TW201138837A

TW201138837A - Method of preparing plant stem cell liposome

Info

Publication number: TW201138837A
Application number: TW99115417A
Authority: TW
Inventors: Chong-Yu Chen; Shi-Fen Chen; Sheng-Feng Wu
Original assignee: Univ Shu Te
Priority date: 2010-05-14
Filing date: 2010-05-14
Publication date: 2011-11-16

Abstract

A method of preparing plant stem cell liposome comprises: using nano-lipid with high cell permeability to wrap CIC2 plant stem cell, preparing phosphatidylcholine and CIC2 with high speed homogenization/high pressure homogenization emulsion method, and carrying out evaluation of average particle size distribution, safetiness, and stability of CIC2 liposome.

Description

201138837 六、發明說明：【發明所屬之技術領域】一種用於植物幹細胞微脂粒製備方法與有效性評估，利用奈米脂質易穿透細胞之特性包覆CIC2植物幹細胞，將磷脂酿膽驗 (Phosphatidylcholine)及CIC2以高速均質/高壓均質乳化法進行CIC2植物幹細胞微脂粒製備，根據微脂粒之平均粒徑分布評估其安定性與安全性’找出使微脂粒配方安定之最佳化條件。 •.【先前技術】幹細胞是原始的尚未特化的細胞，它具有潛力保留特化出其它細胞類型的能力。幹細胞能夠擔當自體的修復系統，植物方面位於莖與根尖端者稱頂端分生組織（Apical meristem)，而頂端生長點包含具有全能性的幹細胞，是植物胚後發育中新的器官產生的源泉。由於生化科技發達，許多各領域的學者們投入精力研發幹細胞，在美膚保養方面，從法國引進海茴香之海洋原生植物， # 從之中取得幹細胞。海茴香（Crithmum maritimum)是布列塔尼海邊之原生植物，可食性植物，茂盛地生長在地中海沿岸的岩壁上。其根可以吸收海霧和海潮的養份去面對嚴苛的陸地環境（失去水分、陽光、紫外線等）。1837年，法國船員出海時都會二著海菌香-同出海’當皮膚曬傷歧傷口時’海茴麵是他們的救命草藥，1874年，正是被法國納入法國國家藥典中，更在一夕之門成為法國女人傳頌的保養秘方。1988年，科學家發現海寸香中的 201138837 ， CIC2成份是對抗乾燥缺水及強烈紫外線最直接有效的方式。海茴香主要成份有多種氨基酸蛋白質（生物體内最重要的活性分子）、抗氧化之化合物（可刺激皮膚細胞再生、修護等功能），是目前美容界新穎保養品之一。 P遺著生物科技時代的來臨，如何使化妝品長效畴及大幅提升化妝品的魏，與如触錄品齡分子細微化、增加渗透性，更成為各國化妝品產業全力投入研發的主要方向。而在眾多強調癱生醫材料與奈米技術的生技公司皆以Γ三高」即是高功效、高安全性及⑤科技之美容美髮產品為主要重點發展。近年來除了醫藥製劑將微脂粒普遍應用在生物體内進行比例居多，其他應用領域也甚為廣泛’如羊毛、絲織物之染色加工、食品科技、動物飼料、民生工業、美容保養品…等。微脂粒是1965年英國學者Alec Bangham發現的。微脂粒 (liposomes)是脂質空心微球，如圖i所示，粒徑約〇〇25 3. 5 ♦微米（卵），懸浮於水相中，脂質膜(球皮）主要是由鱗脂質所構成的脂質雙層（lipid bilayers)。鱗脂分子的磷酸端為親水性，脂質端為疏水性，由此形成磷酸端朝外脂質端在内之雙面親水夾層内為疏水之膜，恰似紅血球膜或細胞膜。水溶性物質之溶液可包在球心，油溶性物質可夾在球皮膜層内（好像三夾板）。因此微脂粒可當做水溶性及油潍藥物之載體。製備微脂粒最常用之脂質為lecithin，其主要磷脂成分為ph〇sphatidylch〇Hne。使用天然構脂質製成之微絲可完全與生物體相容（biQCOmpatible) I ·、ί 4 .201138837 * 碟脂質又稱墙脂酸膽驗(PC，phosphatidylcholine)為生物體的細胞膜主要組成之一’近來利用磷脂質作為微脂粒（Lip〇s〇me) 載體的技術已十分廣泛，但隨著生化科技對於奈米材料的需求增加’微脂粒不穩定等問題隨之而起。影響微脂粒穩定之因素包括物理穩定性及化學穩定性。物理穩定性受到與多因素的影響如，脂質之組成、外在環境、粒徑大小、pH值以及雙層結構之物理狀態的差異，皆會影響微脂粒的物理穩定性。其主要因素有聚集、 • 融合以及包埋物質的滲漏等三項因素；其中以聚集、融合會造成微脂粒平均粒徑大小及分佈的改變，而滲透作用則會導致包覆物質的減少。化學穩定性在微脂粒製備及保存過程中發生氧化或水解的自然反應，其原因在於磷脂質具有不飽和鍵或特殊官能基，會導致脂質分子之碳鏈結構的改變，而影響微脂粒之穩定性。 :、、;而，微知粒在熱力學上處於介穩態(Metastabie)，會因外界衫響而產生結構性的改變’在應用上仍有不穩定的缺點存在， #其包含物理、化學與生物等穩定性。物穩定性⑽加㈤201138837 VI. Description of the invention: [Technical field of invention] A method for evaluating the preparation and effectiveness of plant stem cell microlipids, coating CIC2 plant stem cells with the characteristics of nano-lipid penetrating cells, and concentrating phospholipids ( Phosphatidylcholine) and CIC2 were prepared by high-speed homogenization/high-pressure homogenization emulsification method for CIC2 plant stem cell microlipids. The stability and safety of the lipid particles were evaluated according to the average particle size distribution of the microlipids. condition. • [Prior Art] Stem cells are primitive, unspecialized cells that have the potential to retain the ability to specialize in other cell types. Stem cells can act as an autologous repair system. Plants are located at the tip of the stem and root. They are called Apical meristem, while the apical growth point contains stem cells with pluripotency, which is the source of new organs in plant post-embryonic development. . Due to the development of biochemical technology, many scholars in various fields have invested in research and development of stem cells. In the aspect of skin care, marine native plants of sea fennel have been introduced from France, and stem cells have been obtained from them. Crithmum maritimum is a native plant of Brittany, an edible plant that flourishes on the rocky walls of the Mediterranean coast. Its roots can absorb the nutrients of sea fog and tides to face the harsh land environment (loss of moisture, sunlight, ultraviolet light, etc.). In 1837, when the French crew went out to sea, they would have two seaweed fragrances - the same as the sea 'when the skin was sunburned and wounded', the sea fennel noodles were their life-saving herbs. In 1874, it was included in the French National Pharmacopoeia by France. The door of the evening has become a secret recipe for the French woman. In 1988, scientists discovered that 201138837, CIC2 in Haixiangxiang is the most direct and effective way to combat dry and water shortage and strong ultraviolet rays. The main ingredients of sea fennel are a variety of amino acid proteins (the most important active molecules in the body), antioxidant compounds (which can stimulate skin cell regeneration, repair and other functions), and are one of the new skin care products in the beauty industry. With the advent of the era of biotechnology, how to make cosmetics long-lasting and greatly enhance the cosmetics, and the micro-leveling and increasing permeability of the age-old products have become the main direction of the cosmetics industry in all countries. In many biotech companies that emphasize biomedical materials and nanotechnology, the company focuses on high-efficiency, high-security and 5-tech beauty and hairdressing products. In recent years, in addition to pharmaceutical preparations, microlipids are widely used in living organisms, and other applications are also extensive. For example, wool, silk fabric dyeing, food technology, animal feed, people's livelihood, beauty care products, etc. . The vesicles were discovered by British scholar Alec Bangham in 1965. Liposomes are lipid hollow microspheres, as shown in Figure i, with a particle size of about 25 3. 5 ♦ micron (egg), suspended in the aqueous phase, and the lipid membrane (spheroid) is mainly composed of squamous lipids. The lipid bilayers are composed. The phosphate end of the squamous lipid molecule is hydrophilic, and the lipid end is hydrophobic, thereby forming a hydrophobic membrane in the double-sided hydrophilic interlayer including the phosphoric acid end toward the outer lipid end, just like a red blood cell membrane or a cell membrane. The solution of the water-soluble substance may be contained in the center of the sphere, and the oil-soluble substance may be sandwiched in the spheroidal layer (like a three-ply board). Therefore, the vesicles can be used as a carrier for water-soluble and oil-based drugs. The most commonly used lipid for the preparation of vesicles is lecithin, the main phospholipid component of which is ph〇sphatidylch〇Hne. Microfilaments made with natural constitutive lipids are completely compatible with organisms (biQCOmpatible) I ·, ί 4 .201138837 * Disc lipids, also known as PC, phosphatidylcholine, are one of the major components of the cell membrane of organisms. 'The technology for the recent use of phospholipids as a carrier for liposome (Lip〇s〇me) has been extensive, but with the increasing demand for biomaterials for nanomaterials, problems such as instability of microlipids have arisen. Factors affecting the stability of the liposome include physical stability and chemical stability. Physical stability is affected by multiple factors such as lipid composition, external environment, particle size, pH, and physical state of the bilayer structure, all of which affect the physical stability of the vesicles. The main factors are aggregation, fusion, and leakage of embedded materials. Among them, aggregation and fusion will cause changes in the average particle size and distribution of the vesicles, while osmosis will lead to the reduction of coating materials. . Chemical stability The natural reaction of oxidation or hydrolysis during the preparation and preservation of vesicles. The reason is that phospholipids have unsaturated bonds or special functional groups, which can cause changes in the carbon chain structure of lipid molecules and affect vesicles. Stability. :,,;,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Stability such as biology. Stability (10) plus (5)

Stability)，疋因微脂粒結構自身之聚集與融合 (Fusion)所造成，而化學穩定性(Chemicai lability)，是因鱗月曰質月曰肪自夂鏈之氧化(〇xidati〇n)及水解(jjydr〇lysis)的化學反應其在製備過程及保存期間易受外在的效應(如溫度及邱值）而造成微脂_轉定’微絲的穩定性將影響其包埋物的功效。 201138837 【發明内容】有鑑於此，為解決上述問題，本發明提供—種植物幹細胞微脂粒製備方法，利用奈米脂質易穿透細胞之特性來包覆 ⑽植物幹細胞，以作為载體來包覆⑽植物幹細胞，用向速均質/高壓均質乳化法製備微脂粒(Lip〇s〇me)。另一方面，本發明亦提供將磷脂醯膽鹼 • (Ph〇sphatidylcholine)及CIC2用超音波震遭法及高速均質/高塵均質乳化法兩種製備方法製備，以二種製備方法刊功率、時間或壓力等條件來進行⑽微絲之平均粒徑分布、安定性與安全性評估’汹使微對搶方蚊之最佳化條件，本發明將可提供產業界做為製備奈米材料的參考指標。本發明之有益功效為以磷脂質作為載體來包覆CIC2植物幹細胞形成植物幹細胞微脂粒加入配方中，利系微脂粒所包覆da能 •有保濕、美白、抗氧化等多重的效果，可提升美白及抗氧化功效，較一般市售產品的效果佳、美白及抗氧化功效_他命更好。此外’藉由控制微脂粒粒徑大小，提升微脂粒美白及抗氧化配方產品之有效性及安定性與安全性。【實施方式】有關本發明的特徵與實作，茲以最佳實施例詳細說明如下： 1.材料 6 201138837 BASIS LS-60HR ’ 礙脂醯膽驗(Phosphatidylcholine) 60%〜70% ’ Nisshin Oillio、CIC2(Crithmum maritimum callus culture filtrate) ， Biotech Marine。 2. CIC2植物幹細胞微脂粒製備： 2· 1超音波震盪法 Α.取5毫升(ml)的PC溶液及CIC2溶液，以超音波細胞粉碎機在功率21瓦(W)下震盪即均勻溶解後，即刻使用粒 • 徑分析儀檢測’使粒徑達成50奈米(nm)至200奈米(nm) 之間。 B.再將PC溶液及CIC2溶液互相混合後，以超音波細胞粉碎機在功率21瓦(W)下震盪1〇分鐘，即完成微脂粒的製備 2. 2高速均質/高壓均質乳化法 A. 將100毫升(ml)之PC水溶液及CIC2水溶液，以高速均 • 質機在14000rPm轉速即均勻混合成PC溶液及CIC2溶液後備用。 B. 再將PC溶液及CIC2溶液，以高壓均質機在1〇〇〇bar高壓下’即均勻溶解後，立即使用粒徑分析儀檢測，使粒徑達成50奈米(nm)至200奈米(nm)之間。 C·將PC溶液及CIC2溶液互相混合後，以高壓均質機在壓力1000bar高壓下’來回運轉15回取之，即完成微脂粒的製備 201138837 3·超音波震躲與高速均質/高壓均魏化法之平均粒捏大小及分佈比本實驗PC: CIC2 =3 : 2的重量比例，利用薄超音波震盪法及高速均質法/高壓均質乳化法兩種製備方法，製備⑽微脂粒，並^ 試其平均粒徑大小及分佈，結果如糾标，超音波震盪法製備的微絲，粒徑為352.95奈米(nm);高速均質/高壓均質法製備的微脂粒’粒徑為92,71奈米(㈣。不論是超音波震盪法或高速均質/高 # 壓均質法，粒徑大小分佈均具有顯著性差異。以超音波震盪法製備的微脂粒，所測得的粒徑均在3〇〇奈米（nm)以上，但高速均質/ 高壓均質法製備的微脂粒，其粒徑小於1〇〇奈米（nm)。另外，超音波製備微脂粒為高分子之濃稠狀態，所測粒徑大於3〇〇奈米（nm) 以上，經皮滲透及吸收慢；而高速均質/高壓均質法製備微脂粒為乳化液體’所測粒徑小於3〇〇奈米（nm)以下，粒徑小可快速經皮吸收。 # 經由不同製備方法分析比較結果，高速均質/高壓均質法製備的微脂粒粒徑小於超音波震盪法製備的微脂粒粒徑。 201138837 表1，不同製備方法之粒徑比較分析表脂質配方比例超音波震盪法 ♦均翁:徑(am) pdi值高速均質/‘高壓均質乳化法乎均趣-(0111) PDI值 92P1 ^234 260.90=4.43 352^5 =14.43 PC/3 CIC2/2 PC:CIC2/3:2 0.79 = 0.03 29.48 ± 4.04 0.93 = 0 11 050 =008 Π4.03±158 0.34=0.04 0-88 士 0.02 92.71 ±252 0.51 &〇.〇2 4·超音波震盪法與高速均質/高壓均質乳化法之安定性比本實驗PC: CIC2 =3 : 2的重量比例，利用超音波震盪法或高速均質/高壓均質法兩雜備方法，製備⑽微脂粒，製備完成後儲存於4〇C及常溫環境下，探討其平均粒從大小及分佈在第1、7、14、21、28天内有所變化。結果如表2所示，超音波震盪法之粒徑變化大，貯存於室溫在7天後的粒徑與第1天的粒徑相較之下，粒徑大小有顯著性變化，由659.40奈綠11〇變大為1273.03奈米（11111)，貯存於28天後，其粒徑為4558奈米（nm)變更大，且微脂粒有聚集、融合現象；而其貯存於4Ϊ在7天後之粒徑與第1天的粒獲相較之下，粒徑由 352. 95奈米㈤變為376. 32奈米（咖），粒徑變化不大，但檢刪次之標準差高聊.78 ’貯存於28天後，粒彳il349 5G奈米(nm)亦變大’且微脂《分層縣，依據朗讀齡超音波震紅微脂粒極為不安定，不適用製備微脂粒包覆⑽植物幹細胞之方法。高速/高壓均質法製備之微脂粒貯存於室溫環境下，經由狀 201138837 天的儲存，觀察不同儲存時間下之粒徑變化，其結果如表2所示。貯存於至溫在7天後的粒徑與第丨天的粒徑相較之下，粒徑由原本的92. 71奈米（nm)至110. 70奈米（nm)，經過28天其粒徑增加至 134· 03奈米（nm)，微脂粒酸敗變濃稍；而其貯存於代在7天後粒柽為80. 77奈米（nm)至91. 59奈米（nm)，經過28天其粒徑為92. 94奈米（nm)均無顯著性差異。顯示以高速/高壓均質法製備之微絲貯存於4 C環i兄下，粒徑不會隨著時間的增加而改變極為安定，適用 • 於製備微脂粒包覆CIC2劑型之方法。表2.不同製備法之微脂粒儲存於不同環境下之粒徑變化表Stability), due to the aggregation and fusion of the vesicle structure itself, and the chemical stability (Chemicai lability) is due to the oxidation of the scorpion scorpion Hydrolysis (jjydr〇lysis) chemical reaction during the preparation process and storage period is susceptible to external effects (such as temperature and Qi value) and cause micro-lipid _ transfer 'microfilament stability will affect the efficacy of its embedding . In view of the above, in order to solve the above problems, the present invention provides a method for preparing a plant stem cell microlipid, which utilizes the characteristics of a nanolipid easily penetrating cell to coat (10) a plant stem cell as a carrier. (10) plant stem cells were prepared, and the liposome (Lip〇s〇me) was prepared by a uniform homogenization/high pressure homogenization emulsification method. In another aspect, the present invention also provides the preparation method of the phospholipid choline (Ph〇sphatidylcholine) and the CIC2 by the ultrasonic shock method and the high-speed homogenization/high dust homogenization emulsification method, and the two preparation methods are used for publication, Conditions such as time or pressure are carried out (10) The average particle size distribution, stability and safety evaluation of the microfilaments are optimized for the micro-pairing of the mosquitoes, and the present invention will provide the industry as a preparation of nanomaterials. reference indicator. The beneficial effect of the invention is that the phospholipid is used as a carrier to coat the CIC2 plant stem cells to form the plant stem cell microlipids into the formula, and the microlipids coated with da can have multiple effects of moisturizing, whitening and anti-oxidation. It can improve whitening and anti-oxidation effect, and has better effect, whitening and anti-oxidation effect than other commercially available products. In addition, by controlling the particle size of the vesicles, the effectiveness, stability and safety of the vesicle whitening and antioxidant formulations are enhanced. [Embodiment] The features and implementations of the present invention are described in detail in the following preferred embodiments: 1. Material 6 201138837 BASIS LS-60HR 'Phosphatidylcholine 60%~70% ' Nisshin Oillio, CIC2 (Crithmum maritimum callus culture filtrate), Biotech Marine. 2. Preparation of CIC2 plant stem cell microlipid: 2. 1 ultrasonic shock method. Take 5 ml (ml) of PC solution and CIC2 solution, and pulsate and dissolve evenly under the power of 21 watts (W) by ultrasonic cell pulverizer. Immediately, use a particle size analyzer to detect 'make the particle size between 50 nanometers (nm) and 200 nanometers (nm). B. The PC solution and the CIC2 solution are mixed with each other, and then oscillated by an ultrasonic cell pulverizer at a power of 21 watts (W) for 1 minute to complete the preparation of the vesicles. 2. High-speed homogenization/high-pressure homogenization emulsification method A 100 ml (ml) of PC aqueous solution and CIC2 aqueous solution were uniformly mixed into a PC solution and a CIC2 solution at a speed of 14000 rPm at a high speed homogenizer for use. B. Then, the PC solution and the CIC2 solution are uniformly dissolved in a high-pressure homogenizer under a high pressure of 1 bar, and immediately detected by a particle size analyzer to achieve a particle size of 50 nm (nm) to 200 nm. Between (nm). C·The PC solution and the CIC2 solution are mixed with each other, and then taken back and forth 15 times with a high pressure homogenizer under a pressure of 1000 bar, that is, the preparation of the microlipid is completed. 201138837 3. Ultrasonic wave collision and high-speed homogenization/high pressure uniformization method The average particle size and distribution ratio of the PC: CIC2 = 3: 2 weight ratio, using the thin ultrasonic vibration method and high-speed homogenization method / high-pressure homogenization emulsification two preparation methods, preparation (10) vesicles, and test The average particle size and distribution, the results such as correction, microfilament prepared by ultrasonic vibration method, particle size of 352.95 nanometers (nm); high-speed homogenization / high pressure homogenization method of preparation of micro-lipid particles 'particle size of 92,71 Nano ((4). Whether it is ultrasonic oscillating method or high-speed homogenization/high# pressure homogenization method, the particle size distribution has significant difference. The granules prepared by ultrasonic oscillating method have the measured particle size 3 〇〇 nanometer (nm) or more, but the high-pressure homogenization / high-pressure homogenization method of the preparation of micro-lipid particles, the particle size is less than 1 〇〇 nanometer (nm). In addition, the ultrasonic preparation of the vesicles is thick polymer State, the measured particle size is greater than 3 nanometers (nm) or more, Transdermal permeation and absorption are slow; while high-speed homogenization/high-pressure homogenization method prepares microlipid particles as emulsified liquids. The measured particle size is less than 3 〇〇 nanometer (nm), and the particle size is small and can be quickly percutaneously absorbed. # via different preparation The results of the method analysis and comparison showed that the particle size of the micro-lipids prepared by the high-speed homogenization/high-pressure homogenization method was smaller than that of the micro-lipid particles prepared by the ultrasonic vibration method. 201138837 Table 1. Comparative analysis of particle size of different preparation methods Method ♦ 均翁: Diameter (am) pdi value high-speed homogenization/'high-pressure homogenization emulsification method---(0111) PDI value 92P1 ^234 260.90=4.43 352^5 =14.43 PC/3 CIC2/2 PC:CIC2/3 :2 0.79 = 0.03 29.48 ± 4.04 0.93 = 0 11 050 =008 Π4.03±158 0.34=0.04 0-88 士0.02 92.71 ±252 0.51 &〇.〇2 4·Supersonic oscillation method and high-speed homogenization/high pressure homogenization The stability of the emulsification method is compared with the weight ratio of PC: CIC2 = 3: 2 in this experiment. Ultrasonic shock method or high-speed homogenization/high pressure homogenization method is used to prepare (10) vesicles. After preparation, store at 4〇C. And under normal temperature environment, the average particle size and distribution are discussed in the first, seventh, and fourth The results vary within 21 and 28 days. The results are shown in Table 2. The particle size of the ultrasonic vibration method varies greatly. The particle size stored at room temperature after 7 days is compared with the particle size of the first day. There was a significant change in size from 659.40 Nai 11〇 to 1273.03 nm (11111). After 28 days of storage, the particle size was changed to 4558 nm (nm), and the microlipids were aggregated and fused. And the particle size stored in 4Ϊ after 7 days compared with the grain acquisition on the first day, the particle size changed from 352. 95 nm (five) to 376. 32 nm (coffee), the particle size did not change much. However, the standard deviation of the inspection and deletion is high. 78 'After 28 days of storage, the granule il349 5G nanometer (nm) also became larger' and the micro-fat "stratified county, according to the lang reading age ultrasonic shock red granules Extremely unstable, it is not suitable for the preparation of microlipid coated (10) plant stem cells. The microlipids prepared by the high-speed/high-pressure homogenization method were stored at room temperature, and the particle size changes under different storage times were observed through storage for 201138837 days. The results are shown in Table 2. The particle size stored at 7 days after the temperature is compared with the particle size of the third day, the particle size is from 92.71 nm (nm) to 110.70 nm (nm), after 28 days. The particle size is increased to 134·03 nm (nm), and the oligosaccharide is rancid, and the granules are stored at 7 days after the granules are 80. 77 nm (nm) to 91.59 nm (nm). After 28 days, the particle size was 92.94 nm (nm) and there was no significant difference. It is shown that the microfilament prepared by the high-speed/high-pressure homogenization method is stored under the 4 C ring, and the particle size does not change extremely stably with time, and is suitable for the preparation of the CIP2 dosage form of the microlipid coating. Table 2. Particle size change table of different lipids stored in different environments

5.安全性測試本實驗將CIC2微脂粒溶液加入三組不同配方組合’為 1BG-3G001、2BD-3E003、3BG-027 (沒有 CIC2成份）’其中符號” 為代表沒有包覆CIC2 水溶液；“-2”為慢速包覆CIC2 (高速均質法），-3”為奈米包覆CIC2 (高壓均質法），來進行皮膚過敏性測s式。結果如表3所表示，測試 1 〇位健康皮膚之爻測者’接受48Hrs貼膚測試後， 201138837 二種〉谷液對皮膚反應平均為0 ,表示包覆CIC2之微脂粒對人體皮膚不會產生過敏現象，顯示實驗配方是安全無慮的。表3·皮膚過敏性測試之皮膚反應指數表不同绦#组合求】試時數(hr) 皮膚反應指數 AOl 1BG-3G0011-3^-0020.5% 48 48 細 2 BD-3E003 2-3 + GC2 03% A03 3 B G-027 3-3 -i-CIC2 Oi%5. Safety test This experiment adds CIC2 liposome solution to three different combinations of formulas '1BG-3G001, 2BD-3E003, 3BG-027 (without CIC2) 'where the symbol' is not coated with CIC2 aqueous solution; -2" is a slow-coated CIC2 (high-speed homogenization method), and -3" is a nano-coated CIC2 (high-pressure homogenization method) to perform skin allergy test. The results are shown in Table 3. Test 1 健康 position of healthy skin tester 'after receiving 48Hrs skin test, 201138837 two kinds> gluten solution on skin response average 0, indicating that CIP2 coated vesicles are not for human skin It can cause allergies and show that the experimental formula is safe. Table 3. Skin reaction index of skin allergic test is different 绦#Combination test hours (hr) Skin reaction index AOl 1BG-3G0011-3^-0020.5% 48 48 Fine 2 BD-3E003 2-3 + GC2 03 % A03 3 B G-027 3-3 -i-CIC2 Oi%

6·皮膚保濕測試實驗 12名文試者，年齡為20-40健康肌膚之男女，在溫度保持 25±2C及相對濕度為45%〜50%RH下，測得塗抹含ciC2微脂粒溶液配方，對於正常皮膚角質層水分含量的變化，結果如表4所示。各組配方在經由塗抹3〇分鐘後，測試皮膚角質水分含量，以 BG-027 3-2+CIC2 0.5%皮膚角質水分含量提升比率最高2129〇/〇; 皮膚角質水分含量提升比率最少為BD-3E003 2-2+CIC2 0.5%的配方5. 28%。60分鐘後，測試皮膚角質水分含量，以BD-3E003 Base 配方含水量比率最高為15.8〇%;80-3£003 2-2+(：102 0.5%配方含水量比率最少為4· 45°/。。90分鐘後，測試皮膚角質水分含量，以 BD-3E003 Base配方含水量比率最高為丨4. 〇9% ; BD-3E003 2-3+CIC2 0.5%配方含水量比率最少為丨94%。ι2〇分鐘後，測試皮膚角質水分含量’以BG-027 3-1+CIC2 0.1%之配方含水量比率最高為 11 201138837 12.18% ; BD-3E003 2-2+CIC2 5%為0· 47%。從表3之保濕數據分析，以BG-027配方樣品組之皮膚角質水分含量最好，從3〇分鐘量測平均含水率為21.29%〜14.80%，經過120分鐘量測平均含水率為 12.18%〜5. 20%，為最具有保濕的配方。表4·皮膚角質層水分含量測試分析表6. Skin moisturizing test 12 people in the test, age 20-40 healthy skin for men and women, measured at a temperature of 25 ± 2 C and a relative humidity of 45% ~ 50% RH, measured smear containing ciC2 microlipid solution For the change in the moisture content of the normal skin stratum corneum, the results are shown in Table 4. Each group of formulas tested the skin keratin moisture content after 3 minutes of application, with BG-027 3-2+CIC2 0.5% skin keratin moisture content increase rate of 2129 〇 / 〇; skin keratin moisture content increase ratio of at least BD- 3E003 2-2+CIC2 0.5% of the formula 5. 28%. After 60 minutes, the skin moisture content of the skin was tested. The water content ratio of the BD-3E003 Base formula was 15.8〇%; 80-3£003 2-2+ (:102 0.5% formula water content ratio was at least 4·45°/ After 90 minutes, the skin keratin moisture content was tested, and the water content ratio of the BD-3E003 Base formula was 丨4. 〇9%; BD-3E003 2-3+CIC2 0.5% formula water content ratio was at least 94%. After ι2〇, test the skin keratin moisture content 'BG-027 3-1+CIC2 0.1% formula water content ratio is up to 11 201138837 12.18%; BD-3E003 2-2+CIC2 5% is 0·47%. From the moisture retention data of Table 3, the skin keratin moisture content of the BG-027 formula sample group was the best, the average water content measured from 3 minutes was 21.29%~14.80%, and the average moisture content was 12.18% after 120 minutes. ~5. 20%, the most moisturizing formula. Table 4: Skin cuticle moisture content test analysis table

T«t times A^JStd S分 % at Avs-,: Stl 齡 % ^ai3§ig& A^./Std 筘分 % 1游(却魏 .值 Avs/Sii 2^· % 130300(1 Be% 4i57±3a 15^ 秘 iS-4 1431 5.S5*T^ 1212 3-^im $J7$ 1·1+α€201βΛ 44f3± 纽 141 3.56*7.1 1M 11^5.7 « ι-ι+αα〇5% 4&77±14 3^*5.1 ?.7：S 1怎 1.40= t4 2S9· Uli IL? ι*>αα^ι% 4i03±?^ 顿纖 ϋ虫SJ 1J4 5M xia^ it 4vS2 bh-aci^/o ±7.1 3.27 7.04 1.7€=t-5：2 sm X55±l~ 5_^ 2.23 ± ?,S 樣 ι->αααι% 44SS* 科 4.^6=9.1 娜 4CB ± 102 135土客.1 5JS l.S5i 4.14 ι-^-ααο^/β 45.i3*£i 4.02* 7.S S.S1 330 M 7-23 3.13 i 1.7 錄 IS5± H? 4J7 49.S7±5.2 IgJl T：S5±15,4 li© 14⑤ 142*115 雄 44^1 *S9 4.ITig^ §31 4^±l€i5 鹽 .263=tfJ 5,t? 1,62±5^ 3jS 47^5 ±?.S 4m BSJ±73 '.45 1.72=43 1.53 ώδ.4 3名 ^i+aamve 4S^ilL0 4^i7.4 464 ±m §-箱 ITEilM 5.® 2.02±?„S 4.13 况十aci齢 4£?1±7.S 147*3.0 5】頂 ±3:.:S 4.45 1.45=7.7 3.1© 002*5^ 0.47 4:S1 i 豸 9M- 4.:2^ ±^.5 143 4..06^^.4 :115 1..5:2 ± 11.1 3..S5 40SUI4 4^*5.g 1¾^ 3.©±dl 9.02 0-79^5,5 IM 〇_^)± L4? 4Ut±im 7.B±&g l樣 ^3SilU B25 XIV 116*145 im. 3-1+CK20154 λ^33 = €α 1455 5】土 15 ms 540± 1211 3~1+<3C2S5% 41^6 i £4 HV} isjf 4.07 ±9Λ 915 3i2士 7-4 罐 3^i 12- 7.2S 3-2+aaai% 4404±5i iS-12 觸 4.19*5S &51 3J2±xt 3*2+CK205% 4LS5*a8 191 ±7.3 2L.P ^.35 mi 5.?i*a4 HT4 5.07±4.^ UU 5*3+€K3(kl% 44.44±1% i 5_2 i3.ai 5.4S：±5.0 ms 5JS：il4 a眩 131±15 3^+aC2· 4454 ± £9 E边 iia? 146±ias 7.7? ZSS 土 53 5.47 Z5Ei 72 S,V 7.皮膚美白測試實驗 12名受試者，年齡為20〜40健康肌膚之男女，膚色各有深T«t times A^JStd S points % at Avs-,: Stl Age % ^ai3§ig& A^./Std 筘%% 1 swim (but Wei. Value Avs/Sii 2^· % 130300(1 Be% 4i57±3a 15^ Secret iS-4 1431 5.S5*T^ 1212 3-^im $J7$ 1·1+α€201βΛ 44f3± New 141 3.56*7.1 1M 11^5.7 « ι-ι+αα〇5 % 4&77±14 3^*5.1 ?.7:S 1How 1.40= t4 2S9· Uli IL? ι*>αα^ι% 4i03±?^ ϋ ϋ S SJ 1J4 5M xia^ it 4vS2 bh- Aci^/o ±7.1 3.27 7.04 1.7€=t-5:2 sm X55±l~ 5_^ 2.23 ± ?,S-like ι->αααι% 44SS* Section 4.^6=9.1 Na 4CB ± 102 135客.1 5JS l.S5i 4.14 ι-^-ααο^/β 45.i3*£i 4.02* 7.S S.S1 330 M 7-23 3.13 i 1.7 Record IS5± H? 4J7 49.S7±5.2 IgJl T:S5±15,4 li© 145 142*115 Male 44^1 *S9 4.ITig^ §31 4^±l€i5 Salt.263=tfJ 5,t? 1,62±5^ 3jS 47^5 ±?.S 4m BSJ±73 '.45 1.72=43 1.53 ώδ.4 3名^i+aamve 4S^ilL0 4^i7.4 464 ±m §-Box ITEilM 5.® 2.02±?„S 4.13 Condition 10 Aci齢4£?1±7.S 147*3.0 5] Top ±3:.:S 4.45 1.45=7.7 3.1© 002*5^ 0.47 4:S1 i 豸9M- 4.:2^ ±^.5 143 4..06^^.4 :115 1..5:2 ± 11.1 3..S5 40SUI4 4^*5.g 13⁄4^ 3.©±dl 9.02 0-79^5,5 IM 〇_^)± L4? 4Ut±im 7.B±&gl-like^3SilU B25 XIV 116 *145 im. 3-1+CK20154 λ^33 = €α 1455 5] soil 15 ms 540± 1211 3~1+<3C2S5% 41^6 i £4 HV} isjf 4.07 ±9Λ 915 3i2士 7-4 Tank 3^i 12- 7.2S 3-2+aaai% 4404±5i iS-12 Touch 4.19*5S &51 3J2±xt 3*2+CK205% 4LS5*a8 191 ±7.3 2L.P ^.35 mi 5 .i*a4 HT4 5.07±4.^ UU 5*3+€K3(kl% 44.44±1% i 5_2 i3.ai 5.4S:±5.0 ms 5JS:il4 a glare 131±15 3^+aC2· 4454 ± £9 E side iia? 146±ias 7.7? ZSS soil 53 5.47 Z5Ei 72 S, V 7. Skin whitening test 12 subjects, aged 20 to 40 healthy men and women with deep complexion

Γ C 12 201138837 淺，为別將21瓶實驗樣品塗抹於左右手臂内側，分別每天塗抹一次實驗樣品，每星期進行檢測美白从值之深淺度，觀測膚色的變化’結果如表5所示。經由表5分析，第7天的檢測美白从值之皮膚深淺度，以3 BG-027 Base之μ值最高，比原始膚色淺127 ;以BD-3E003 Base 之δι值最低，比原始膚色深1· 34。第14天的檢測美白δι值之皮膚深淺度’以BG-3G001BaseM值最高，比原始膚色淺丨66 ;以BG_〇27 φ 3-1+CIC2 0. 5%δι值最低，為〇. 〇4，以14天之檢測值得知，本實驗 21組配方樣品到14天皆能讓膚色變淺。第21天的檢測美白μ值之皮膚深淺度，以BD-3E003 2-2+CIC2 0.1%之Δΐ值最高，為丨.38 ; 以BD-3E003 Base之膚色μ值反加深為〇. 29。第28天的檢測美白M 值之皮膚深淺度，以BG-3G001 1-3+CIC2 0.5%之μ值最高，膚色變淺為1.57 ;而BD-3E003 Base之膚色δζ值加深為0. 51。依28天之整體觀測，以BG-3G001配方的樣品最具有美白效 φ 果，又以CIC2 0. 5%效果最好，依初步判斷分析CIC2含量越高美白效果越好。 13 201138837 表5.皮膚測試分析表Γ C 12 201138837 Slightly, do not apply 21 bottles of experimental samples to the inside of the left and right arms, apply the test samples once a day, and test the whiteness of the skin from the value of the skin color every week. The results are shown in Table 5. According to the analysis in Table 5, the skin whitening value of the whitening value from the 7th day was the highest in the 3 BG-027 Base, which was 127 lighter than the original skin color; the BD-3E003 Base had the lowest δι value, which was 1 deeper than the original skin color. · 34. On the 14th day, the skin whiteness of the whitening δι value was the highest in BG-3G001BaseM, which was shallower than the original skin color 66; the lowest value was BG_〇27 φ 3-1+CIC2 0. 5%δι, 〇. 〇 4. According to the detection value of 14 days, the 21 groups of formula samples in this experiment can make the skin lighter to 14 days. On the 21st day, the skin whiteness of the whitening value was measured, and the Δΐ value of BD-3E003 2-2+CIC2 0.1% was the highest, which was 丨.38; the darkening value of the skin color of BD-3E003 Base was 〇.29. On the 28th day, the skin whiteness of the whitening M value was measured, and the B value of BG-3G001 1-3+CIC2 0.5% was the highest, and the skin color became 1.57, while the skin color δζ value of BD-3E003 Base was deepened to 0.51. According to the overall observation of 28 days, the sample with BG-3G001 formula has the most whitening effect φ fruit, and the best effect is CIC2 0.5%. According to the preliminary judgment, the higher the CIC2 content, the better the whitening effect. 13 201138837 Table 5. Skin test analysis table

tines /San^pla Av3./SEi. 7(天推化值 A^/ Std. 14{天度化值 Ava./ Std 21(天>铤化值 Ave；Std 2㈣狄值 Ava.·* SEi. 1KJ-3G501 Bssa 62S4 = 4 -0.36±1.&5 !.6£i3 27 0.73^3.75 0.20 = 4.13 l-i^aczaisb £6 i 2^S 024 ±0M O^3± 1J07 0.92± 124 O.SU1.07 i-wac2〇iSi> ¢4^4 i 2.SS 1.^5 = 2.42 1.14= li2 £*il±li4 1.17il.4? l^aaca% €5.14 = 2Sl 0.45 = 0.44 0i?±1.41 0.4^ i 1.2$ 1-2—00205¾ 64.S1 ± 2 〇7 1.01 = 0,^7 0.45== 1J4 0.€7± 126 1.52*1.32 i-3—a<2ca% 64.S3 i 3.42 -0.01 ± 0.55 0.13 i 0.7# 0JS±U# 0.72 i €>.91 l-3^a<ZC6% 6Sm^2M 0.§Si〇J§ 131 ㈣ 1.51 ±0M 2BI>3Ea〇3 Bsm ^3.47 = 3^3 -1.34i3.73 O.iS -㈣ ±2.1S -0.51 i 2.49 24+acai% ^4.67 ±331 ^.17 = 0.75 ϋ.21^ύ21 0.42 i 13^ 4U2±1.2S M+aC205% ^431 i 3.65 S.3'5sl.4S 0.^= OiS _ ± 1.02 0.S4i〇.95 2^^aC20.1% i53il=4J5 -1.02s：2.32 1.47=324 1_3S±2.股 ^.1δ±3.3^ 2-2^aa〇i^ 64.53 ±3.72 ^>.21 = 0.71 ύΜ±ύ.Ώ, QMS ± 122 1.03 ±1.51 2^-^aC20.1^ 6313=:3^ l.n± 155 0.01 ±1.49 2-3^a〇0i'2^ 44.71=326 0.4# i〇,鈴 0.15= 1.Π -±125 -0.4? ϋ.εε 3BG^27 64£S = 323 1.21 ±2M §.T〇iI.n D.lf ilil 0.37=1.82 3-l^-aC20.Bo ¢5.40^237 1.05 ±1.47 0.24 0.42 ±0.61 0.4E±0.S3 3·ι—acza^b 64.S4±325 -0.^5 = 1.19 ©.04=0.77 0.12 = 126 0.3© i 1.14 3-2-r-aC2&l=/〇 ^525±2.<53 l.€-3 = 1.71 033 = 0.^ 0.45±0.S7 0.4¾ ±1.03 3-2+(30205¾ 64i5i3.46 0.02=.1.45 0.14= 0.7S 0.17 i 1.15 0.j7±l.eS 3-3-^ac2〇.m -64.21=3.19 0.44s=〇.$3 0.49^0^4 0.14 * 0.45*0.SB 8.自由基之還原力檢測參考Oyaizu等（1986)之方法，將（維生素C、MAP、CIC2、Tines /San^pla Av3./SEi. 7 (day push value A^/ Std. 14{天度化值Ava./ Std 21(天>铤化值Ave; Std 2(四)Di value Ava.·* SEi 1KJ-3G501 Bssa 62S4 = 4 -0.36±1.&5 !.6£i3 27 0.73^3.75 0.20 = 4.13 li^aczaisb £6 i 2^S 024 ±0M O^3± 1J07 0.92± 124 O. SU1.07 i-wac2〇iSi> ¢4^4 i 2.SS 1.^5 = 2.42 1.14= li2 £*il±li4 1.17il.4? l^aaca% €5.14 = 2Sl 0.45 = 0.44 0i?± 1.41 0.4^ i 1.2$ 1-2—002053⁄4 64.S1 ± 2 〇7 1.01 = 0,^7 0.45== 1J4 0.€7± 126 1.52*1.32 i-3—a<2ca% 64.S3 i 3.42 -0.01 ± 0.55 0.13 i 0.7# 0JS±U# 0.72 i €>.91 l-3^a<ZC6% 6Sm^2M 0.§Si〇J§ 131 (4) 1.51 ±0M 2BI>3Ea〇3 Bsm ^3.47 = 3^3 -1.34i3.73 O.iS -(iv) ±2.1S -0.51 i 2.49 24+acai% ^4.67 ±331 ^.17 = 0.75 ϋ.21^ύ21 0.42 i 13^ 4U2±1.2S M+aC205 % ^431 i 3.65 S.3'5sl.4S 0.^= OiS _ ± 1.02 0.S4i〇.95 2^^aC20.1% i53il=4J5 -1.02s:2.32 1.47=324 1_3S±2. .1δ±3.3^ 2-2^aa〇i^ 64.53 ±3.72 ^>.21 = 0.71 ύΜ±ύ.Ώ, QMS ± 122 1.03 ±1.51 2^-^aC20.1^ 6313=:3^ ln± 155 0.01 1.49 2-3^a〇0i'2^ 44.71=326 0.4# i〇, bell 0.15= 1.Π -±125 -0.4? ϋ.εε 3BG^27 64£S = 323 1.21 ±2M §.T〇iI .n D.lf ilil 0.37=1.82 3-l^-aC20.Bo ¢5.40^237 1.05 ±1.47 0.24 0.42 ±0.61 0.4E±0.S3 3·ι—acza^b 64.S4±325 -0.^ 5 = 1.19 ©.04=0.77 0.12 = 126 0.3© i 1.14 3-2-r-aC2&l=/〇^525±2.<53 l.€-3 = 1.71 033 = 0.^ 0.45±0 .S7 0.43⁄4 ±1.03 3-2+(302053⁄4 64i5i3.46 0.02=.1.45 0.14= 0.7S 0.17 i 1.15 0.j7±l.eS 3-3-^ac2〇.m -64.21=3.19 0.44s=〇 .$3 0.49^0^4 0.14 * 0.45*0.SB 8. Reducing power detection of free radicals. Refer to Oyaizu et al. (1986) for (vitamin C, MAP, CIC2,

14 201138837 微脂粒包覆CIC2之各取1%、l. 5%、2°/〇，使用UV-Vis分光光度計測其在700奈米（nm)之吸收值，吸收值越高表示樣品的還原力越強。結果如圖2所示’吸收值最高為CIC2由2.79〜2.87> PC+CIC2:2· 62~2. 85> 維生命c:2.12〜2. 4G>MAP: 1· 64〜2. 〇〇，可得知CIC2本S具有抗氧化效果，且濃度越高效果越多。雖然已說明且描述了本發明之實施例，但是熟悉此項 • 技術者可作各種修改及改良。並不意欲將本發明限制於如所說明之特殊形式，且所有不背離本發明之精神及範圍的修改都屬於如隨附之申請專利範圍中所界定之範園内。綜觀上述，本發明以其整體之組合與特徵而言，既未曾見諸於同類產品中，申請前亦未公開，誠已符合專利法之法定要件，依法提出發明專利之申請。【圖式簡單說明】 ® 圖1微脂粒模型；以及圖2 還原抗氧化之圖示。【主要元件符號說明】無 1514 201138837 The lipid-coated CIC2 was taken at 1%, 1.5%, 2°/〇, and its absorbance at 700 nm (nm) was measured using a UV-Vis spectrophotometer. The stronger the reducing power. The results are shown in Fig. 2. The highest absorption value is CIC2 from 2.79 to 2.87>PC+CIC2:2·62~2.85> Vital life c: 2.12~2. 4G>MAP: 1·64~2. 〇〇 It can be known that CIC2 has an antioxidant effect, and the higher the concentration, the more the effect. While the embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by those skilled in the art. The invention is not intended to be limited to the particular forms disclosed, and all modifications may be made without departing from the spirit and scope of the invention. Looking at the above, the present invention, in terms of its overall combination and characteristics, has not been seen in similar products, and has not been disclosed before the application. It has already complied with the statutory requirements of the Patent Law and has filed an application for an invention patent according to law. [Simple description of the diagram] ® Figure 1 shows the microlipid model; and Figure 2 shows the reduction of antioxidants. [Main component symbol description] None 15

Claims

201138837 VII. Patent application scope: l A method for preparing plant stem cell microlipids, which is to coat plant stem cells with the characteristics that nanolipids are easy to penetrate and permeabilize cells. 2. The method for preparing a plant stem cell liposome according to claim 1, wherein the method uses a high-speed homogenization/high pressure homogenization emulsification method. 3. The method for preparing a plant stem cell vesicle according to the invention of claim 2, wherein the plant stem cell microlipid system prepared by the high-speed homogenization/high-pressure homogenization emulsification method is an emulsified liquid. 4. The method for preparing a plant stem cell liposome according to the invention of claim 2, wherein the plant stem cell microlipid diameter prepared by the high-speed homogenization/high pressure homogenization emulsification method is less than 300 nm. 5. The method for producing a plant stem cell liposome according to claim 1, wherein the nanolipid is phospholipid choline. 6. The method for preparing a plant stem cell microlipid according to claim 1, wherein the plant stem cell line is selected from the sea fennel plant stem cell. 7. The sea fennel plant stem cell according to claim 6 of the patent application scope, The stem cell component of the sea fennel plant is CIC2.