TWI564031B - Nanoparticulate composition having butylidenephthalide, preparation process and pharmaceutical use thereof - Google Patents
Nanoparticulate composition having butylidenephthalide, preparation process and pharmaceutical use thereof Download PDFInfo
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本發明尤指一種透過均勻混合表面穩定劑，使其包覆於正-丁烯基苯酞之表面的含有正-丁烯基苯酞的奈米顆粒組合物、製備方法及其用途。 In particular, the present invention relates to a nanoparticle composition containing n-butenylquinone which is coated on a surface of n-butenylbenzoquinone by uniformly mixing a surface stabilizer, a preparation method, and use thereof.
奈米科技為近年研究重心之一，因許多材料(或稱物質)在奈米尺寸下，會呈現與一般狀態下完全不同之物理特性、化學特性及生物特性，奈米科技可應用於各領域，以生物醫藥產業中的藥物傳輸系統(Drug delivery system)為例，透過將分子奈米化的設計，能有效改善它的輸送模式，提高其生物利用度(Bioavailability)，與製劑的均勻性、分散性及吸收性，藉以提升藥品的療效，其中，將分子藉由聚合物或脂質的包覆，在適當的製備條件下形成微脂體(Liposomes)、固體脂質奈米顆粒(Solid lipid nanoparticles)或聚合物奈米顆粒(Polymeric nanoparticles)，更已被廣為應用，如中華民國專利公告案第I325315號「含有非諾貝特之 奈米級粒子的製造方法、奈米級粒子」及中國專利公告案第CN102838760號「一種聚合物納米顆粒的製備方法」。 Nanotechnology is one of the research focuses in recent years. Because many materials (or substances) exhibit nanoscopic physical properties, chemical properties and biological properties under normal conditions, nanotechnology can be applied to various fields. Taking the drug delivery system in the biomedical industry as an example, through the design of molecular nanocrystallization, it can effectively improve its delivery mode, improve its bioavailability, and uniformity of preparation, Dispersibility and absorption, in order to enhance the efficacy of the drug, wherein the molecules are coated with a polymer or a lipid to form a liposome, solid lipid nanoparticles under appropriate preparation conditions. Or polymer nano particles, which have been widely used, such as the Republic of China Patent Notice No. I325315 "containing fenofibrate Method for producing nano-sized particles, nano-sized particles" and Chinese Patent Publication No. CN102838760 "Method for preparing a polymer nanoparticle".
正-丁烯基苯酞(Butylidenephthalide，簡稱BP)已知為中草藥，如當歸(Angelica sinensis)及川芎(Ligusticum chuanxiong)等植物之萃取物的主要成分之一(Wen-Wan Chao,Bi-Fong Lin,2011,Bioactivities of major constituents isolated from Angelica sinensis,Chinese Medicine,6：29)，且已被許多研究證實對於癌細胞有抑制作用，如中華民國專利公告案第I298259號「用於抑制/治療腦腫瘤之醫藥組成物」，揭露一種當歸之丙酮萃取物、氯仿萃取物或己烷萃取物，以及由其所純化之成分，即正-丁烯基苯酞，可有效抑制癌細胞之端粒酶(Telomerase)活性，且進一步誘發其細胞凋亡(Apoptosis)，而可使用於治療腦腫瘤，或如美國專利公開案第US20110165201號「Anticancer formulation」，揭露一種含有正丁烯基苯酞及多元酸酐的醫藥組合物，透過混合有聚合物之方式，可使有效成分依特定期間，緩慢且持續性地釋放至特定組織內，達成有效治療多形性成膠質細胞瘤、肺癌、肝細胞癌、結腸癌、黑色素瘤、乳腺癌、神經胚細胞瘤、畸胎瘤或人類白血病之功效，又，有其他研究亦證實正-丁烯基苯酞對於相關疾病有治療效果，如中華民國發明專利公開案第TW201326147號「含正-亞丁基苯酞之醫藥組成物用於治療肝損傷及改善肝功能」，揭露一種包含治療有效量之正-丁烯基苯酞化合物的醫藥組合物，並可用 於治療如肝纖維化、肝硬化或肝發炎等肝損傷，其他如抗發炎(如中華民國專利公告案第I331033號)及治療牛皮癬(如中國專利公開案第CN103505535號)等。 Buttylidenephthalide (BP) is known as a Chinese herbal medicine, such as one of the main components of extracts from plants such as Angelica sinensis and Ligusticum chuanxiong (Wen-Wan Chao, Bi-Fong Lin). , 2011, Bioactivities of major constituents isolated from Angelica sinensis, Chinese Medicine, 6:29), and has been confirmed by many studies to inhibit cancer cells, such as the Republic of China Patent Notice No. I298259 "for inhibition / treatment of brain tumors The pharmaceutical composition discloses an angelic extract of Angelica, a chloroform extract or a hexane extract, and a component purified therefrom, n-butenylquinone, which is effective for inhibiting telomerase of cancer cells ( Telomerase), and further induces apoptosis (Apoptosis), which can be used for the treatment of brain tumors, or as disclosed in US Patent Publication No. US20110165201 "Anticancer formulation", which discloses a method comprising n-butenyl phenylhydrazine and a polybasic anhydride. The pharmaceutical composition can be slowly and continuously released into a specific tissue according to a specific period by mixing a polymer. Effective in the treatment of pleomorphic glioblastoma, lung cancer, hepatocellular carcinoma, colon cancer, melanoma, breast cancer, neuroblastoma, teratoma or human leukemia, and other studies have confirmed positive-butyl Alkenyl phenylhydrazine has therapeutic effects on related diseases, such as the Republic of China Invention Patent Publication No. TW201326147 "Pharmaceutical composition containing n-butylidene benzoquinone for treating liver damage and improving liver function", revealing a therapeutically effective amount a pharmaceutical composition of a n-butenyl phenylhydrazine compound, and is available For the treatment of liver damage such as liver fibrosis, cirrhosis or liver inflammation, others such as anti-inflammatory (such as the Republic of China Patent Publication No. I331033) and the treatment of psoriasis (such as Chinese Patent Publication No. CN103505535).
由上所述可知，已有許多前案揭露正-丁烯基苯酞 在癌症及相關疾病上，具有極高的抑制與治療效果，然而，相關前案多著重於正-丁烯基苯酞可用於治療何種疾病的研究上，對於正-丁烯基苯酞有不易溶於水(Poor aqueous solubility)及滲透穿透率不佳(Poor permeability)等特性，造成正-丁烯基苯酞在生理環境內的溶解度極低，且傳輸效率低，使得其具有有限的生物利用度，進而使治療效果受到限制的問題上，皆未多有著墨，是以，亟需一種將正-丁烯基苯酞奈米化，藉以加強生物利用度，進而減少使用劑量的解決辦法。 As can be seen from the above, many prior cases have revealed n-butenyl phenylhydrazine. It has a very high inhibitory and therapeutic effect on cancer and related diseases. However, the related pre-existing cases focus on the study of which diseases can be used for the treatment of n-butenyl phenylhydrazine. Poor aqueous solubility and poor permeability, causing n-butenyl phenylhydrazine to have very low solubility in physiological environment and low transmission efficiency, making it limited. Bioavailability, and thus the treatment effect is limited, there is not much ink, so there is a need for a solution of n-butenyl benzoquinone to enhance bioavailability and reduce the dosage. Method.
再者，由上可知，多數之前案文獻係強調含有正- 丁烯基苯酞之組合物可用於治療癌症(如腦腫瘤)及其他疾病(如肝硬化與牛皮癬)，在促進毛髮生長之用途上，除了如中國專利公開案第CN101268992號「植物防脫洗髮液及其製法」，揭露有一種包含有何首烏、女貞子、白鮮皮、當歸、使君子、訶子、人參和藏紅花等中草藥萃取物的洗髮液，可滋養頭髮並助於頭髮生長，然，其為由多種中草藥之萃取物所組成，實質上是哪個有效活性成分能促進毛髮生長，係並未具體揭露，換言之，並未有相關前案具體提出 正-丁烯基苯酞可用於促進毛髮生長之功效。 Furthermore, it can be seen from the above that most of the previous literatures emphasize that they contain positive - The composition of butenyl benzoquinone can be used for the treatment of cancer (such as brain tumors) and other diseases (such as cirrhosis and psoriasis), in the use of hair growth promotion, in addition to, for example, Chinese Patent Publication No. CN101268992 The hair liquid and its preparation method disclose a shampoo containing Chinese herbal extracts such as Shouwu, Ligustrum lucidum, white fresh skin, Chinese angelica, gentian, medlar, ginseng and saffron, which can nourish hair and help hair growth. However, it is composed of a variety of Chinese herbal medicine extracts, which is actually an effective active ingredient that can promote hair growth, which is not specifically disclosed. In other words, there is no relevant prior case. N- Butenylphenylhydrazine can be used to promote hair growth.
有鑑於上述的問題，本發明人係依據多年來從事相關行業的經驗，針對現有的組合物及製備方法進行研究及分析，期能研發出較佳的正-丁烯基苯酞組合物；緣此，本發明之主要目的在於提供一種粒徑大小分佈均勻，且具有較高之生物利用度的含有正-丁烯基苯酞的奈米顆粒組合物及其製備方法。 In view of the above problems, the present inventors have researched and analyzed existing compositions and preparation methods based on years of experience in related industries, and have been able to develop better n-butenyl benzoquinone compositions; Therefore, the main object of the present invention is to provide a nanoparticle composition containing n-butenylphenylhydrazine having a uniform particle size distribution and high bioavailability and a preparation method thereof.
為達上述的目的，本發明之含有正-丁烯基苯酞的奈米顆粒組合物，係包括有一正-丁烯基苯酞及至少一表面穩定劑，且所述之含有正-丁烯基苯酞的奈米顆粒組合物的有效平均粒徑小於約2200奈米，較佳情況下，有效平均粒徑為5~230奈米，而較佳實施例的製備方法，包括以下步驟：(1)將定量的一正-丁烯基苯酞與至少一表面穩定劑，溶解於一有機溶劑，並均勻混合；(2)去除有機溶劑；(3)加入定量的一水相溶液。 For the above purposes, the n-butenyl phenylhydrazine-containing nanoparticle composition of the present invention comprises a n-butenyl phenylhydrazine and at least one surface stabilizer, and the n-butene is contained. The effective particle size of the bismuth quinone nanoparticle composition is less than about 2200 nm, preferably, the effective average particle size is 5 to 230 nm, and the preparation method of the preferred embodiment comprises the following steps: 1) Dissolving a quantity of n-butenyl phenylhydrazine with at least one surface stabilizer in an organic solvent and uniformly mixing; (2) removing the organic solvent; and (3) adding a quantitative aqueous phase solution.
又，本發明之含有正-丁烯基苯酞的奈米顆粒組合物的另一實施例的製備方法，包括以下步驟：(1)將定量的一正-丁烯基苯酞與至少一表面穩定劑，溶解於一有機溶劑，並均勻混合； (2)加入定量的一水相溶液；(3)去除有機溶劑。 Further, a preparation method of another embodiment of the n-butenylquinone-containing nanoparticle composition of the present invention comprises the steps of: (1) quantifying a n-butenylbenzoquinone with at least one surface a stabilizer dissolved in an organic solvent and uniformly mixed; (2) adding a quantitative aqueous phase solution; (3) removing the organic solvent.
其中，所述之表面穩定劑包括有陰離子表面穩定劑、陽離子表面穩定劑、兩性離子表面穩定劑、非離子表面穩定劑，以及離子表面穩定劑。 Wherein, the surface stabilizer comprises an anionic surface stabilizer, a cationic surface stabilizer, a zwitterionic surface stabilizer, a nonionic surface stabilizer, and an ionic surface stabilizer.
具體而言，所述之表面穩定劑可為聚乙二醇、聚甲基丙烯酸甲酯、聚乙交酯、聚丙交酯、聚己內酯、聚丙交酯-聚乙二醇、聚己內酯-聚乙二醇、維生素E聚乙二醇琥珀酸酯、由聚環氧乙烷及聚環氧丙烷所聚合而成的泊洛沙姆、聚氧乙烯氫化蓖麻油，以及由脂質所聚合而成的微脂體，如二棕櫚醯基卵磷脂等。 Specifically, the surface stabilizer may be polyethylene glycol, polymethyl methacrylate, polyglycolide, polylactide, polycaprolactone, polylactide-polyethylene glycol, poly-caprol Ester-polyethylene glycol, vitamin E polyethylene glycol succinate, poloxamer polymerized from polyethylene oxide and polypropylene oxide, polyoxyethylene hydrogenated castor oil, and polymerized by lipids A micro-lipid, such as dipalmitosyl lecithin.
於一些實施例中，所述之表面穩定劑係為分子量為400~5000的聚乙二醇、分子量為1000~10000的聚丙交酯，以及分子量為5000~8000的聚己內酯，其中，較佳情況下，表面穩定劑為聚丙交酯-聚乙二醇的分子量為6500-2000，表面穩定劑為聚己內酯-聚乙二醇的分子量為5000-2000。 In some embodiments, the surface stabilizer is polyethylene glycol having a molecular weight of 400 to 5000, polylactide having a molecular weight of 1000 to 10000, and polycaprolactone having a molecular weight of 5000 to 8000, wherein In a good case, the surface stabilizer is a molecular weight of polyglycolide-polyethylene glycol of 6500-2000, and the surface stabilizer is polycaprolactone-polyethylene glycol having a molecular weight of 5000-2000.
其中，所述之有機溶劑可為正己烷、乙醇、異丙醇、丙酮、甲酸、醋酸、乙腈、二氯甲烷、二甲基甲醯胺、四氫呋喃等，又，所述之水相溶液可為去離子水，但不以此為限。 Wherein, the organic solvent may be n-hexane, ethanol, isopropanol, acetone, formic acid, acetic acid, acetonitrile, dichloromethane, dimethylformamide, tetrahydrofuran, etc., and the aqueous phase solution may be Deionized water, but not limited to this.
再者，本發明之另一目的在於提供一種可用於促進 毛髮生長的含有正-丁烯基苯酞的奈米顆粒組合物之用途，其中，所述之含有正-丁烯基苯酞的奈米顆粒組合物， 係包括有一正-丁烯基苯酞及至少一表面穩定劑。 Furthermore, another object of the present invention is to provide an improvement that can be used to promote Use of a hair-grown nano-butenyl benzoquinone-containing nanoparticle composition, wherein the n-butenylquinone-containing nanoparticle composition is The system comprises a n-butenyl phenylhydrazine and at least one surface stabilizer.
為使 貴審查委員得以清楚了解本發明之目的、技術特徵及其實施後之功效，茲以下列各實施例，並搭配圖示進行說明，敬請參閱。 In order to give a clear understanding of the purpose, technical features and effects of the present invention, the following examples will be described with reference to the drawings.
第1圖，為本發明之第一實施例的奈米顆粒粒徑分佈圖。 Fig. 1 is a view showing the particle size distribution of nanoparticles in the first embodiment of the present invention.
第2圖，為本發明之第一實施例的奈米顆粒穿透式電子顯微鏡影像圖。 Fig. 2 is a view showing a nanoparticle penetrating electron microscope image of the first embodiment of the present invention.
第3圖，為本發明之第一實施例的奈米顆粒粒徑變化圖。 Fig. 3 is a graph showing changes in particle size of nanoparticles according to the first embodiment of the present invention.
第4圖，為本發明之第二實施例的奈米顆粒粒徑分佈圖。 Fig. 4 is a view showing the particle size distribution of nanoparticles in the second embodiment of the present invention.
第5圖，為本發明之第三實施例的奈米顆粒粒徑分佈圖。 Fig. 5 is a view showing the particle size distribution of nanoparticles in the third embodiment of the present invention.
第6圖，為本發明之第四實施例的奈米顆粒粒徑分佈圖。 Fig. 6 is a view showing the particle size distribution of nanoparticles in the fourth embodiment of the present invention.
第7圖，為本發明之用於促進毛髮生長的對照組與實驗組比較示意圖。 Fig. 7 is a schematic view showing a comparison between a control group for promoting hair growth and an experimental group of the present invention.
首先，闡明本發明於說明文字中所使用之各定義，包括：“有效平均粒徑小於約2200奈米”一詞，係指利用動態光散射儀(Dynamic light scattering，簡稱DLS)，或熟習本技術者所知的其他方法進行有效平均粒徑(Z-average)的測 定時，可得到至少約50%的含有正-丁烯基苯酞的奈米顆粒組合物具有小於約2200奈米的粒徑，其中，本發明所使用之動態光散射儀為Malvern Instruments Ltd.的Zetasizer Nano ZS90，其係利用雷射光射入含有粒子的溶液中，當雷射光撞擊到粒子後會產生散射光，而散射光會隨時間產生變化，因此量測散射光而計算出粒子之粒徑分佈。 First, the definitions used in the description of the present invention include: "the effective average particle diameter is less than about 2200 nm", which means using a dynamic light scattering (DLS), or a familiar book. Other methods known to the skilled person for measuring the effective average particle size (Z-average) Timing, at least about 50% of the nanoparticle composition containing n-butenylquinone has a particle size of less than about 2200 nm, wherein the dynamic light scattering instrument used in the present invention is Malvern Instruments Ltd. Zetasizer Nano ZS90, which uses laser light to inject into a solution containing particles. When laser light hits the particle, it will produce scattered light, and the scattered light will change with time. Therefore, the scattered light is measured to calculate the particle size. distributed.
“約”一詞，係指某種程度的變動與誤差，當熟習本技術者無法確認本發明所用之“約”一詞時，則其代表所述之值的±10%。 The term "about" refers to a certain degree of variation and error, and when the person skilled in the art is unable to confirm the term "about" as used in the present invention, it represents ±10% of the stated value.
“多分散係數(Polydispersity index，簡稱PDI)”一詞，係常用於粒徑大小或高分子之分子量的測定中，指一種描述粒子是否分佈均一的指標，PDI數值在0~1之間，亦即PDI的數值越小，代表粒子的均一度越高，則越趨向單分散性，換言之，PDI的數值越大，代表粒子的均一度越低，則越趨向多分散性。 The term "polydispersity index (PDI)" is commonly used in the determination of particle size or molecular weight of a polymer. It refers to an index that describes whether particles are uniformly distributed. The PDI value is between 0 and 1, That is, the smaller the value of PDI, the higher the uniformity of the representative particles, the more monodispersive, in other words, the larger the value of PDI, the lower the uniformity of the representative particles, and the more polydispersive.
“穩定”一詞，係指含有正-丁烯基苯酞的奈米顆粒組合物具有下列一項或多項特性：(1)不會因顆粒間的吸引力，而產生絮凝作用(Flocculation)或黏聚作用(Agglomeration)，因而使粒徑大小隨時間明顯增加；(2)物理結構不因時間而改變，例如從非晶相轉為結晶相；以及/或(3)具有化學穩定性。 The term "stable" means that the nanoparticle composition containing n-butenylphenylhydrazine has one or more of the following characteristics: (1) does not cause flocculation due to attraction between particles or Agglomeration, thus causing a significant increase in particle size over time; (2) physical structure does not change over time, such as from an amorphous phase to a crystalline phase; and/or (3) is chemically stable.
“治療有效劑量”一詞，係指將含有正-丁烯基苯酞 的奈米顆粒組合物，投與特定數量至待治療的生物體時，能產生特定之藥理反應的劑量。 The term "therapeutically effective dose" means that it will contain n-butenyl phenylhydrazine. A nanoparticle composition that, when administered in a specific amount to the organism to be treated, produces a dose of a particular pharmacological response.
本發明提供一種含有正-丁烯基苯酞的奈米顆粒組合物，其係包括有一正-丁烯基苯酞(BP)及至少一表面穩定劑(Surface stabilizer)，其中，正-丁烯基苯酞可由植物萃取而得，所述之植物包括當歸或川芎，但不以此為限，特先陳明，又，正-丁烯基苯酞的萃取方法已由諸多前案所揭，本發明在此不加贅述，再者，表面穩定劑為物理性地黏附於正-丁烯基苯酞顆粒，或與正-丁烯基苯酞顆粒的表面相結合，但表面穩定劑不會與正-丁烯基苯酞顆粒，或其本身產生化學反應，其中，所述之表面穩定劑包括有陰離子(Anionic)表面穩定劑、陽離子(Cationic)表面穩定劑、兩性離子(Zwitterionic)表面穩定劑、非離子(Nonionic)表面穩定劑，以及離子(Ionic)表面穩定劑之其中一種或其組合，在一些具體實施例中，表面穩定劑可為聚乙二醇(Polyethylene glycol，簡稱PEG)、聚甲基丙烯酸甲酯(PMMA)、聚丙交酯(PLA)、聚乙交酯(PGA)、聚己內酯(PCL)、聚丙交酯(PLA)-聚乙二醇(PLA-PEG)、聚己內酯-聚乙二醇(PCL-PEG)、維生素E聚乙二醇琥珀酸酯(D-α-tocopheryl polyethylene glycol succinate，或稱Vitamin E TPGS，簡稱TPGS)、由聚環氧乙烷(Polyethylene oxide，簡稱PEO)及聚環氧丙烷(Polypropylene oxide，簡稱PPO)所聚合而成的泊洛沙姆(Poloxamer)，其商品名稱為Pluronic®、聚氧乙烯氫化蓖麻油 (PEG-40 Hydrogenated Castor Oil)，其商品名稱為Cremophor® RH40，以及由脂質(Lipids)所聚合而成的微脂體，如二棕櫚醯基卵磷脂(Dipalmitoylphosphatidylcholine，簡稱DPPC)之其中一種或其組合，其中，表面穩定劑的選擇包括，但不限於上述所列，特先陳明。 The present invention provides a nanoparticle composition containing n-butenylphenyl hydrazine, which comprises a n-butenyl phenylhydrazine (BP) and at least one surface stabilizer, wherein n-butene The phenyl hydrazine can be obtained by extracting from plants, including the angelica or Chuanxiong, but not limited thereto, and the extraction method of n-butyl benzoquinone has been revealed by many previous cases. The present invention will not be described herein. Further, the surface stabilizer is physically adhered to the n-butenyl phenylhydrazine particles or combined with the surface of the n-butenyl phenylhydrazine particles, but the surface stabilizer does not. Chemically reacting with n-butenyl phenylhydrazine particles, or the surface stabilizer thereof, including an anionic surface stabilizer, a Cationic surface stabilizer, and a zwitterionic surface stability One or a combination of a nonionic surfactant, a nonionic surface stabilizer, and an ionic surface stabilizer. In some embodiments, the surface stabilizer may be polyethylene glycol (PEG). Polymethyl methacrylate (PMMA), Lactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), polylactide (PLA)-polyethylene glycol (PLA-PEG), polycaprolactone-polyethylene glycol ( PCL-PEG), vitamin E polyethylene glycol succinate (D-α-tocopheryl polyethylene glycol succinate, or Vitamin E TPGS, TPGS for short), polyethylene oxide (PEO) and polycyclic ring Poloxamer polymerized from Polypropylene oxide (PPO) under the trade name Pluronic ® and PEG-40 Hydrogenated Castor Oil under the trade name Cremophor ® RH40, and a liposome polymerized by a lipid (Lipids), such as one of dipalmitoylphosphatidylcholine (DPPC) or a combination thereof, wherein the choice of surface stabilizer includes, but is not limited to, the above Listed, special first Chen Ming.
本發明所述之含有正-丁烯基苯酞的奈米顆粒組合物的製備方法，於較佳實施例下，包括有以下步驟：(1)將定量的一正-丁烯基苯酞與至少一表面穩定劑，溶解於一有機溶劑，並均勻混合；(2)將有機溶劑去除；(3)加入定量的一水相溶液。 The preparation method of the n-butenyl phenylhydrazine-containing nanoparticle composition of the present invention comprises, in a preferred embodiment, the following steps: (1) quantitatively determining a n-butenyl benzoquinone At least one surface stabilizer dissolved in an organic solvent and uniformly mixed; (2) removing the organic solvent; (3) adding a quantitative aqueous phase solution.
以下係舉多個實施例，具體說明本發明，但本發明之實施態樣不限於此。 The invention will be specifically described below by way of a plurality of examples, but the embodiment of the invention is not limited thereto.
<第一實施例> <First Embodiment>
將正-丁烯基苯酞(BP)，與聚氧乙烯氫化蓖麻油及聚乙二醇(PEG)依照等比例之重量比，溶解於1毫升(mL)的乙醇(Ethanol)，使其均勻混合後，透過旋轉濃縮機，將乙醇去除，最後，加入2毫升的去離子水配製成溶液，即完成本發明之含有正-丁烯基苯酞的奈米顆粒組合物的製備；其中，所述之等比例的重量比，係正-丁烯基苯酞與聚氧乙烯氫化蓖麻油的重量比為1：2.5至1：8之間，較佳情況下，正-丁烯基苯酞與聚氧乙烯氫化蓖麻油的重量比可為 1：2.5至1：5之間，又，正-丁烯基苯酞與聚乙二醇的重量比為1：0.02至1：10之間，較佳情況下，正-丁烯基苯酞與聚乙二醇的重量比可為1：0.05至1：5之間，亦即，若以正-丁烯基苯酞、聚氧乙烯氫化蓖麻油及聚乙二醇之重量比為1：3：0.2為例，當正-丁烯基苯酞為10毫克時，則聚氧乙烯氫化蓖麻油及聚乙二醇分別為30毫克與2毫克；其中，聚氧乙烯氫化蓖麻油係購自惠民製藥股份有限公司的Cremophor® RH40，聚乙二醇係購自Showa Chemical Industry Co.,Ltd.(Tokyo,Japan)，且為分子量於400~5000之間。 The n-butenyl phenylhydrazine (BP), in a weight ratio of polyoxyethylene hydrogenated castor oil and polyethylene glycol (PEG), is dissolved in 1 ml (mL) of ethanol (Ethanol) to make it uniform. After mixing, the ethanol is removed by a rotary concentrator, and finally, 2 ml of deionized water is added to prepare a solution, that is, the preparation of the nano-butenyl benzoquinone-containing nanoparticle composition of the present invention is completed; The weight ratio of the ratio is n-butenyl phenylhydrazine and polyoxyethylene hydrogenated castor oil in a weight ratio of 1:2.5 to 1:8, preferably n-butenyl phenylhydrazine The weight ratio of polyoxyethylene hydrogenated castor oil may be between 1:2.5 and 1:5, and the weight ratio of n-butenyl phenylhydrazine to polyethylene glycol is between 1:0.02 and 1:10. Preferably, the weight ratio of n-butenyl phenylhydrazine to polyethylene glycol may be between 1:0.05 and 1:5, that is, if n-butenyl phenylhydrazine or polyoxyethylene is hydrogenated The weight ratio of sesame oil to polyethylene glycol is 1:3:0.2. When n-butenyl phenylhydrazine is 10 mg, polyoxyethylene hydrogenated castor oil and polyethylene glycol are 30 mg and 2 mg, respectively. ; , A polyoxyethylene hydrogenated castor oil available from Waste Management Pharmaceutical Co., Ltd. Cremophor ® RH40, a polyethylene glycol available from Showa Chemical Industry Co., Ltd. ( Tokyo, Japan), and a molecular weight of 400 to 5000 between.
承上，並請搭配參閱「第1圖」，圖中所示為本發明之第一實施例的奈米顆粒粒徑分佈圖，將第一實施例所製備而成之含有正-丁烯基苯酞的奈米顆粒組合物，透過動態光散射儀(DLS)，進行粒徑大小的測定，由圖所示之訊號強弱(Intensity)，可得知粒徑大小分佈的範圍在10至50奈米之間，又，動態光散射儀所測得之粒徑大小的平均值(即有效平均粒徑)約為20.67奈米，又，多分散係數(PDI)為0.068；再請搭配參閱「第2圖」，圖中所示為本發明之第一實施例的奈米顆粒穿透式電子顯微鏡影像圖，如圖，將本發明所製備而得之含有正-丁烯基苯酞的奈米顆粒組合物，進行乾燥後，透過穿透式電子顯微鏡(Transmission electron microscopy，簡稱TEM)的觀察，並根據顆粒數目及比例尺計算後，可得有效平均粒徑為5.39±0.08奈米；再請搭配參閱 「第3圖」，圖中所示為本發明之第一實施例的奈米顆粒粒徑變化圖，由圖可發現，將本發明所製備而得之含有正-丁烯基苯酞的奈米顆粒組合物靜置一段時間(圖中所示為四天)後，其粒徑大小不會隨著時間，而有大太的變化(變動範圍僅為10%至15%之間)，且此變動不會造成奈米結構上的崩散，換言之，由本發明之製備方法，所製備而得之含有正-丁烯基苯酞的奈米顆粒組合物為穩定結構之狀態。 Please refer to "Figure 1" for reference. The figure shows the particle size distribution of the nanoparticles according to the first embodiment of the present invention. The first embodiment has a n-butenyl group. The nanoparticle composition of benzoquinone is subjected to dynamic light scattering (DLS) to measure the particle size. From the intensity of the signal shown in the figure, the particle size distribution can be found in the range of 10 to 50 nm. Between the meters, the average value of the particle size measured by the dynamic light scattering instrument (ie, the effective average particle diameter) is about 20.67 nm, and the polydispersity coefficient (PDI) is 0.068; 2, the figure shows a nanoparticle penetrating electron microscope image of the first embodiment of the present invention, as shown in the figure, the nanometer containing n-butenylquinone prepared by the present invention. The granule composition is dried and observed by transmission electron microscopy (TEM), and calculated according to the number of particles and the scale, the effective average particle size is 5.39±0.08 nm; See "Fig. 3", which shows a variation of the particle size of the nanoparticles of the first embodiment of the present invention, and it can be seen from the figure that the naphthalene containing n-butenylbenzoquinone prepared by the present invention is obtained. After the rice granule composition is allowed to stand for a period of time (four days shown in the figure), the particle size does not change with time, but there is a change in the size (the variation range is only between 10% and 15%), and This change does not cause collapse of the nanostructure, in other words, the nanoparticle composition containing n-butenylquinone prepared by the production method of the present invention is in a state of stable structure.
<第二實施例> <Second embodiment>
首先，係取等比例之重量比的正-丁烯基苯酞與二棕櫚醯基卵磷脂(DPPC)，其中，所述之正-丁烯基苯酞與二棕櫚醯基卵磷脂的重量比可為1：3至1：10之間，而較佳情況下為1：4至1：6之間，又，二棕櫚醯基卵磷脂係購自Avanti Polar Lipids,Inc.(Birmingham,AL)，於本實施例中，係以正-丁烯基苯酞與二棕櫚醯基卵磷脂的重量比為1：5為例，將4毫克之正-丁烯基苯酞及20毫克之二棕櫚醯基卵磷脂，溶解於1毫升的二氯甲烷(Dichloromethane，簡稱DCM)，並均勻混合後，透過旋轉濃縮機，將二氯甲烷去除，最後，加入10毫升的去離子水配製成溶液，即完成本發明之含有正-丁烯基苯酞的奈米顆粒組合物之製備；請搭配參閱「第4圖」，圖中所示為本發明之第二實施例的奈米顆粒粒徑分佈圖，由圖所示之訊號強弱，可得知粒徑大小分佈的範圍較分散，在800至2000奈米之間，又，經動態光散射儀所測得的有效平均粒徑約為1581奈米，且多分散係數(PDI)為0.370。 First, an equal ratio by weight of n-butenyl phenylhydrazine and dipalmitosylphosphatidylcholine (DPPC) is obtained, wherein the weight ratio of n-butenyl phenylhydrazine to dipalmitosyl phosphatidylcholine It may be between 1:3 and 1:10, and preferably between 1:4 and 1:6. Further, dipalmitosyl lecithin is purchased from Avanti Polar Lipids, Inc. (Birmingham, AL). In the present embodiment, the weight ratio of n-butenyl phenylhydrazine to dipalmitosyl lecithin is 1:5, 4 mg of n-butenyl phenylhydrazine and 20 mg of two palms are used. The sulfhydryl lecithin is dissolved in 1 ml of Dichloromethane (DCM) and uniformly mixed. The dichloromethane is removed through a rotary concentrator. Finally, 10 ml of deionized water is added to prepare a solution. That is, the preparation of the nanoparticle composition containing n-butenylquinone of the present invention is completed; please refer to "Fig. 4" for matching, and the particle size distribution of the nanoparticles of the second embodiment of the present invention is shown. Figure, the signal shown in the figure is strong and weak, it can be seen that the range of particle size distribution is more dispersed, between 800 and 2000 nm, and the dynamic light Effective average particle size measured by the exit device is about 1581 nm, and the polydispersity index (PDI) of 0.370.
又，本發明所述之含有正-丁烯基苯酞的奈米顆粒組合物的製備方法，於另一實施例下，係包括有以下步驟：(1)將定量的一正-丁烯基苯酞與至少一表面穩定劑，溶解於一有機溶劑，並均勻混合；(2)加入定量的一水相溶液；(3)去除有機溶劑。 Further, the method for preparing a nanoparticle composition containing n-butenylphenylhydrazine according to the present invention, in another embodiment, comprises the steps of: (1) quantifying a n-butenyl group; Phenylhydrazine and at least one surface stabilizer are dissolved in an organic solvent and uniformly mixed; (2) a quantitative aqueous solution is added; and (3) the organic solvent is removed.
以下同樣舉多個實施例，具體說明本發明，但本發明之實施態樣不限於此。 Hereinafter, the present invention will be specifically described by way of a plurality of examples, but the embodiment of the present invention is not limited thereto.
<第三實施例> <Third embodiment>
將正-丁烯基苯酞與泊洛沙姆(Poloxamer)依照等比例之重量比溶於四氫呋喃(Tetrahydrofuran，簡稱THF)，並透過攪拌使其均勻混合，且可選擇性地，以較低的溫度(約35℃~55℃)進行加熱，藉以加速其溶解，接續，將四氫呋喃溶液加入10毫升的去離子水中，形成一混合溶液，最後，將混合溶液加熱至較高的溫度(約75℃~90℃)，使四氫呋喃揮發後，即完成本發明之含有正-丁烯基苯酞的奈米顆粒組合物之製備；其中，所述之等比例之重量，係正-丁烯基苯酞與泊洛沙姆的重量比為1：0.2至1：2之間，較佳情況下可為1：0.5至1：1之間，於本實施例中，係以正-丁烯基苯酞與泊洛沙姆之重量比為1：0.5為例，即取10毫克的正-丁烯基苯酞與5毫克的泊洛沙姆；其中，本實施例中所使用的泊洛沙姆係選用惠民製藥 股份有限公司的Pluracare® F127，亦可選用Sigma-Aldrich Corporation的Pluronic® F-127；承上，並請搭配參閱「第5圖」，圖中所示為本發明之第三實施例的奈米顆粒粒徑分佈圖，將第二實施例所製備之含有正-丁烯基苯酞的奈米顆粒組合物，透過動態光散射儀的測定，由圖所示之訊號強弱，可得知粒徑大小分佈的範圍為90至700奈米之間，又，經動態光散射儀所測得的有效平均粒徑約為204.9奈米，且多分散係數(PDI)為0.157。 The n-butenyl phenylhydrazine and the poloxamer are dissolved in tetrahydrofuran (Tetrahydrofuran, THF for short) in an equal ratio by weight, and uniformly mixed by stirring, and optionally, at a lower ratio. The temperature (about 35 ° C ~ 55 ° C) is heated to accelerate its dissolution, and then, the tetrahydrofuran solution is added to 10 ml of deionized water to form a mixed solution, and finally, the mixed solution is heated to a higher temperature (about 75 ° C ~90 ° C), after the tetrahydrofuran is volatilized, the preparation of the nano-butenyl benzoquinone-containing nanoparticle composition of the present invention is completed; wherein the equal proportion of the weight is n-butenyl phenylhydrazine The weight ratio to poloxamer is between 1:0.2 and 1:2, preferably between 1:0.5 and 1:1. In this example, n-butenyl phenylhydrazine is used. For example, a weight ratio of poloxamer of 1:0.5 is taken as 10 mg of n-butenyl phenylhydrazine and 5 mg of poloxamer; wherein the poloxamer used in this embodiment Pluracare ® F127 from Huimin Pharmaceutical Co., Ltd., or Pluronic ® F- from Sigma-Aldrich Corporation 127; 上上, and please refer to "figure 5", which shows the particle size distribution map of the third embodiment of the present invention, and the n-butene group prepared in the second embodiment The nanoparticle composition of benzoquinone is measured by a dynamic light scattering instrument, and the signal intensity shown in the figure shows that the particle size distribution ranges from 90 to 700 nm, and the dynamic light scattering instrument The measured effective average particle size was about 204.9 nm and the polydispersity coefficient (PDI) was 0.157.
<第四實施例> <Fourth embodiment>
將正-丁烯基苯酞、維生素E聚乙二醇琥珀酸酯(TPGS)與泊洛沙姆依照等比例之重量比溶於四氫呋喃，並透過攪拌使其均勻混合，且可選擇性地，以較低的溫度(約35℃~55℃)進行加熱，藉以加速其溶解，接續，將四氫呋喃溶液加入10毫升的去離子水中，形成混合溶液，最後，將混合溶液加熱至較高的溫度(約75℃~90℃)，使四氫呋喃揮發後，即完成本發明之含有正-丁烯基苯酞的奈米顆粒組合物之製備；其中，所述之等比例的重量比，係正-丁烯基苯酞與TPGS的重量比為1：0.2至1：2之間，較佳情況下，正-丁烯基苯酞與TPGS的重量比可為1：0.5至1：1之間，又，正-丁烯基苯酞與泊洛沙姆的重量比為1：0.5至1：2之間，較佳情況下，正-丁烯基苯酞與泊洛沙姆的重量比可為1：0.5至1：1之間，於本實施例中，係以正-丁烯基苯酞、TPGS及泊洛 沙姆之重量比為1：0.5：0.5為例，即取10毫克的正-丁烯基苯酞、5毫克的TPGS及5毫克的泊洛沙姆；其中，本實施例中所使用的維生素E聚乙二醇琥珀酸酯(TPGS)係購自巴斯夫股份公司(BASF Corporation)，又，泊洛沙姆係選用惠民製藥股份有限公司的Pluracare® F127，亦可選用Sigma-Aldrich Corporation的Pluronic® F-127；承上，再請搭配參閱「第6圖」，圖中所示為本發明之第四實施例的奈米顆粒粒徑分佈圖，將本實施例所製備之含有正-丁烯基苯酞的奈米顆粒組合物，透過動態光散射儀的測定，由圖所示之訊號強弱，可得知粒徑大小分佈的範圍分為兩個區間，包括有80至700奈米之間及2000至3000奈米之間的兩個分佈區間，又，經動態光散射儀所測得的有效平均粒徑約為173.3奈米，且多分散係數(PDI)為0.555。 N-butenyl phenylhydrazine, vitamin E polyethylene glycol succinate (TPGS) and poloxamer are dissolved in tetrahydrofuran in an equal ratio by weight, and uniformly mixed by stirring, and optionally, Heating at a lower temperature (about 35 ° C ~ 55 ° C), thereby accelerating its dissolution, followed by adding tetrahydrofuran solution to 10 ml of deionized water to form a mixed solution, and finally, heating the mixed solution to a higher temperature ( After the tetrahydrofuran is volatilized, the preparation of the nanoparticle composition containing n-butenylphenylhydrazine of the present invention is completed; wherein the ratio by weight is n-butyl The weight ratio of alkenyl phenylhydrazine to TPGS is between 1:0.2 and 1:2. Preferably, the weight ratio of n-butenyl phenylhydrazine to TPGS may be between 1:0.5 and 1:1. The weight ratio of n-butenyl phenylhydrazine to poloxamer is between 1:0.5 and 1:2. Preferably, the weight ratio of n-butenyl phenylhydrazine to poloxamer can be 1. Between 0.5 and 1:1, in the present embodiment, the weight ratio of n-butenyl phenylhydrazine, TPGS and poloxamer is 1:0.5:0.5, for example, 10 mg of n-butenyl phenylhydrazine, 5 mg of TPGS and 5 mg of poloxamer; wherein the vitamin E polyethylene glycol succinate (TPGS) used in this example was purchased from BASF The company (BASF Corporation), in addition, poloxamer is selected from Pluracare ® F127 of Huimin Pharmaceutical Co., Ltd., or Pluronic ® F-127 of Sigma-Aldrich Corporation can be used; please refer to "Figure 6" The figure shows a particle size distribution diagram of a nanoparticle according to a fourth embodiment of the present invention, and the nanoparticle composition containing n-butenylphenylhydrazine prepared in the present embodiment is transmitted through a dynamic light scattering instrument. The measurement, the signal strength shown in the figure, can be seen that the range of particle size distribution is divided into two intervals, including two distribution intervals between 80 and 700 nm and between 2000 and 3000 nm. The effective average particle diameter measured by a dynamic light scattering instrument was about 173.3 nm, and the polydispersity coefficient (PDI) was 0.555.
<第五實施例> <Fifth Embodiment>
將正-丁烯基苯酞與聚丙交酯-聚乙二醇(PLA-PEG)依照等比例之重量比，溶於四氫呋喃，並透過攪拌使其均勻混合，接續，將均勻混合之四氫呋喃溶液，加入10毫升的去離子水中，形成混合溶液，最後，將混合溶液加熱至一特定溫度(75℃~90℃)，使四氫呋喃揮發後，即完成本發明之含有正-丁烯基苯酞的奈米顆粒組合物之製備；其中，正-丁烯基苯酞與聚丙交酯-聚乙二醇的重量比可為1：2至1：6之間，較佳情況下可為1：3至1：5之間，又，聚丙交酯-聚乙二醇係參考相關文獻，自行合成製備而得， 即所述之合成方法可由相關前案得知，在此不詳加贅述，另，不同分子量之聚丙交酯與聚乙二醇所形成的表面穩定劑，會使所製備出之含有正-丁烯基苯酞的奈米顆粒組合物，具有不同大小的有效平均粒徑，請參閱表一所示，其係列出多個由不同分子量之組合的聚丙交酯-聚乙二醇，分別與正-丁烯基苯酞，透過本發明之製備方法，而生成的含有正-丁烯基苯酞的奈米顆粒組合物，由表一可知，若使用分子量為1000至10000的聚丙交酯與分子量為2000至5000所形成的聚丙交酯-聚乙二醇，作為表面穩定劑，可得到有效平均粒徑在215.6至2211.0奈米之間的奈米顆粒，且多分散係數在0.232至0.859之間，而當使用分子量為6500-2000的聚丙交酯-聚乙二醇，可得到較佳的有效平均粒徑，約為215.6奈米(PDI為0.232)。 The n-butenyl phenylhydrazine and the polylactide-polyethylene glycol (PLA-PEG) are dissolved in tetrahydrofuran in an equal proportion by weight ratio, and uniformly mixed by stirring, followed by uniformly mixing the tetrahydrofuran solution. Add 10 ml of deionized water to form a mixed solution. Finally, heat the mixed solution to a specific temperature (75 ° C to 90 ° C) to volatilize the tetrahydrofuran to complete the n-butenylbenzoquinone-containing naphthalene of the present invention. The preparation of the rice granule composition; wherein the weight ratio of n-butenyl phenylhydrazine to polylactide-polyethylene glycol may be between 1:2 and 1:6, preferably 1:3 to Between 1:5, in addition, polylactide-polyethylene glycol is referenced to the relevant literature, which is prepared by self-synthesis. That is, the synthesis method can be known from the related prior case, and the details of the above-mentioned surface stabilizers formed by polylactide and polyethylene glycol of different molecular weights will result in the preparation of n-butene containing The cyanophthalide nanoparticle composition has different effective average particle sizes, as shown in Table 1, which is a series of polylactide-polyethylene glycol with different molecular weight combinations, respectively Butyl alkenyl benzoquinone, a nano granule composition containing n-butenyl phenylhydrazine formed by the preparation method of the present invention, as shown in Table 1, if a polylactide having a molecular weight of 1,000 to 10,000 and a molecular weight of Polylactide-polyethylene glycol formed from 2000 to 5000, as a surface stabilizer, can obtain nano particles with an effective average particle diameter of 215.6 to 2211.0 nm, and the polydispersity coefficient is between 0.232 and 0.859. When a polylactide-polyethylene glycol having a molecular weight of 6500-2000 is used, a preferred effective average particle diameter of about 215.6 nm (PDI of 0.232) can be obtained.
<第六實施例> <Sixth embodiment>
將正-丁烯基苯酞與聚己內酯-聚乙二醇(PCL-PEG)依照等比例之重量比，溶於四氫呋喃，並透過攪拌使其均勻混合，接續，將均勻混合之四氫呋喃溶液，加入10毫升的去離子水中，形成混合溶液，最後，將混合溶液加熱至特定溫度(75℃~90℃)，使四氫呋喃揮發後，即完成本發明之含有正-丁烯基苯酞的奈米顆粒組合物之製備；其中，正-丁烯基苯酞與聚己內酯-聚乙二醇的重量比可為1：2至1：6之間，較佳情況下可為1：3至1：5之間，又，聚己內酯-聚乙二醇係參考相關文獻，自行合成製備而得，即所述之合成方法可由相關前案得知，在此不詳加贅述，又，不同分子量之聚己內酯-聚乙二醇所形成的表面穩定劑，會使所製備出之含有正-丁烯基苯酞的奈米顆粒組合物，具有不同大小的有效平均粒徑，請參閱表二所示，其係列出兩種由不同分子量之組合的聚己內酯-聚乙二醇作為表面穩定劑，分別與正-丁烯基苯酞，透過本發明之製備方法，而生成的含有正-丁烯基苯酞的奈米顆粒組合物，由表二可知，若使用分子量為8000-2000的聚己內酯-聚乙二醇，可得到有效平均粒徑約為229.7奈米(PDI為0.375)的奈米顆粒，而當使用分子量為5000-2000的聚己內酯-聚乙二醇，則可得到較佳的有效平均粒徑，約為214.3奈米(PDI為0.253)。 The n-butenyl phenylhydrazine and polycaprolactone-polyethylene glycol (PCL-PEG) are dissolved in tetrahydrofuran in an equal ratio by weight, and uniformly mixed by stirring, followed by uniformly mixed tetrahydrofuran solution. Adding 10 ml of deionized water to form a mixed solution, and finally, heating the mixed solution to a specific temperature (75 ° C to 90 ° C) to volatilize the tetrahydrofuran, thereby completing the n-butenylbenzoquinone-containing naphthalene of the present invention. The preparation of the rice granule composition; wherein the weight ratio of n-butenyl phenylhydrazine to polycaprolactone-polyethylene glycol may be between 1:2 and 1:6, preferably 1:3. Between 1:5, and again, polycaprolactone-polyethylene glycol is obtained by self-synthesis and preparation by reference to related literatures, that is, the synthetic method described above can be known from the related prior case, and is not described in detail herein. The surface stabilizer formed by polycaprolactone-polyethylene glycol of different molecular weights will produce the nanoparticle composition containing n-butenylphenylhydrazine having different effective average particle sizes, please Referring to Table 2, there are two series of polycaprolactone-polymers with different molecular weight combinations. The diol as a surface stabilizer, and the n-butenyl phenylhydrazine, respectively, are produced by the preparation method of the present invention, and the n-butenyl benzoquinone-containing nanoparticle composition is produced. As shown in Table 2, if molecular weight is used. A polycaprolactone-polyethylene glycol of 8000-2000 can obtain a nanoparticle having an effective average particle diameter of about 229.7 nm (PDI of 0.375), and when a polycaprolactone having a molecular weight of 5000-2000 is used - With polyethylene glycol, a preferred effective average particle size of about 214.3 nm (PDI of 0.253) is obtained.
<使用含有正-丁烯基苯酞的奈米顆粒組合物，促進公鼠毛髮生長的實驗> <Experiment to promote hair growth of male rats using a nanoparticle composition containing n-butenylphenylhydrazine>
毛髮生長的實驗，係使用購自樂斯科生物科技公司之六至八週齡的10隻公鼠(其所屬命名為C57BL/6NCrlBltw)進行測試，於實驗前一天，先利用電動剃毛刀將公鼠背部至近尾端處的毛髮剃除，並利用除毛膏將殘餘短毛移除，以使公鼠背部露出面積約2×2公分的光滑表皮，因此面積的毛囊皆達到同樣的基準，故作為本實驗的實驗部位，其中，將10隻公鼠分成對照組與實驗組，每組各為5隻公鼠，對照組係使用磷酸鹽緩衝生理食鹽水(Phosphate buffered saline，簡稱PBS)塗抹於實驗部位，實驗組則塗抹由前述之第一實施例(即正-丁烯基苯酞、聚氧乙烯氫化蓖麻油及聚乙二醇)所製備而得的含有正-丁烯基苯酞的奈米顆粒組合物於實驗部位，各組皆須每日塗抹，並持續塗抹14天，且塗抹後須待實驗部位完成乾燥，期間，公鼠係飼養於恆溫恆濕且日夜光照循環之環境，再者，定期(如每日)觀察各組公鼠之實驗部位的毛髮生長情況，以及公鼠的體重、活力、 食量及外觀有無異常；請參閱「第7圖」，圖中所示為本發明之用於促進毛髮生長的對照組與實驗組比較示意圖，如圖，在塗抹第14天後，可觀察到塗抹有本發明之含有正-丁烯基苯酞的奈米顆粒組合物的實驗組，有顯著的毛髮生長，雖對照組亦有毛髮生長，然而，從毛髮之密度與生長速度而言，可判斷其為自然生長，是以，在對動物投與治療有效劑量之含有正-丁烯基苯酞的奈米顆粒組合物後，確實可發現公鼠之實驗部位具有明顯之毛髮生長，需先陳明的是，雖本實驗僅使用由第一實施例所製備而得之含有正-丁烯基苯酞的奈米顆粒組合物進行塗抹，然，由其他實施例所製備之本發明的奈米顆粒組合物，同樣具有促進毛髮生長之功效，再者，雖然本實驗的結果係根據公鼠以顯示含有正-丁烯基苯酞的奈米顆粒組合物促進動物毛髮生長的新用途，但本發明的範圍並非應侷限於此，熟習本領域之技術者應可瞭解本發明同樣可應用在任何具有毛囊或生長毛髮之毛囊表皮細胞的溫血動物，包括人類、哺乳類動物，如牛、豬、馬、羊、狗、兔子、與寵物、禽鳥類，如雞、鴨、鵝及火雞等。 The hair growth experiment was carried out using 10 male rats (named C57BL/6NCrlBltw) from 6 to 8 weeks old from Lesco Biotech, and the electric razor was used one day before the experiment. The hair of the male rat to the near end is shaved, and the residual short hair is removed by using the hair removal cream, so that the back of the male mouse exposes a smooth skin of about 2×2 cm, so the hair follicles of the area reach the same benchmark. Therefore, as the experimental part of the experiment, 10 male rats were divided into a control group and an experimental group, each group was 5 male rats, and the control group was smeared with Phosphate buffered saline (PBS). In the experimental part, the experimental group was coated with the n-butenyl benzoquinone prepared by the first embodiment (i.e., n-butenyl phenylhydrazine, polyoxyethylene hydrogenated castor oil and polyethylene glycol). The nanoparticle composition is applied to the experimental site, and each group must be applied daily for 14 days, and the tissue is dried after the application. During the period, the male mouse is kept in a constant temperature and humidity environment. , again, on a regular basis Daily) Experiment hair growth was observed in each portion of the male mice and male rats of the body weight, activity, Whether there is any abnormality in food intake and appearance; please refer to "Fig. 7", which shows a comparison of the control group for promoting hair growth of the present invention and the experimental group, as shown in the figure, after the 14th day of application, smear can be observed. The experimental group having the n-butenylquinone-containing nanoparticle composition of the present invention has significant hair growth, and although the control group also has hair growth, it can be judged from the density and growth rate of the hair. It is natural growth, so that after administering a therapeutically effective dose of a nanoparticle composition containing n-butenylphenylhydrazine to an animal, it is indeed found that the experimental part of the male mouse has obvious hair growth, and it is necessary to first It is to be noted that although the experiment was carried out using only the nanoparticle composition containing n-butenylquinone prepared by the first embodiment, the nanoparticle of the present invention prepared by other examples was used. The granular composition also has the effect of promoting hair growth, and further, although the results of the experiment are based on the male mouse to show a new use of a nanoparticle composition containing n-butenylquinone to promote hair growth in animals, invention The scope of the invention is not limited thereto, and those skilled in the art should understand that the present invention can also be applied to any warm-blooded animal having hair follicle epidermal cells of hair follicles or growing hair, including humans, mammals, such as cattle, pigs, and horses. , sheep, dogs, rabbits, and pets, birds, such as chickens, ducks, geese and turkeys.
由上所述可知，本發明所述之含有正-丁烯基苯酞 的奈米顆粒組合物，係包括有一正-丁烯基苯酞及至少一表面穩定劑，其中，在一些實施例中，係可包括有正-丁烯基苯酞及兩種表面穩定劑，而較佳實施例的製備方法，包括以下步驟： (1)將定量的一正-丁烯基苯酞與至少一表面穩定劑，溶解於一有機溶劑，並均勻混合；(2)去除有機溶劑；(3)加入定量的一水相溶液。 It can be seen from the above that the present invention contains n-butenyl phenylhydrazine. The nanoparticle composition comprises a n-butenyl phenylhydrazine and at least one surface stabilizer, wherein, in some embodiments, the n-butenyl phenylhydrazine and the two surface stabilizers may be included. The preparation method of the preferred embodiment includes the following steps: (1) A quantitative amount of n-butenyl phenylhydrazine and at least one surface stabilizer are dissolved in an organic solvent and uniformly mixed; (2) an organic solvent is removed; and (3) a quantitative aqueous phase solution is added.
又，本發明之含有正-丁烯基苯酞的奈米顆粒組合物的另一實施例的製備方法，包括以下步驟：(1)將定量的一正-丁烯基苯酞與至少一表面穩定劑，溶解於一有機溶劑，並均勻混合；(2)加入定量的一水相溶液；(3)去除有機溶劑。 Further, a preparation method of another embodiment of the n-butenylquinone-containing nanoparticle composition of the present invention comprises the steps of: (1) quantifying a n-butenylbenzoquinone with at least one surface a stabilizer, dissolved in an organic solvent, and uniformly mixed; (2) adding a quantitative aqueous phase solution; (3) removing the organic solvent.
其中，所述之表面穩定劑包括有陰離子表面穩定劑、陽離子表面穩定劑、兩性離子表面穩定劑、非離子表面穩定劑，以及離子表面穩定劑。 Wherein, the surface stabilizer comprises an anionic surface stabilizer, a cationic surface stabilizer, a zwitterionic surface stabilizer, a nonionic surface stabilizer, and an ionic surface stabilizer.
具體而言，所述之表面穩定劑可為聚乙二醇、聚甲基丙烯酸甲酯、聚丙交酯、聚乙交酯、聚己內酯、聚丙交酯-聚乙二醇、聚己內酯-聚乙二醇、維生素E聚乙二醇琥珀酸酯、由聚環氧乙烷及聚環氧丙烷所聚合而成的泊洛沙姆、聚氧乙烯氫化蓖麻油，以及由脂質所聚合而成的微脂體，如二棕櫚醯基卵磷脂等。 Specifically, the surface stabilizer may be polyethylene glycol, polymethyl methacrylate, polylactide, polyglycolide, polycaprolactone, polylactide-polyethylene glycol, poly-caprol Ester-polyethylene glycol, vitamin E polyethylene glycol succinate, poloxamer polymerized from polyethylene oxide and polypropylene oxide, polyoxyethylene hydrogenated castor oil, and polymerized by lipids A micro-lipid, such as dipalmitosyl lecithin.
於一些實施例中，所述之表面穩定劑係為分子量為400、600或2000~5000的聚乙二醇、分子量為1000~10000的聚丙交酯，以及分子量為5000~8000的聚己內酯，其中，較佳情況下，表面穩定劑為聚丙交酯-聚乙二醇的分子量為6500-2000，表面穩定劑為聚己內酯-聚乙二醇的分子量為 5000-2000。 In some embodiments, the surface stabilizer is polyethylene glycol having a molecular weight of 400, 600 or 2000-5000, polylactide having a molecular weight of 1000-10000, and polycaprolactone having a molecular weight of 5000-8000. Wherein, preferably, the surface stabilizer is a molecular weight of polylactide-polyethylene glycol of 6500-2000, and the surface stabilizer is a molecular weight of polycaprolactone-polyethylene glycol. 5000-2000.
其中，所述之有機溶劑可為正己烷、乙醇、異丙醇、丙酮、甲酸、醋酸、乙腈、二氯甲烷、二甲基甲醯胺、四氫呋喃等，又，所述之水相溶液可為去離子水，但不以此為限。 Wherein, the organic solvent may be n-hexane, ethanol, isopropanol, acetone, formic acid, acetic acid, acetonitrile, dichloromethane, dimethylformamide, tetrahydrofuran, etc., and the aqueous phase solution may be Deionized water, but not limited to this.
承上，由本發明所製備而得之含有正-丁烯基苯酞的奈米顆粒組合物，經粒徑大小測定後，有效平均粒徑小於約2200奈米，在一些實施例中，有效平均粒徑可小於約1600奈米、小於約1000奈、小於約800奈米或小於約300奈米，而於較佳情況下，有效平均粒徑可為5~230奈米，且均一度高及穩定度佳，具體而言，本發明之優點有三：(一)提高正-丁烯基苯酞的生物利用度：奈米載體具有奈米的尺度，是比體細胞還小的分子載體，因此能夠以流體的形式給藥，又，毛細血管的直徑大約是數微米，故，奈米分子載體可以輕而易舉地進入毛細血管，在血液循環系統中自由流動；(二)提高水溶性不佳之正-丁烯基苯酞的溶解度：奈米載體具有較高的比表面積(Specific surface area)，可以提高水溶性不佳的分子在奈米載體中的溶解度，解決無法藉由傳統方法製備成製劑的難題；(三)減少使用次數：對於半衰期短的活性成份，病人可能需要每天 重複使用，但奈米分子載體能大幅延長活性成份的半衰期，如此，可解決長期治療之病人，需頻繁地使用的問題。 The nanoparticle composition containing n-butenylphenylhydrazine prepared by the present invention has an effective average particle diameter of less than about 2200 nm after being determined by particle size, and in some embodiments, an effective average The particle size may be less than about 1600 nanometers, less than about 1000 nanometers, less than about 800 nanometers, or less than about 300 nanometers, and preferably, the effective average particle diameter may be from 5 to 230 nanometers, and the uniformity is high. The stability is good. Specifically, the advantages of the present invention are three: (1) improving the bioavailability of n-butenyl phenylhydrazine: the nano carrier has a nanometer scale and is a molecular carrier smaller than the somatic cell, thus It can be administered in the form of a fluid, and the diameter of the capillaries is about several micrometers. Therefore, the nano molecular carrier can easily enter the capillaries and flow freely in the blood circulation system; (2) improve the water solubility poorly - Solubility of butenyl benzoquinone: The nano carrier has a high specific surface area, which can improve the solubility of molecules with poor water solubility in the nano carrier, and solve the problem that the preparation cannot be prepared by the conventional method. ;(three) Reduced use: For active ingredients with a short half-life, the patient may need to take it daily Reusable, but the nano molecular carrier can greatly extend the half-life of the active ingredient, thus solving the problem of frequent use of patients for long-term treatment.
是以，本發明據以實施後，確實可達到本發明之主要目的，即提供一種粒徑大小分佈均勻，且具有較高之生物利用度的含有正-丁烯基苯酞的奈米顆粒組合物及其製備方法。 Therefore, after the present invention is implemented, the main object of the present invention can be achieved by providing a nanoparticle composition containing n-butenylbenzoquinone with uniform particle size distribution and high bioavailability. And its preparation method.
再者，經使用含有正-丁烯基苯酞的奈米顆粒組合物，促進公鼠毛髮生長的實驗，可發現塗抹有含有正-丁烯基苯酞的奈米顆粒組合物的公鼠，相較於對照組而言，期毛髮生長速度明顯增加，是以，本發明據以實施後，確實可達到本發明之另一目的，即提供一種可用於促進毛髮生長的含有正-丁烯基苯酞的奈米顆粒組合物之用途。 Further, by using a nanoparticle composition containing n-butenylphenylhydrazine to promote hair growth in male rats, a male mouse coated with a nanoparticle composition containing n-butenylphenylhydrazine can be found. Compared with the control group, the hair growth rate is significantly increased. Therefore, after the present invention is implemented, it is indeed another object of the present invention to provide a n-butenyl group which can be used for promoting hair growth. Use of a benzoquinone nanoparticle composition.
唯，以上所述者，僅為本發明之較佳之實施例而已，並非用以限定本發明實施之範圍；任何熟習此技藝者，在不脫離本發明之精神與範圍下所作之均等變化與修飾，皆應涵蓋於本發明之專利範圍內。 The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; any changes and modifications made by those skilled in the art without departing from the spirit and scope of the invention All should be covered by the patent of the present invention.
綜上所述，本發明之功效，係具有發明之「產業可利用性」、「新穎性」與「進步性」等專利要件；申請人爰依專利法之規定，向 鈞局提起發明專利之申請。 In summary, the effects of the present invention are patents such as "industry availability," "novelty," and "progressiveness" of the invention; the applicant filed an invention patent with the bureau in accordance with the provisions of the Patent Law. Application.
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