Injectables were prepared by using the extract according to the present invention.
[조성][Furtherance]
박새 추출물 100 mg, 만니톨 180 mg, 인산일수소나트륨 25 mg, 주사용 물 2974 mgMedicinal Herb Extract 100 mg, mannitol 180 mg, sodium dihydrogen phosphate 25 mg, water for injection 2974 mg
실시예 7 : 화장료의 제조Example 7 Preparation of Cosmetics
본 발명의 박새 추출물을 이용하여 다음과 같은 조성으로 에센스를 제조하였다.Essence was prepared by the following composition using the extract according to the invention.
[조성][Furtherance]
박새 추출물 10.0 mg, 글리세린 3.0 mg, EDTA 0.05 mg, 벤조페논-9 0.04 mg, 카르복시비닐 폴리머 0.2 mg, 트리에탄올아민 0.18 mg, 옥시도테세스-25 0.6 mg, 글리세릴모노스테아레이트 1.0 mg, 방부제 0.01 mg, 향료 0.01 mg, 정제수 잔량Methanol Extract 10.0 mg, Glycerine 3.0 mg, EDTA 0.05 mg, Benzophenone-9 0.04 mg, Carboxyvinyl Polymer 0.2 mg, Triethanolamine 0.18 mg, Oxidothecese-25 0.6 mg, Glyceryl monostearate 1.0 mg, Preservative 0.01 mg, fragrance 0.01 mg, remaining amount of purified water
[독성 실험 결과]Toxicity Test Results
본 발명이 박새 식물로부터 얻은 추출물 및 활성물질은 천연물로서 독성에 대한 염려가 전혀 없다. 독성을 알아보기 위하여, 활성물질 1 ∼ 20 ㎎을 24 마리의 생쥐에게 복강내 투여하여 행동 관찰 후 24시간 생존 여부를 확인하였다. 그 결과 20 ㎎을 투여한 6 마리중 3 마리가 생존하고 나머지 3 마리는 희생당하였음을 알 수 있었다. 반면에, 20 ㎎ 미만의 용량을 투여한 생쥐의 경우는 모두 생존하였으며, 행동 관찰상 약물을 투여하지 않았던 생쥐와 비교하여 통계학상의 유의성 있는 차이를 보이지 않았다. 이상의 결과를 고려할 때, 생쥐에서 화학식 1의 화합물의 대략적인 반수가 생존할 독성 용량(TD50)은 20 mg(1 mg/g)으로 판단된다.The extracts and active substances of the present invention from the nectarine plants are natural and have no concern about toxicity. In order to determine the toxicity, 1 to 20 mg of the active substance was intraperitoneally administered to 24 mice to confirm survival after 24 hours of behavior observation. As a result, it was found that 3 out of 6 mice administered 20 mg survived and the remaining 3 were sacrificed. On the other hand, all mice that received the dose of less than 20 mg survived, and showed no statistically significant difference compared to the mice that did not receive the drug in behavioral observation. In view of the above results, a toxic dose (TD 50 ) at which approximately half of the compound of formula 1 will survive in the mouse is judged to be 20 mg (1 mg / g).
본 발명은 상기의 특정 실시예들에 의해 상세히 설명되었지만, 특허 청구범위에 정의된 본 발명의 진의 및 범위를 벗어나지 않은 많은 변형 및 치환이 가능하다는 것은 당분야의 숙련자들에게 자명하며, 그러한 변형 및 치환 역시 본 발명의 범위에 포함된다. While the invention has been described in detail by the specific embodiments thereof, it will be apparent to those skilled in the art that many modifications and substitutions are possible without departing from the spirit and scope of the invention as defined in the claims. Substitutions are also included within the scope of the present invention.
이상에서 설명한 바와 같이, 본 발명에 따른 박새 추출물 및 화학식 1로 표시되는 화합물은 타이로시네이즈 억제하는 활성 성분으로서 피부미백제의 새로운 약제의 개발에 유용하다.As described above, the extract according to the present invention and the compound represented by the formula (1) is useful for the development of a new drug of the skin whitening agent as an active ingredient to inhibit tyrosinase.
도 1은 화학식 1로 표시되는 화합물의 수소 핵자기공명(1H-NMR) 스펙트럼을 나타낸 것이고,Figure 1 shows the hydrogen nuclear magnetic resonance ( 1 H-NMR) spectrum of the compound represented by Formula 1,
도 2는 화학식 1로 표시되는 화합물의 탄소 핵자기공명(13C-NMR) 스펙트럼을 나타낸 것이다.Figure 2 shows the carbon nuclear magnetic resonance ( 13 C-NMR) spectrum of the compound represented by Formula 1.
Claims (6)
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KR970005241A (en) * | 1995-07-26 | 1997-02-19 | 장택희 | Electronic stethoscope |
JP2000281567A (en) * | 1999-01-29 | 2000-10-10 | Sunstar Inc | Medicinal and food composition containing stilbene-based compound |
JP2001072583A (en) * | 1999-09-07 | 2001-03-21 | Sunstar Inc | Prophylactic or therapeutic composition for hyperlipemia |
JP2001069948A (en) * | 1999-09-07 | 2001-03-21 | Sunstar Inc | Food for prevention of senescence |
US6361815B1 (en) * | 1998-12-21 | 2002-03-26 | Pure World Botanicals, Inc. | Products comprising trihydroxystilbenes and derivatives thereof and methods for their manufacture and use |
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KR970005241A (en) * | 1995-07-26 | 1997-02-19 | 장택희 | Electronic stethoscope |
US6361815B1 (en) * | 1998-12-21 | 2002-03-26 | Pure World Botanicals, Inc. | Products comprising trihydroxystilbenes and derivatives thereof and methods for their manufacture and use |
JP2000281567A (en) * | 1999-01-29 | 2000-10-10 | Sunstar Inc | Medicinal and food composition containing stilbene-based compound |
JP2001072583A (en) * | 1999-09-07 | 2001-03-21 | Sunstar Inc | Prophylactic or therapeutic composition for hyperlipemia |
JP2001069948A (en) * | 1999-09-07 | 2001-03-21 | Sunstar Inc | Food for prevention of senescence |
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