JP2021080212A - Cell proliferation promoter, mmp-2 inhibitor, topical skin preparation, pharmaceutical and internal preparation - Google Patents
Cell proliferation promoter, mmp-2 inhibitor, topical skin preparation, pharmaceutical and internal preparation Download PDFInfo
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- JP2021080212A JP2021080212A JP2019209598A JP2019209598A JP2021080212A JP 2021080212 A JP2021080212 A JP 2021080212A JP 2019209598 A JP2019209598 A JP 2019209598A JP 2019209598 A JP2019209598 A JP 2019209598A JP 2021080212 A JP2021080212 A JP 2021080212A
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- extract
- mmp
- agrimonia pilosa
- cell proliferation
- agrimonia
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Landscapes
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Abstract
Description
本発明は、細胞増殖促進剤、ＭＭＰ−２阻害剤、皮膚外用剤、医薬品及び内用剤に関する。 The present invention relates to cell proliferation promoters, MMP-2 inhibitors, external preparations for skin, pharmaceuticals and internal preparations.
加齢と共に表皮細胞の増殖・***能は低下し、表皮層自体は薄くなる（非特許文献１）。生体因子であるＥｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ（ＥＧＦ／上皮細胞成長因子）や女性ホルモン（エストロゲン）は皮膚の表皮細胞増殖に働きかけるが、加齢と共にその分泌は低下する。このような加齢による表皮細胞代謝機能の低下は、皮膚のターンオーバー速度を遅らせ、肌荒れや皮膚の老化の原因となる。また、角層表面から剥がれ落ちる角層細胞が滞留することで、表皮内メラニンの***がスムーズに行われなくなり、色素沈着や肌のくすみの原因となる。さらに表皮の創傷治癒が遅くなること等も知られている。これらの現象の進行を防止あるいは改善するために、表皮細胞の増殖を促進させる成分の探索や、多くの皮膚外用剤の提案がなされてきた。 With aging, the proliferation and division ability of epidermal cells decreases, and the epidermal layer itself becomes thin (Non-Patent Document 1). The biological factors Epidermal Growth Factor (EGF / epidermal growth factor) and female hormone (estrogen) act on the proliferation of epidermal cells in the skin, but their secretion decreases with aging. Such a decrease in epidermal cell metabolic function due to aging slows down the turnover rate of the skin and causes rough skin and aging of the skin. In addition, the retention of stratum corneum cells that peel off from the surface of the stratum corneum prevents smooth excretion of melanin in the epidermis, which causes pigmentation and dullness of the skin. It is also known that wound healing of the epidermis is delayed. In order to prevent or improve the progression of these phenomena, the search for components that promote the proliferation of epidermal cells and the proposal of many external preparations for skin have been made.
マトリックスメタロプロテアーゼ（以下ＭＭＰとする）に属するゼラチナーゼ（ＭＭＰ−２）は、線維芽細胞や内皮細胞、ガン細胞等が産生する酵素であり、コラーゲン、ゼラチン、エラスチン（動脈、腱、皮膚など弾性組織の特殊成分をなす構造タンパク質）等の基質や基底膜の主要構成成分であるラミニン５などを分解する。その発現及び活性は、紫外線の照射により大きく増加し、紫外線による基底膜成分の減少、基底膜の構造変化の原因となり、皮膚におけるシワやたるみの形成等の大きな要因となることが明らかとなっている（非特許文献２）。従って、ゼラチナーゼに対して阻害活性を有する物質は、皮膚におけるシワやたるみの予防だけでなく、ガン組織における血管新生やガンの転移を抑制する効果が期待され、ガン疾患の予防、治療に有用であると考えられる。さらに、ＭＭＰ−２は、潰瘍形成、慢性関節リュウマチ、骨粗鬆症、歯周炎等の種々の病態での細胞外基質の分解に関与していることが報告されている。よって、ＭＭＰ−２の阻害活性を有すれば、ガンの転移、潰瘍形成、慢性関節リュウマチ、骨粗鬆症、歯周炎等、ＭＭＰ−２の亢進が原因で起こる各種疾患の治療及び改善に有用である。 Zelatinase (MMP-2), which belongs to matrix metalloproteinase (hereinafter referred to as MMP), is an enzyme produced by fibroblasts, endothelial cells, cancer cells, etc., and is an elastic tissue such as collagen, gelatin, and elastin (arteries, tendons, skin, etc.). Decomposes substrates such as (structural proteins that form a special component of) and laminin 5, which is a major constituent of the basement membrane. It has been clarified that its expression and activity are greatly increased by irradiation with ultraviolet rays, cause a decrease in basement membrane components due to ultraviolet rays, cause structural changes in the basement membrane, and become a major factor such as formation of wrinkles and sagging in the skin. (Non-Patent Document 2). Therefore, a substance having an inhibitory activity on gelatinase is expected to have an effect of suppressing angiogenesis and cancer metastasis in cancer tissue as well as preventing wrinkles and sagging in the skin, and is useful for prevention and treatment of cancer diseases. It is believed that there is. Furthermore, MMP-2 has been reported to be involved in the degradation of extracellular matrix in various pathological conditions such as ulceration, rheumatoid arthritis, osteoporosis, and periodontitis. Therefore, if it has an inhibitory activity of MMP-2, it is useful for treating and ameliorating various diseases caused by an increase in MMP-2, such as cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis, and periodontitis. ..
バラ目バラ科キンミズヒキ属のキンミズヒキ（Ａｇｒｉｍｏｎｉａ ｐｉｌｏｓａ）は、その全草の抽出物が、チロシナーゼ活性阻害作用や活性酸素阻害作用を有し、美白及び老化防止化粧料に適用されること（特許文献１）や、メラニン生成抑制作用やＭＭＰ−１活性阻害作用、コラーゲン産生促進作用を有し、皮膚化粧料に適用されること（特許文献２）、ドーパオキシダーゼ活性抑制作用を有し美白剤に適用されること（特許文献３）が知られている。しかしながら、これらのキンミズヒキが、細胞増殖促進作用及びＭＭＰ−２阻害作用を有することは知られていなかった。 Agrimonia pilosa, which belongs to the genus Agrimonia of the Rosales family, has a tyrosinase activity-inhibiting effect and an active oxygen-inhibiting effect, and is applied to whitening and antiaging cosmetics (Patent Document 1). ), It has melanin production inhibitory action, MMP-1 activity inhibitory action, collagen production promoting action, and is applied to skin cosmetics (Patent Document 2), and it has dopaoxidase activity inhibitory action and is applied to whitening agents. (Patent Document 3) is known. However, it has not been known that these Agrimonia pilosa have cell proliferation promoting action and MMP-2 inhibitory action.
安全で安定性に優れ、細胞増殖促進作用及びＭＭＰ−２阻害作用に優れた素材が望まれているが、未だ十分満足し得るものが提供されていないのが現状である。 Materials that are safe, have excellent stability, and have excellent cell proliferation promoting action and MMP-2 inhibitory action are desired, but at present, materials that are sufficiently satisfactory have not been provided.
このような事情により、本発明者らは鋭意検討した結果、キンミズヒキの抽出物が優れた細胞増殖促進作用及びＭＭＰ−２阻害作用を持ち、安定性においても優れていることを見出した。さらに、その抽出物を含有する外用剤又は内用剤が、安全で安定であり、細胞増殖促進作用及びＭＭＰ−２阻害作用に優れており、多機能性美容・健康用素材・医薬品と成り得ることを見いだし、本発明を完成するに至った。 Under these circumstances, the present inventors have diligently studied and found that the extract of Agrimonia pilosa has excellent cell proliferation promoting action and MMP-2 inhibitory action, and is also excellent in stability. Furthermore, the external or internal preparation containing the extract is safe and stable, has excellent cell proliferation promoting action and MMP-2 inhibitory action, and can be a multifunctional beauty / health material / pharmaceutical product. We found that and came to complete the present invention.
本発明に用いるキンミズヒキは、バラ目バラ科キンミズヒキ属の植物で、日本から東ヨーロッパにかけて広く分布している多年草である。日本では、本州、四国、九州などの林や原野、路傍などに生息している。草丈は１ｍ程にまで伸び、表面には全体的に長毛が密生している。夏から秋にかけ、長く伸びた茎の上部に、黄色の小さな花を穂状につける。この黄色い小花を細長く穂のように咲かせる姿を「金色の水引」に見立てて、「キンミズヒキ」の名がついたと言われている。生薬名は、「仙鶴草」もしくは「龍牙草」という。 Agrimonia pilosa used in the present invention is a plant belonging to the genus Agrimonia of the Rosales family, and is a perennial plant widely distributed from Japan to Eastern Europe. In Japan, it inhabits forests, wilderness, and roadsides in Honshu, Shikoku, and Kyushu. The plant height grows to about 1 m, and long hairs are densely grown on the surface. From summer to autumn, small yellow flowers are sprouted on the top of the long stems. It is said that the name "Kinmizuhiki" was given to the appearance of these yellow florets blooming like elongated spikes, as if they were "golden mizuhiki". The crude drug name is "Senzuru grass" or "Ryuga grass".
本発明におけるキンミズヒキの抽出物とは、バラ目バラ科キンミズヒキ属のキンミズヒキ（学名：Ａｇｒｉｍｏｎｉａ ｐｉｌｏｓａ）が用いられ、部位としては、花、実、種子、葉、茎、根等の植物体の一部又は全草から抽出したものである。その抽出方法は特に限定されず、例えば、加熱抽出したものであってもよいし、常温抽出したものであってもよい。また、抽出には、植物体をそのまま使用してもよく、乾燥、粉砕、細切等の処理を行ってもよい。 As the extract of Agrimonia in the present invention, Agrimonia pilosa (scientific name: Agrimonia pilosa) of the genus Agrimonia of the Rosales family is used, and the site is a part of a plant such as flowers, fruits, seeds, leaves, stems and roots. Or it is extracted from whole plant. The extraction method is not particularly limited, and for example, it may be heat-extracted or room-temperature extracted. In addition, the plant body may be used as it is for extraction, or may be subjected to treatments such as drying, crushing, and shredding.
抽出方法は、特に限定されないが、水もしくは熱水、又は水と有機溶媒の混合溶媒を用い、撹拌又はカラム抽出する方法等により行うことができる。抽出溶媒としては、例えば、水、低級アルコール類（メタノール、エタノール、１−プロパノール、２−プロパノール、１−ブタノール、２−ブタノール等）、液状多価アルコール類（１，３−ブチレングリコール、プロピレングリコール、グリセリン等）、ケトン類（アセトン、メチルエチルケトン等）、アセトニトリル、エステル類（酢酸エチル、酢酸ブチル等）、炭化水素類（ヘキサン、ヘプタン、流動パラフィン等）、エーテル類（ジエチルエーテル、テトラヒドロフラン、プロピルエーテル等）が挙げられる。好ましくは、水、低級アルコール及び液状多価アルコール等の極性溶媒が良く、特に好ましくは、水、エタノール、１，３−ブチレングリコール及びプロピレングリコールがよい。これらの溶媒は一種でも二種以上を混合して用いてもよい。特に好ましい抽出溶媒としては、水、又は水−エタノール系の混合極性溶媒が挙げられる。溶媒の使用量については、特に限定はなく、例えばキンミズヒキの全草（乾燥重量）に対し、１０倍以上、好ましくは２０倍以上であればよいが、抽出後に濃縮を行ったり、単離したりする場合の操作の便宜上１００倍以下であることが好ましい。また、抽出温度や時間は、用いる溶媒の種類や抽出時の圧力等によって適宜選択できる。 The extraction method is not particularly limited, but can be carried out by a method of stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.) and liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, etc.). , Glycerin, etc.), Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (diethyl ether, tetrahydrofuran, propyl ether, etc.) Etc.). Polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferred, and water, ethanol, 1,3-butylene glycol and propylene glycol are particularly preferred. These solvents may be used alone or in admixture of two or more. Particularly preferable extraction solvents include water and water-ethanol mixed polar solvents. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, based on the whole plant (dry weight) of Agrimonia pilosa, but may be concentrated or isolated after extraction. It is preferably 100 times or less for convenience of operation in the case. The extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure at the time of extraction, and the like.
上記抽出物は、抽出した溶液のまま用いてもよいが、必要に応じて、本発明の効果を奏する範囲で、濃縮（減圧濃縮、膜濃縮等による濃縮）、希釈、濾過、活性炭等による脱色、脱臭、エタノール沈殿等の処理を行ってから用いてもよい。さらには、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理を行い、乾燥物として用いてもよい。 The above extract may be used as it is in the extracted solution, but if necessary, concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc. is performed within the range in which the effect of the present invention is exhibited. , Deodorization, ethanol precipitation, etc. may be performed before use. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze drying and the like, and used as a dried product.
本発明は、上記抽出物をそのまま使用しても良く、抽出物の効果を損なわない範囲内で、化粧品、医薬部外品、医薬品及び食品等に用いられる成分である油脂類、ロウ類、炭化水素類、脂肪酸類、アルコール類、エステル類、界面活性剤、金属石鹸、ｐＨ調整剤、防腐剤、香料、保湿剤、粉体、紫外線吸収剤、増粘剤、色素、酸化防止剤、美白剤、キレート剤、賦形剤、皮膜剤、甘味料、酸味料等の成分が含有されていてもよい。 In the present invention, the above extract may be used as it is, and oils and fats, waxes, and carbonized components used in cosmetics, non-pharmaceutical products, pharmaceuticals, foods, etc., as long as the effect of the extract is not impaired. Hydrogens, fatty acids, alcohols, esters, surfactants, metal soaps, pH regulators, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents , Chelating agents, excipients, filming agents, sweeteners, acidity agents and the like may be contained.
本発明は、化粧品、医薬部外品、医薬品、食品のいずれにも用いることができ、その剤形としては、例えば、化粧水、クリーム、乳液、ゲル剤、エアゾール剤、エッセンス、パック、洗浄剤、浴用剤、ファンデーション、打粉、口紅、軟膏、パップ剤、錠菓、カプセル剤、チョコレート、ガム、飴、飲料、散剤、顆粒剤、錠剤、糖衣錠剤、カプセル剤、シロップ剤、丸剤、懸濁剤、液剤、乳剤、坐剤、注射用溶液等が挙げられる。 The present invention can be used for any of cosmetics, non-pharmaceutical products, pharmaceuticals, and foods, and the dosage forms thereof include, for example, cosmetics, creams, emulsions, gels, aerosols, essences, packs, and cleaning agents. , Baths, foundations, dusting, lipsticks, ointments, poultices, tablets, capsules, chocolates, gums, candy, beverages, powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions Examples include agents, solutions, emulsions, suppositories, solutions for injection and the like.
外用の場合、本発明に用いる上記抽出物の含有量は、固形物に換算して０．０００１重量％以上が好ましく、０．００１〜１０重量％がより好ましい。さらに、０．０１〜５重量％が最も好ましい。０．０００１重量％未満では十分な効果は望みにくい。１０重量％を越えると、効果の増強は認められにくく不経済である。 In the case of external use, the content of the extract used in the present invention is preferably 0.0001% by weight or more, more preferably 0.001 to 10% by weight in terms of solid matter. Further, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If it exceeds 10% by weight, the enhancement of the effect is hardly recognized and it is uneconomical.
内用の場合、摂取量は年齢、体重、症状、治療効果、投与方法、処理時間等により異なる。通常、成人１人当たりの１日の摂取量としては、５ｍｇ以上が好ましく、１０ｍｇ〜５ｇがより好ましい。さらに、２０ｍｇ〜２ｇが最も好ましい。 For internal use, the intake varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, and the like. Usually, the daily intake per adult is preferably 5 mg or more, and more preferably 10 mg to 5 g. Further, 20 mg to 2 g is most preferable.
次に本発明を詳細に説明するため、実施例として本発明に用いる抽出物の製造例、実験例及び処方例を挙げるが、本発明はこれに限定されるものではない。製造例に示す％とは重量％を、処方例に示す含有量の部とは重量部を示す。 Next, in order to explain the present invention in detail, examples of production, experiment, and formulation of the extract used in the present invention will be given as examples, but the present invention is not limited thereto. The% shown in the production example means% by weight, and the content part shown in the formulation example means a weight part.
キンミズヒキの抽出物の製造例
キンミズヒキの抽出物を以下のとおり製造した。製造例１〜４において抽出材料にはキンミズヒキの全草を用いた。
Example of production of Agrimonia pilosa extract An extract of Agrimonia pilosa was produced as follows. In Production Examples 1 to 4, the whole plant of Agrimonia pilosa was used as the extraction material.
（製造例１）キンミズヒキの熱水抽出物の調製
キンミズヒキの乾燥物１０ｇに２００ｍＬの水を加え、９５〜１００℃で２時間抽出した。得られた抽出液を濾過し、その濾液を濃縮し、凍結乾燥してキンミズヒキの熱水抽出物を０．９ｇ得た。
(Production Example 1) Preparation of hot water extract of Agrimonia pilosa 200 mL of water was added to 10 g of a dried product of Agrimonia pilosa, and the mixture was extracted at 95 to 100 ° C. for 2 hours. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 0.9 g of a hot water extract of Agrimonia pilosa.
（製造例２）キンミズヒキの５０％エタノール抽出物の調製
キンミズヒキの乾燥物１０ｇを２００ｍＬの５０％エタノール水溶液に室温で７日間浸漬し抽出を行った。得られた抽出液を濾過した後、エバポレーターで濃縮乾固してキンミズヒキの５０％エタノール抽出物を１．０ｇ得た。
(Production Example 2) Preparation of 50% ethanol extract of Agrimonia pilosa 10 g of a dried product of Agrimonia pilosa was immersed in 200 mL of a 50% ethanol aqueous solution at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 1.0 g of a 50% ethanol extract of Agrimonia pilosa.
（製造例３）キンミズヒキのエタノール抽出物の調製
キンミズヒキの乾燥物１０ｇを２００ｍＬのエタノールに室温で７日間浸漬し抽出を行った。得られた抽出液を濾過した後、エバポレーターで濃縮乾固してキンミズヒキのエタノール抽出物を０．２ｇ得た。
(Production Example 3) Preparation of ethanol extract of Agrimonia pilosa 10 g of a dried product of Agrimonia pilosa was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 0.2 g of an ethanol extract of Agrimonia pilosa.
（製造例４）キンミズヒキの１，３−ブチレングリコール抽出物の調製
キンミズヒキの乾燥物１０ｇを２００ｍＬの１，３−ブチレングリコールに室温で７日間浸漬し抽出を行った。得られた抽出液を濾過してキンミズヒキの１，３−ブチレングリコール抽出物を１９０ｇ得た。
(Production Example 4) Preparation of 1,3-butylene glycol extract of Agrimonia pilosa 10 g of a dried product of Agrimonia pilosa was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction. The obtained extract was filtered to obtain 190 g of 1,3-butylene glycol extract of Agrimonia pilosa.
（処方例１） 化粧水
処方 含有量（部）
１．キンミズヒキの熱水抽出物（製造例１） ２．０
２．１，３−ブチレングリコール ８．０
３．グリセリン ２．０
４．キサンタンガム ０．０２
５．クエン酸 ０．０１
６．クエン酸ナトリウム ０．１
７．エタノール ５．０
８．パラオキシ安息香酸メチル ０．１
９．ポリオキシエチレン硬化ヒマシ油（４０Ｅ．Ｏ．） ０．１
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１〜６及び１１と、成分７〜１０をそれぞれ均一に溶解し、両者を混合し濾過して製品とする。
(Prescription example 1) Toner prescription content (part)
1. 1. Hot water extract of Agrimonia pilosa (Production Example 1) 2.0
2.1,3-butylene glycol 8.0
3. 3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40EO) 0.1
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 6 and 11 and components 7 to 10 are uniformly dissolved in purified water to make the total amount 100, and both are mixed and filtered to obtain a product.
（比較処方例１） 従来の化粧水
処方例１において、キンミズヒキの熱水抽出物を精製水に置き換えたものを、従来の化粧水とした。
(Comparative Formulation Example 1) Conventional Toner In Formulation Example 1, the hot water extract of Agrimonia pilosa was replaced with purified water, and the conventional lotion was used.
（処方例２） クリーム
処方 含有量（部）
１．キンミズヒキの５０％エタノール抽出物（製造例２） １．０
２．スクワラン ５．５
３．オリーブ油 ３．０
４．ステアリン酸 ２．０
５．ミツロウ ２．０
６．ミリスチン酸オクチルドデシル ３．５
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ベヘニルアルコール １．５
９．モノステアリン酸グリセリン ２．５
１０．香料 ０．１
１１．パラオキシ安息香酸メチル ０．２
１２．１，３−ブチレングリコール ８．５
１３．精製水にて全量を１００とする
［製造方法］成分２〜９を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１１〜１３を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分１０を加え、さらに３０℃まで冷却して製品とする。
(Prescription example 2) Cream prescription content (part)
1. 1. 50% ethanol extract of Agrimonia pilosa (Production Example 2) 1.0
2. Squalene 5.5
3. 3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Behenyl alcohol 1.5
9. Glycerin monostearate 2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12.1,3-butylene glycol 8.5
13. [Manufacturing method] Ingredients 2 to 9 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1 and 11 to 13 are heated and dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 10 is added at 45 ° C., and the mixture is further cooled to 30 ° C. to obtain a product.
（比較処方例２） 従来のクリーム
処方例２において、キンミズヒキの５０％エタノール抽出物を精製水に置き換えたものを、従来のクリームとした。
(Comparative Formulation Example 2) Conventional Cream In Formulation Example 2, a cream obtained by replacing 50% ethanol extract of Agrimonia pilosa with purified water was used as a conventional cream.
（処方例３） 乳液
処方 含有量（部）
１．キンミズヒキのエタノール抽出物（製造例３） ０．０１
２．スクワラン ５．０
３．オリーブ油 ５．０
４．ホホバ油 ５．０
５．セタノール １．５
６．モノステアリン酸グリセリン ２．０
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ポリオキシエチレンソルビタンモノオレエート（２０Ｅ．Ｏ．） ２．０
９．香料 ０．１
１０．プロピレングリコール １．０
１１．グリセリン ２．０
１２．パラオキシ安息香酸メチル ０．２
１３．精製水にて全量を１００とする
［製造方法］成分１〜８を加熱溶解して混合し、７０℃に保ち油相とする。成分１０〜１３を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分９を加え、さらに３０℃まで冷却して製品とする。
(Prescription example 3) Emulsion prescription content (part)
1. 1. Ethanol extract of Agrimonia pilosa (Production Example 3) 0.01
2. Squalene 5.0
3. 3. Olive oil 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glycerin monostearate 2.0
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Polyoxyethylene sorbitan monooleate (20EO) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl paraoxybenzoate 0.2
13. [Manufacturing method] Ingredients 1 to 8 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 10 to 13 are heated and dissolved and mixed to prepare an aqueous phase at 75 ° C. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 9 is added at 45 ° C., and the mixture is further cooled to 30 ° C. to obtain a product.
（処方例４） ゲル剤
処方 含有量（部）
１．キンミズヒキの１，３−ブチレングリコール抽出物（製造例４） １．０
２．エタノール ５．０
３．パラオキシ安息香酸メチル ０．１
４．ポリオキシエチレン硬化ヒマシ油（６０Ｅ．Ｏ．） ０．１
５．香料 適量
６．１，３−ブチレングリコール ５．０
７．グリセリン ５．０
８．キサンタンガム ０．１
９．カルボキシビニルポリマー ０．２
１０．水酸化カリウム ０．２
１１．精製水にて全量を１００とする
［製造方法］成分２〜５と、成分１及び６〜１１をそれぞれ均一に溶解し、両者を混合して製品とする。
(Prescription example 4) Gel preparation Prescription content (part)
1. 1. Agrimonia pilosa 1,3-butylene glycol extract (Production Example 4) 1.0
2. Ethanol 5.0
3. 3. Methyl paraoxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60EO) 0.1
5. Appropriate amount of fragrance 6.1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Manufacturing method] Ingredients 2 to 5 and components 1 and 6 to 11 having a total amount of 100 in purified water are uniformly dissolved, and the two are mixed to obtain a product.
（処方例５） パック
処方 含有量（部）
１．キンミズヒキの熱水抽出物（製造例１） １．０
２．キンミズヒキの１，３−ブチレングリコール抽出物（製造例４） ５．０
３．ポリビニルアルコール １２．０
４．エタノール ５．０
５．１，３−ブチレングリコール ８．０
６．パラオキシ安息香酸メチル ０．２
７．ポリオキシエチレン硬化ヒマシ油（２０Ｅ．Ｏ．） ０．５
８．クエン酸 ０．１
９．クエン酸ナトリウム ０．３
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１〜１１を均一に溶解し製品とする。
(Prescription example 5) Pack prescription content (part)
1. 1. Hot water extract of Agrimonia pilosa (Production Example 1) 1.0
2. Agrimonia pilosa 1,3-butylene glycol extract (Production Example 4) 5.0
3. 3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5.1,3-butylene glycol 8.0
6. Methyl paraoxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20EO) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 11 having a total amount of 100 in purified water are uniformly dissolved to prepare a product.
（処方例６） ファンデーション
処方 含有量（部）
１．キンミズヒキの５０％エタノール抽出物（製造例２） １．０
２．ステアリン酸 ２．４
３．ポリオキシエチレンソルビタンモノステアレート（２０Ｅ．Ｏ．） １．０
４．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ２．０
５．セタノール １．０
６．液状ラノリン ２．０
７．流動パラフィン ３．０
８．ミリスチン酸イソプロピル ６．５
９．カルボキシメチルセルロースナトリウム ０．１
１０．ベントナイト ０．５
１１．プロピレングリコール ４．０
１２．トリエタノールアミン １．１
１３．パラオキシ安息香酸メチル ０．２
１４．二酸化チタン ８．０
１５．タルク ４．０
１６．ベンガラ １．０
１７．黄酸化鉄 ２．０
１８．香料 適量
１９．精製水にて全量を１００とする
［製造方法］成分２〜８を加熱溶解し、８０℃に保ち油相とする。成分１９に成分９をよく膨潤させ、続いて、成分１及び１０〜１３を加えて均一に混合する。これに粉砕機で粉砕混合した成分１４〜１７を加え、ホモミキサーで撹拌し７５℃に保ち水相とする。油相に水相をかき混ぜながら加え、乳化する。その後、冷却し、４５℃で成分１８を加え、かき混ぜながら３０℃まで冷却して製品とする。
(Prescription example 6) Foundation prescription content (part)
1. 1. 50% ethanol extract of Agrimonia pilosa (Production Example 2) 1.0
2. Stearic acid 2.4
3. 3. Polyoxyethylene sorbitan monostearate (20EO) 1.0
4. Polyoxyethylene cetyl ether (20EO) 2.0
5. Cetanol 1.0
6. Liquid lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Sodium Carboxymethyl Cellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Appropriate amount of fragrance 19. [Manufacturing method] Ingredients 2 to 8 having a total amount of 100 in purified water are dissolved by heating and kept at 80 ° C. to prepare an oil phase. Ingredient 9 is well swollen with ingredient 19, followed by addition of ingredients 1 and 10-13 and mixed uniformly. Ingredients 14 to 17 pulverized and mixed by a pulverizer are added thereto, and the mixture is stirred with a homomixer and kept at 75 ° C. to prepare an aqueous phase. Add the aqueous phase to the oil phase while stirring to emulsify. Then, the mixture is cooled, component 18 is added at 45 ° C., and the product is cooled to 30 ° C. with stirring to obtain a product.
（処方例７） 浴用剤
処方 含有量（部）
１．キンミズヒキのエタノール抽出物（製造例３） １．０
２．炭酸水素ナトリウム ５０．０
３．黄色２０２号（１） 適量
４．香料 適量
５．硫酸ナトリウムにて全量を１００とする
［製造方法］成分１〜５を均一に混合し製品とする。
(Prescription example 7) Prescription content for bath (part)
1. 1. Ethanol extract of Agrimonia pilosa (Production Example 3) 1.0
2. Sodium bicarbonate 50.0
3. 3. Yellow No. 202 (1) Appropriate amount 4. Appropriate amount of fragrance 5. [Manufacturing method] Ingredients 1 to 5 having a total amount of 100 with sodium sulfate are uniformly mixed to prepare a product.
（処方例８） 軟膏
処方 含有量（部）
１．キンミズヒキの熱水抽出物（製造例１） ５．０
２．キンミズヒキの１，３−ブチレングリコール抽出物（製造例４） １．０
３．ポリオキシエチレンセチルエーテル（３０Ｅ．Ｏ．） ２．０
４．モノステアリン酸グリセリン １０．０
５．流動パラフィン ５．０
６．セタノール ６．０
７．パラオキシ安息香酸メチル ０．１
８．プロピレングリコール １０．０
９．精製水にて全量を１００とする
［製造方法］成分３〜６を加熱溶解して混合し、７０℃に保ち油相とする。成分１、２及び７〜９を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら３０℃まで冷却して製品とする。
(Prescription example 8) Ointment prescription content (part)
1. 1. Hot water extract of Agrimonia pilosa (Production Example 1) 5.0
2. Agrimonia pilosa 1,3-butylene glycol extract (Production Example 4) 1.0
3. 3. Polyoxyethylene cetyl ether (30EO) 2.0
4. Glycerin monostearate 10.0
5. Liquid paraffin 5.0
6. Cetanol 6.0
7. Methyl paraoxybenzoate 0.1
8. Propylene glycol 10.0
9. [Manufacturing method] Ingredients 3 to 6 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1, 2 and 7 to 9 are heated, dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring.
（処方例９） 散剤
処方 含有量（部）
１．キンミズヒキの熱水抽出物（製造例１） １．０
２．乾燥コーンスターチ ３９．０
３．微結晶セルロース ６０．０
［製造方法］成分１〜３を混合し、散剤とする。
(Prescription example 9) Powder prescription content (part)
1. 1. Hot water extract of Agrimonia pilosa (Production Example 1) 1.0
2. Dried cornstarch 39.0
3. 3. Microcrystalline Cellulose 60.0
[Manufacturing method] Ingredients 1 to 3 are mixed to prepare a powder.
（処方例１０） 錠剤
処方 含有量（部）
１．キンミズヒキのエタノール抽出物（製造例３） ５．０
２．乾燥コーンスターチ ２５．０
３．カルボキシメチルセルロースカルシウム ２０．０
４．微結晶セルロース ４０．０
５．ポリビニルピロリドン ７．０
６．タルク ３．０
［製造方法］成分１〜４を混合し、次いで成分５の水溶液を結合剤として加えて顆粒成型する。成型した顆粒に成分６を加えて打錠する。１錠０．５２ｇとする。
(Prescription example 10) Tablet prescription content (part)
1. 1. Ethanol extract of Agrimonia pilosa (Production Example 3) 5.0
2. Dried cornstarch 25.0
3. 3. Carboxymethyl Cellulose Calcium 20.0
4. Microcrystalline Cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6. Talc 3.0
[Manufacturing method] Ingredients 1 to 4 are mixed, and then an aqueous solution of ingredient 5 is added as a binder to form granules. Ingredient 6 is added to the molded granules and the mixture is locked. One tablet weighs 0.52 g.
（処方例１１） 錠菓
処方 含有量（部）
１．キンミズヒキのエタノール抽出物（製造例３） ２．０
２．乾燥コーンスターチ ４９．８
３．エリスリトール ４０．０
４．クエン酸 ５．０
５．ショ糖脂肪酸エステル ３．０
６．香料 ０．１
７．精製水にて全量を１００とする
［製造方法］成分１〜４及び７を混合し、顆粒成型する。成型した顆粒に成分５及び６を加えて打錠する。１粒１．０ｇとする。
(Prescription example 11) Tablet confectionery prescription content (part)
1. 1. Ethanol extract of Agrimonia pilosa (Production Example 3) 2.0
2. Dried cornstarch 49.8
3. 3. Erythritol 40.0
4. Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6. Fragrance 0.1
7. Set the total amount to 100 with purified water
[Manufacturing method] Ingredients 1 to 4 and 7 are mixed and granulated. Ingredients 5 and 6 are added to the molded granules and the mixture is locked. One grain is 1.0 g.
（処方例１２） 飲料
処方 含有量（部）
１．キンミズヒキの熱水抽出物（製造例１） ０．０５
２．ステビア ０．０５
３．リンゴ酸 ５．０
４．香料 ０．１
５．精製水にて全量を１００とする
［製造方法］成分２及び３を少量の水に溶解する。次いで、成分１、４及び５を加えて混合する。
(Prescription example 12) Beverage prescription content (part)
1. 1. Hot water extract of Agrimonia pilosa (Production Example 1) 0.05
2. Stevia 0.05
3. 3. Malic acid 5.0
4. Fragrance 0.1
5. [Manufacturing method] Components 2 and 3 having a total amount of 100 in purified water are dissolved in a small amount of water. Then, ingredients 1, 4 and 5 are added and mixed.
次に、本発明の効果を詳細に説明するため、実験例を挙げる。 Next, in order to explain the effect of the present invention in detail, an experimental example will be given.
実験例１ 細胞増殖促進試験
ＨａＣａＴ細胞を、０．１％ＦＢＳを含むＤＭＥＭ培養液にて、９６ｗｅｌｌプレートに１ｗｅｌｌあたり５×１０３個播種し、各試料を添加した後、３７℃、５％ＣＯ２条件下にて４日間培養した。細胞数の測定は、染色法により行った。すなわち、培養終了後、培養液を除き、メタノールを用いて細胞を固定した。続いて、０．１％メチレンブルーを加え、１時間細胞の染色を行った。乾燥させた後、０．１Ｎ ＨＣｌを各ｗｅｌｌに１００μＬずつ加えてよく撹拌させ、マイクロプレートリーダーを用いて６５０ｎｍにおける吸光度を測定した。
Experimental Example 1 Cell proliferation promotion test HaCaT cells were seeded in DMEM culture medium containing 0.1% FBS in 5 × 10 3 cells per 1 well on a 96-well plate, and after adding each sample, the temperature was 37 ° C. and 5% CO. The cells were cultured under 2 conditions for 4 days. The number of cells was measured by the staining method. That is, after the completion of the culture, the culture solution was removed and the cells were fixed with methanol. Subsequently, 0.1% methylene blue was added, and the cells were stained for 1 hour. After drying, 100 μL of 0.1N HCl was added to each well and stirred well, and the absorbance at 650 nm was measured using a microplate reader.
これらの実験結果を表１に示した。その結果、本発明のキンミズヒキの熱水抽出物（製造例１）、５０％エタノール抽出物（製造例２）及びエタノール抽出物（製造例３）は、優れた細胞増殖促進作用を示した。
実験例２ ＭＭＰ−２ ｍＲＮＡ発現量の測定
ヒト線維芽細胞ＮＢ１ＲＧＢをφ６０ｍｍ ｄｉｓｈに１×１０５個播種し、コンフルエントになった時点で、終濃度が１μｇ／ｍＬになるように試料を添加した。コントロールには、試料を希釈した溶媒を添加した。２４時間培養後、総ＲＮＡの抽出を行った。細胞からの総ＲＮＡの抽出はＲＮＡｉｓｏ Ｐｌｕｓ（タカラバイオ）を用いて行い、総ＲＮＡ量は分光光度計（ＮａｎｏＤｒｏｐ）を用いて２６０ｎｍにおける吸光度により求めた。ｍＲＮＡ発現量の測定は、細胞から抽出した総ＲＮＡを基にしてリアルタイムＲＴ−ＰＣＲ法により行った。リアルタイムＲＴ−ＰＣＲ法には、Ｈｉｇｈ Ｃａｐａｃｉｔｙ ＲＮＡ−ｔｏ−ｃＤＮＡ Ｋｉｔ（アプライドバイオシステムズ）及びＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用いた。すなわち、５００ｎｇの総ＲＮＡを逆転写反応後、ＰＣＲ反応（９５℃：１５秒間、６０℃：６０秒間、４０ｃｙｃｌｅｓ）を行った。その他の操作は定められた方法に従い、ＭＭＰ−２ ｍＲＮＡの発現量を、内部標準であるβ−ａｃｔｉｎ ｍＲＮＡの発現量に対する割合として求めた。ＭＭＰ−２発現率は、コントロールのＭＭＰ−２ ｍＲＮＡの発現量に対する試料添加群のＭＭＰ−２ ｍＲＮＡの発現量の比率として算出した。尚、各遺伝子の発現量の測定に使用したプライマーは次の通りである。
Experimental Example 2 Measurement of MMP-2 mRNA expression level Human fibroblasts NB1RGB were seeded in 1 × 10 5 pieces in a φ60 mm dish, and when they became confluent, samples were added so that the final concentration was 1 μg / mL. A solvent obtained by diluting the sample was added to the control. After culturing for 24 hours, total RNA was extracted. Extraction of total RNA from cells was performed using RNAiso Plus (Takara Bio), and the total amount of RNA was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). The mRNA expression level was measured by the real-time RT-PCR method based on the total RNA extracted from the cells. For the real-time RT-PCR method, High Capacity RNA-to- cDNA Kit (Applied Biosystems) and SYBR Select Master Mix (Life Technologies) were used. That is, after a reverse transcription reaction of 500 ng of total RNA, a PCR reaction (95 ° C: 15 seconds, 60 ° C: 60 seconds, 40 cycles) was performed. For other operations, the expression level of MMP-2 mRNA was determined as a ratio to the expression level of β-actin mRNA, which is an internal standard, according to a predetermined method. The MMP-2 expression rate was calculated as the ratio of the expression level of MMP-2 mRNA in the sample-added group to the expression level of the control MMP-2 mRNA. The primers used to measure the expression level of each gene are as follows.
ＭＭＰ−２用のプライマーセット
ＣＣＧＴＣＧＣＣＣＡＴＣＡＴＣＡＡ（配列番号１）
ＣＴＴＣＴＧＣＡＴＣＴＴＣＴＴＴＡＧＴＧＴＧＴＣＣＴＴ（配列番号２）
β−Ａｃｔｉｎ用のプライマーセット
ＣＡＣＴＣＴＴＣＣＡＧＣＣＴＴＣＣＴＴＣＣ（配列番号３）
ＧＴＧＴＴＧＧＣＧＴＡＣＡＧＧＴＣＴＴＴＧ（配列番号４）
Primer set for MMP-2 CCGTCGCCCATCATCAA (SEQ ID NO: 1)
CTTTGCATCTTCTTTAGTGTGTCCTT (SEQ ID NO: 2)
Primer set for β-Actin CACTCTTCCAGCCTTCCTCC (SEQ ID NO: 3)
GTGTTGGGCGTACAGGTCTTG (SEQ ID NO: 4)
これらの実験結果を表２に示した。その結果、本発明のキンミズヒキの抽出物には、顕著なＭＭＰ−２発現抑制効果（ＭＭＰ−２阻害効果）が認められた。さらに、キンミズヒキの５０％エタノール抽出物（製造例２）のＭＭＰ−２発現抑制効果が特に高かった。
実験例５ 使用試験
処方例１の化粧水及び比較処方例１の従来の化粧水を用いて、シワ、たるみがある５人（２７〜６５才）を対象に１ヶ月間の使用試験を行った。使用後、シワ、たるみの程度をアンケートにより判定した。
Experimental Example 5 Use test Using the lotion of Formulation Example 1 and the conventional lotion of Comparative Formulation 1, a one-month use test was conducted on 5 people (27-65 years old) with wrinkles and sagging. .. After use, the degree of wrinkles and sagging was judged by a questionnaire.
その結果、本発明の抽出物を含有する皮膚外用剤により、シワ、たるみが軽減した。尚、試験期間中、皮膚トラブルは一人もなく、安全性においても問題なかった。また、処方成分の劣化についても問題なかった。 As a result, wrinkles and sagging were alleviated by the external preparation for skin containing the extract of the present invention. During the test period, there were no skin problems and there was no problem in terms of safety. In addition, there was no problem with the deterioration of the prescription ingredients.
以上のことから、本発明のキンミズヒキの抽出物は、優れた細胞増殖促進効果及びＭＭＰ−２阻害作用を有し、安定性にも優れていた。よって、本発明のキンミズヒキの抽出物は、皮膚の老化といった美容分野だけでなく、老化による機能低下の抑制、ガンの予防、治療等といった医療分野にも利用でき、食品、化粧品、医薬部外品及び医薬品等への応用が期待される。 From the above, the extract of Agrimonia pilosa of the present invention had an excellent cell proliferation promoting effect and an MMP-2 inhibitory effect, and was also excellent in stability. Therefore, the extract of Kinmizuhiki of the present invention can be used not only in the beauty field such as skin aging but also in the medical field such as suppression of functional deterioration due to aging, cancer prevention, treatment, etc., and can be used for foods, cosmetics, quasi-drugs, etc. And is expected to be applied to pharmaceutical products.
Claims (6)
An internal product for the prevention and improvement of various diseases caused by an increase in MMP-2, which is characterized by containing an extract of Agrimonia pilosa.
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