技术领域technical field
本发明属于抗菌剂及药物制剂或者化妆品领域，具体涉及一种高稳定性的茶树油纳米脂质体抗菌剂及其制备方法。The invention belongs to the field of antibacterial agents and pharmaceutical preparations or cosmetics, and in particular relates to a highly stable tea tree oil nano liposome antibacterial agent and a preparation method thereof.
背景技术Background technique
茶树油，系唇形科茶树油属植物，常绿灌木，因含有大量天然抗氧化成分而备受人们关注，其中茶树油茎和叶提取得到的挥发油具有很好的抗菌消炎功效；茶树油无色或淡黄色，主要化学成分为桉叶油素、樟脑、龙脑、α-蒎烯、莰烯、乙酸龙脑酯、马鞭草烯酮等，茶树油全草可入药，有健胃发汗功效，精油对金黄色葡萄球菌、大肠杆菌、霍乱弧菌等有较好的抗菌作用，茶树油作为香辛料可广泛地应用于食品工业，而且经研究表明茶树油还是一种高效理想的抗氧化剂。Tea tree oil is a plant of the genus Tea tree oil in the Labiatae family, an evergreen shrub, and has attracted much attention because it contains a large amount of natural antioxidant components. Among them, the volatile oil extracted from the stems and leaves of tea tree oil has good antibacterial and anti-inflammatory effects; tea tree oil has no The main chemical components are eucalyptol, camphor, borneol, α-pinene, camphene, bornyl acetate, verbenone, etc. The whole plant of tea tree oil can be used as medicine, which has the effect of invigorating the stomach and sweating , essential oils have good antibacterial effects on Staphylococcus aureus, Escherichia coli, Vibrio cholerae, etc. Tea tree oil can be widely used in the food industry as a spice, and research shows that tea tree oil is also an efficient and ideal antioxidant.
国内有很多关于茶树油在食品、保健品、医药的专利申请；CN103788494A公开了一种果蔬保鲜薄膜及其制备方法，添加茶树油作为活性成分抑制微生物繁殖及起到抗氧化作用；CN104365822A公开了一种用茶树油保鲜南方葡萄的方法；CN103316158A发明提供了一种包括茶树油的复方精油以达到减肥瘦身效果；CN101506106公开了一种含草药成分的饮用水的配方及方法，配方包括茶树油以稳定成分，从而延长保存期；CN101402864公开了属于高活性抗氧化产品制备技术领域的一种从茶树油中制备抗氧化产品的方法；CN103031210A公开了一种可兼制茶树油的茶树油脱水方法，最大化利用茶树油之良好经济效果；CN102899170A公开了一种采用亚临界流体技术萃取茶树油的方法，适合工业化推广。There are many domestic patent applications about tea tree oil in food, health products, and medicine; CN103788494A discloses a kind of fruit and vegetable fresh-keeping film and its preparation method, adding tea tree oil as an active ingredient to inhibit microbial reproduction and play an antioxidant role; CN104365822A discloses a A method for keeping fresh southern grapes with tea tree oil; CN103316158A invention provides a compound essential oil including tea tree oil to achieve weight loss; CN101506106 discloses a formula and method for drinking water containing herbal ingredients, the formula includes tea tree oil to stabilize composition, thereby prolonging the shelf life; CN101402864 discloses a method for preparing antioxidant products from tea tree oil, which belongs to the technical field of preparation of high-activity antioxidant products; The good economic effect of utilizing tea tree oil; CN102899170A discloses a method for extracting tea tree oil using subcritical fluid technology, which is suitable for industrial promotion.
虽然茶树油在医学，食品和化妆品行业被广泛应用，具有较好的杀菌、增香等特点，但是茶树油易挥发，暴露在空气中不稳定，所以要寻找有效地方法降低茶树油在使用过程中的挥发程度，延长保存期。Although tea tree oil is widely used in medicine, food, and cosmetics, and has good sterilization and aroma enhancement characteristics, tea tree oil is volatile and unstable when exposed to air, so it is necessary to find effective ways to reduce the temperature of tea tree oil during use. The degree of volatility in the medium increases the shelf life.
纳米脂质体能将茶树油包裹在内，并且纳米脂质体不会对茶树油的主要活性成分造成破坏；纳米脂质体使茶树油中的活性成分与外界环境隔绝开来，可以降低茶树油挥发性，延长保存期，但由于脂质体的缓释作用，包裹精油的脂质体保质期受限，所以，选用壳聚糖和明胶制成稳定性高的多层脂质体，此外，纳米脂质体由于它们的亚细胞尺寸，能加强脂质体的被动吸收机制，减少物质运输阻力，从而增强精油的抗菌等效果。Nano-liposomes can encapsulate tea tree oil, and nano-liposomes will not damage the main active ingredients of tea tree oil; nano-liposomes isolate the active ingredients in tea tree oil from the external environment, which can reduce the Volatility, prolong shelf life, but due to the sustained release of liposomes, the shelf life of liposomes encapsulating essential oils is limited, so chitosan and gelatin are used to make multilayer liposomes with high stability. In addition, nano Due to their subcellular size, liposomes can strengthen the passive absorption mechanism of liposomes and reduce the resistance of material transport, thereby enhancing the antibacterial and other effects of essential oils.
发明内容Contents of the invention
本发明的目的是公开一种高稳定的茶树油纳米脂质体及其制备方法，通过将茶树油包裹在多层纳米脂质体中，以实现减少茶树油在使用过程中的挥发，从而减少茶树油的浪费，达到长效抗菌与高效利用的目的。The purpose of the present invention is to disclose a kind of highly stable tea tree oil nano liposome and preparation method thereof, by wrapping tea tree oil in the multilayer nano liposome, to realize reducing the volatilization of tea tree oil in use process, thereby reducing The waste of tea tree oil achieves the purpose of long-acting antibacterial and efficient utilization.
一种高稳定性的茶树油纳米脂质体，茶树油包裹在磷脂双分子层中，其特征在于：磷脂双分子层为第一层纳米脂质体，还设有第二层纳米脂质体，第二层由壳聚糖组成。A highly stable tea tree oil nano-liposome, tea tree oil wrapped in a phospholipid bilayer, characterized in that: the phospholipid bilayer is the first layer of nano-liposomes, and a second layer of nano-liposomes is also provided , the second layer is composed of chitosan.
进一步地，所述的一种高稳定性的茶树油纳米脂质体，其特征在于：还设有第三层纳米脂质体，第三层由明胶组成。Further, the tea tree oil nano-liposome with high stability is characterized in that: a third layer of nano-liposome is also provided, and the third layer is composed of gelatin.
进一步地，第二层纳米脂质体中壳聚糖的浓度为0.2mg/mL。Further, the concentration of chitosan in the second layer of nanoliposomes is 0.2 mg/mL.
进一步地，第三层纳米脂质体中明胶的浓度为0.4mg/mL。Further, the concentration of gelatin in the third layer of nanoliposomes is 0.4 mg/mL.
本发明由茶树油，大豆卵磷脂，胆固醇和表面活性剂，壳聚糖和明胶制成茶树油纳米脂质体。The invention prepares tea tree oil nano liposomes from tea tree oil, soybean lecithin, cholesterol, surfactant, chitosan and gelatin.
大豆卵磷脂、胆固醇是构成脂质体的组要成分，胆固醇均有调节膜流动性的作用，所以考察其两者的配比变化，对构成的脂质体分散性等参数有无影响；表面活性剂是增加脂质体稳定性；茶树油浓度是根据包封率来确定的；醋酸盐溶液的pH是为了最佳溶解壳聚糖；壳聚糖和明胶可以增加稳定性。Soybean lecithin and cholesterol are the main components of liposomes, and cholesterol has the effect of regulating membrane fluidity. Therefore, it is investigated whether the ratio of the two has any effect on the parameters such as the dispersibility of liposomes; the surface The active agent is to increase liposome stability; the tea tree oil concentration is determined according to the encapsulation efficiency; the pH of the acetate solution is for optimal dissolution of chitosan; chitosan and gelatin can increase stability.
本发明的制备方法是将茶树油，大豆卵磷脂，胆固醇混合于有机溶剂，减压蒸干形成光滑的薄膜，加入水相介质和表面活性剂组成的混合溶液溶解薄膜并超声成乳、离心后取上层液体过滤得到单层茶树油纳米脂质体，其特征在于：将单层茶树油纳米脂质体与壳聚糖溶液搅拌均匀，通过离心和微孔滤膜过滤，得到粒径为纳米级的双层茶树油纳米脂质体。The preparation method of the present invention is to mix tea tree oil, soybean lecithin, and cholesterol with an organic solvent, evaporate to dryness under reduced pressure to form a smooth film, add a mixed solution composed of an aqueous medium and a surfactant to dissolve the film, and ultrasonically emulsify and centrifuge Get the upper liquid and filter to obtain the single-layer tea tree oil nano-liposome, which is characterized in that: the single-layer tea tree oil nano-liposome and chitosan solution are stirred evenly, and the obtained particle size is nano-scale by centrifugal and microporous membrane filtration. bilayer tea tree oil nanoliposomes.
进一步地，将双层茶树油纳米脂质体与明胶溶液搅拌均匀，通过离心和微孔滤膜过滤，得到粒径为纳米级的多层茶树油纳米脂质体。Further, the double-layered tea tree oil nanoliposome and the gelatin solution were evenly stirred, and then centrifuged and filtered through a microporous membrane to obtain a multilayered tea tree oil nanoliposome with a nanoscale particle size.
进一步地，本发明的大豆卵磷脂和胆固醇的质量比例为5:1；表面活性剂、茶树油与胆固醇的质量比为：1:4:4，此条件下可以得到最高的包封率。Further, the mass ratio of soybean lecithin and cholesterol of the present invention is 5:1; the mass ratio of surfactant, tea tree oil and cholesterol is 1:4:4, and the highest encapsulation efficiency can be obtained under this condition.
进一步地，表面活性剂为PVP，混合溶液中PVP的浓度为1.0mg/mL。Further, the surfactant is PVP, and the concentration of PVP in the mixed solution is 1.0 mg/mL.
单层茶树油纳米脂质体与壳聚糖溶液的体积比为1:10；双层茶树油纳米脂质体与明胶溶液的体积比为1:10。The volume ratio of the single-layer tea tree oil nano-liposome to the chitosan solution is 1:10; the volume ratio of the double-layer tea tree oil nano-liposome to the gelatin solution is 1:10.
本发明中所述的有机溶剂是氯仿。The organic solvent described in the present invention is chloroform.
本发明中所用的水相介质是根据中国药典2000版标准配制的醋酸盐缓冲溶液，pH值3.5~4.0。The aqueous phase medium used in the present invention is an acetate buffer solution prepared according to the standard of the Chinese Pharmacopoeia 2000 edition, with a pH value of 3.5-4.0.
所述的壳聚糖溶液为壳聚糖的醋酸盐溶液，浓度为0.2mg/mL，醋酸盐溶液是根据中国药典2000版标准配制的醋酸盐缓冲溶液，pH值3.5~4.0，优选3.6，能够最佳溶解壳聚糖。Described chitosan solution is the acetate solution of chitosan, and concentration is 0.2mg/mL, and acetate solution is the acetate buffer solution prepared according to Chinese Pharmacopoeia 2000 edition standard, and pH value is 3.5～4.0, preferably 3.6, can dissolve chitosan optimally.
所述明胶溶液为明胶的醋酸盐溶液，浓度为0.4mg/mL，醋酸盐溶液是根据中国药典2000版标准配制的醋酸盐缓冲溶液，pH值3.5~4.0。The gelatin solution is an acetate solution of gelatin with a concentration of 0.4 mg/mL, and the acetate solution is an acetate buffer solution prepared according to the standards of the Chinese Pharmacopoeia 2000 edition, with a pH value of 3.5-4.0.
本发明中脂质体的第一层是磷脂双分子层，即人工细胞膜层。The first layer of the liposome in the present invention is a phospholipid bilayer, that is, an artificial cell membrane layer.
本发明中脂质体的第二层由壳聚糖组成，浓度为0.2mg/mL。The second layer of the liposome in the present invention is composed of chitosan, and the concentration is 0.2mg/mL.
本发明中脂质体的第三层由明胶组成，浓度为0.4mg/mL。The third layer of the liposome in the present invention is composed of gelatin at a concentration of 0.4 mg/mL.
附图说明Description of drawings
图1为茶树油纳米脂质体的包封率。Fig. 1 is the encapsulation efficiency of tea tree oil nano liposome.
图2为茶树油纳米脂质体的粒径与多分散系数PDI。Fig. 2 is the particle diameter and polydispersity index PDI of tea tree oil nano liposome.
图3为多层茶树油纳米脂质体荧光显微图。Fig. 3 is the fluorescent micrograph of multilayer tea tree oil nano liposome.
图4为多层茶树油纳米脂质体原子力显微图。Figure 4 is an atomic force micrograph of the multilayered tea tree oil nanoliposome.
图5多层茶树油纳米脂质体对大肠杆菌的抗菌性能。Figure 5 Antibacterial properties of multilayered tea tree oil nanoliposomes against Escherichia coli.
图6多层茶树油纳米脂质体对金黄色葡萄球菌的抗菌性能。Figure 6 Antibacterial properties of multilayered tea tree oil nanoliposomes against Staphylococcus aureus.
表1为茶树油纳米脂质体的Zeta电位。Table 1 is the Zeta potential of tea tree oil nano liposomes.
具体实施方式detailed description
通过下面实例说明本发明的具体实施方式，但本发明的保护内容，不仅局限于此The specific embodiment of the present invention is illustrated by the following examples, but the protection content of the present invention is not limited thereto
实施例1多层茶树油纳米脂质体的包封率The encapsulation efficiency of embodiment 1 multilayer tea tree oil nano liposome
1实验材料1 Experimental materials
大豆卵磷脂；BR；国药集团化学试剂有限公司。Soybean lecithin; BR; Sinopharm Chemical Reagent Co., Ltd.
胆固醇；AR；国药集团化学试剂有限公司。Cholesterol; AR; Sinopharm Chemical Reagent Co., Ltd.
三氯甲烷；AR；国药集团化学试剂有限公司。Chloroform; AR; Sinopharm Chemical Reagent Co., Ltd.
茶树油；AR；法国florihana精油。Tea tree oil; AR; French florihana essential oil.
PVP；GR；国药集团化学试剂有限公司。PVP; GR; Sinopharm Chemical Reagent Co., Ltd.
壳聚糖；BR；国药集团化学试剂有限公司。Chitosan; BR; Sinopharm Chemical Reagent Co., Ltd.
明胶；BR；国药集团化学试剂有限公司。Gelatin; BR; Sinopharm Chemical Reagent Co., Ltd.
2实验方法2 Experimental methods
1)单层茶树油纳米脂质体的制备1) Preparation of monolayer tea tree oil nanoliposomes
① 称取1g大豆卵磷脂，0.2g胆固醇和200mg的茶树油，加50mL氯仿使其溶解。① Weigh 1g of soybean lecithin, 0.2g of cholesterol and 200mg of tea tree oil, add 50mL of chloroform to dissolve.
② 在旋转蒸发仪中蒸发至溶剂蒸干，蒸发温度为10~30℃，圆底烧瓶内壁会形成光滑的薄膜；然后将所得产品放入真空干燥箱中，30℃，真空状态下干燥24小时。② Evaporate in a rotary evaporator until the solvent is evaporated to dryness. The evaporation temperature is 10~30°C, and a smooth film will be formed on the inner wall of the round bottom flask; .
③ 称取0.05g的PVP于50mL醋酸盐缓冲液中，超声波条件下扩散，然后将PVP的醋酸盐缓冲液加入圆底烧瓶中超声波条件下进行水化。③ Weigh 0.05g of PVP into 50mL acetate buffer, diffuse under ultrasonic conditions, then add the PVP acetate buffer into a round bottom flask for hydration under ultrasonic conditions.
④ 将水化后的混合液于细胞超微粉碎仪中以工作10s,间隙5s的频率粉碎30min来进一步使脂质体乳状液粒径更均匀细小一些。④Put the hydrated mixture in the cell ultrafine pulverizer for 30min at a frequency of 10s with a gap of 5s to further make the particle size of the liposome emulsion more uniform and finer.
⑤ 将所得产品进行离心，4000rpm，15min，取上层液体。⑤ Centrifuge the obtained product at 4000rpm for 15min, and take the upper layer liquid.
⑥ 将所得液体用0.22μm滤膜进行过滤，得滤液，为单层茶树油纳米脂质体。⑥ Filter the resulting liquid with a 0.22 μm filter membrane to obtain the filtrate, which is a single-layer tea tree oil nanoliposome.
2)双层茶树油纳米脂质体的制备2) Preparation of bilayer tea tree oil nanoliposomes
① 按照上述单层茶树油纳米脂质体的制备方法，制备含200mg茶树油的单层纳米脂质体。① According to the preparation method of the above-mentioned single-layer tea tree oil nano-liposome, prepare the single-layer nano-liposome containing 200mg tea tree oil.
② 将单层茶树油脂质体分散在含0.2mg/mL壳聚糖的醋酸盐溶液中混合均匀；单层茶树油纳米脂质体与壳聚糖的醋酸盐溶液的体积比1：10。② Disperse the monolayer tea tree oil liposomes in the acetate solution containing 0.2mg/mL chitosan and mix evenly; the volume ratio of the monolayer tea tree oil nanoliposomes to the acetate solution of chitosan is 1:10 .
③ 将所得混合液于细胞超微粉碎仪中以工作10s,间隙5s的频率粉碎30min。③ The resulting mixture was pulverized for 30 minutes in a cell ultrafine pulverizer at a frequency of 10 seconds and a gap of 5 seconds.
④ 将所得产品进行离心，4000rpm，15min，取上层液体；④ Centrifuge the obtained product at 4000rpm for 15min, and take the upper liquid;
⑤ 将所得液体用0.22μm滤膜进行过滤，得滤液，为双层茶树油纳米脂质体。⑤ Filter the resulting liquid with a 0.22 μm filter membrane to obtain the filtrate, which is a double-layer tea tree oil nanoliposome.
3)多层茶树油纳米脂质体的制备3) Preparation of multilayered tea tree oil nanoliposomes
① 按照上述双层茶树油纳米脂质体的制备方法，制备含200mg茶树油的双层纳米脂质体。① According to the preparation method of the above-mentioned double-layer tea tree oil nanoliposome, prepare the double-layer nanoliposome containing 200mg tea tree oil.
② 将双层茶树油纳米脂质体分散在含0.4mg/mL明胶的醋酸盐溶液中使其混合均匀；双层茶树油纳米脂质体与明胶的醋酸盐溶液的体积比为1：10。② Disperse the double-layer tea tree oil nanoliposome in the acetate solution containing 0.4mg/mL gelatin to make it evenly mixed; the volume ratio of the double-layer tea tree oil nanoliposome to the acetate solution of gelatin is 1: 10.
③ 将所得混合液于细胞超微粉碎仪中以工作10s,间隙5s的频率粉碎30min；③ Pulverize the obtained mixed solution in the cell ultrafine pulverizer for 30 minutes at a frequency of 10 seconds and a gap of 5 seconds;
④ 将所得产品进行离心，4000rpm，15min，取上层液体；④ Centrifuge the obtained product at 4000rpm for 15min, and take the upper liquid;
⑤ 将所得液体用0.22μm滤膜进行过滤，得滤液，为多层茶树油纳米脂质体。⑤ Filter the resulting liquid with a 0.22 μm filter membrane to obtain the filtrate, which is multilayered tea tree oil nanoliposomes.
4）包封率的测定4) Determination of Encapsulation Efficiency
用无水乙醇稀释茶树油，逐级稀释成浓度分别为0.1、0.2、0.4、0.6、0.8mg/mL的标准溶液。然后，分别吸取1μL标准溶液进行GC-MS分析，对其主要成分1,8-桉叶素的谱峰面积进行自动积分，绘制1,8-桉叶素峰面积-茶树油浓度标准曲线。首先取1mL茶树油纳米脂质体样品，13500rpm，离心3h，倒掉上清液。接着加入1mL乙醇破乳剂，超声3h，最后以10000rpm的转速离心15min，取上清液，用于GC-MS分析；制备好的脂质体样品，对精油主成分的峰面进行自动积分，再根据步骤1中绘制的标准曲线，计算出脂质体中植物精油的含量，则：Dilute tea tree oil with absolute ethanol, and gradually dilute into standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, and 0.8 mg/mL. Then, draw 1 μL of the standard solution for GC-MS analysis, automatically integrate the peak area of its main component 1,8-cineole, and draw the standard curve of 1,8-cineole peak area-tea tree oil concentration. First take 1mL tea tree oil nano liposome sample, centrifuge at 13500rpm for 3h, and pour off the supernatant. Then add 1mL ethanol demulsifier, sonicate for 3h, and finally centrifuge at 10000rpm for 15min, take the supernatant for GC-MS analysis; According to the standard curve drawn in step 1, calculate the content of plant essential oil in the liposome, then:
。 .
3茶树油纳米脂质体的包封率3 Encapsulation efficiency of tea tree oil nanoliposomes
包封率是评价脂质体制剂质量好坏的最重要的指标，也是脂质体能否发挥较普通制剂高效、低毒等特点的关键；由图1可看出，单层茶树油纳米脂质体的包封率为20.7%，双层茶树油纳米脂质体的包封率为37.3%，多层茶树油纳米脂质体的包封率最大，为78.9%。因此制备多层茶树油纳米脂质体可以明显提高脂质体的包封率。Encapsulation efficiency is the most important indicator of evaluating the quality of liposome preparations, and it is also the key to whether liposomes can bring into play the characteristics of high efficiency and low toxicity compared with common preparations; as can be seen from Figure 1, the monolayer tea tree oil nano-lipid The encapsulation efficiency of the bilayer tea tree oil nanoliposome was 20.7%, the encapsulation efficiency of the bilayer tea tree oil nanoliposome was 37.3%, and the encapsulation efficiency of the multilayer tea tree oil nanoliposome was the largest, which was 78.9%. Therefore, the preparation of multilayered tea tree oil nanoliposomes can significantly improve the encapsulation efficiency of liposomes.
实施例2多层茶树油纳米脂质体的粒径与多分散系数PDIParticle diameter and polydispersity index PDI of embodiment 2 multilayered tea tree oil nano liposomes
1实验材料1 Experimental materials
① 单层茶树油纳米脂质体。① Monolayer tea tree oil nanoliposomes.
② 双层茶树油纳米脂质体。② Double layer tea tree oil nano liposome.
③ 多层茶树油纳米脂质体。③ Multi-layer tea tree oil nano liposome.
2实验方法2 Experimental methods
用美国布鲁克海文仪器公司产的，型号为BI-9000的高浓度激光粒度仪测定茶树油纳米脂质体的粒径和多分散系数PDI值，所测样品放入样品池直接测量即可。The particle size and the polydispersity index PDI value of the tea tree oil nanoliposomes were measured with a high-concentration laser particle size analyzer model BI-9000 produced by Brookhaven Instruments in the United States, and the measured samples were put into the sample cell for direct measurement.
3茶树油纳米脂质体的粒径与多分散系数PDI3 Particle size and polydispersity index PDI of tea tree oil nanoliposomes
多分散系数PDI直接反映茶树油纳米脂质体的稳定性，因此是主要参考指标，脂质体的PDI在0~0.3范围内属于最好，在0.3~0.7范围内较差，但可以接受，当PDI>0.8时，不予考虑。如图2所示，单层茶树油纳米脂质体的粒径为176.4nm、PDI为0.353，双层茶树油纳米脂质体的粒径为243.5nm、PDI为0.307，多层茶树油纳米脂质体的粒径为294.8nm、PDI为0.214。多层茶树油纳米脂质体的多分散系数PDI最小，因此制备多层茶树油纳米脂质体可以明显提高脂质体的稳定性。The polydispersity index PDI directly reflects the stability of tea tree oil nano-liposomes, so it is the main reference index. The PDI of liposomes is the best in the range of 0-0.3, and it is poor in the range of 0.3-0.7, but it is acceptable. When PDI>0.8, it will not be considered. As shown in Figure 2, the particle diameter of monolayer tea tree oil nanoliposome is 176.4nm, PDI is 0.353, the particle diameter of bilayer tea tree oil nanoliposome is 243.5nm, PDI is 0.307, and multilayer tea tree oil nanoliposome The particle diameter of the plastid was 294.8 nm, and the PDI was 0.214. The polydispersity index PDI of multilayered tea tree oil nanoliposomes is the smallest, so the preparation of multilayered tea tree oil nanoliposomes can obviously improve the stability of liposomes.
实施例3茶树油纳米脂质体的Zeta电位The Zeta potential of embodiment 3 tea tree oil nano liposomes
1实验材料1 Experimental materials
① 单层茶树油纳米脂质体。① Monolayer tea tree oil nanoliposomes.
② 双层茶树油纳米脂质体。② Double layer tea tree oil nano liposome.
③ 多层茶树油纳米脂质体。③ Multi-layer tea tree oil nano liposome.
2实验方法2 Experimental methods
用英国马尔文仪器有限公司生产的型号为ZetasirernanoZSZeta的电位仪测量，直接将待测脂质体样品放入电位仪中测量即可。It is measured by a potentiometer ZetasirernanoZSZeta produced by Malvern Instrument Co., Ltd. in the United Kingdom, and the liposome sample to be tested is directly put into the potentiometer for measurement.
3茶树油纳米脂质体的Zeta电位3 Zeta potential of tea tree oil nanoliposomes
表1茶树油纳米脂质体的Zeta电位The Zeta potential of table 1 tea tree oil nano liposome
Zeta电位也可以直接反映茶树油纳米脂质体的稳定性，因此也是主要参考指标，脂质体的Zeta电位的绝对值越大说明脂质体越稳定性，Zeta电位的绝对值在0~30范围内属于不稳定，在大于30时脂质体较稳定性。如表1所示，三种茶树油纳米脂质体均带负电，单层茶树油纳米脂质体的Zeta电位为-23.7mV，其绝对值小于30脂质体不稳定，双层茶树油纳米脂质体的Zeta电位为-32.1mV，其绝对值大于30脂质体较稳定，多层茶树油纳米脂质体的Zeta电位为-44.2mV，其绝对值最大，脂质体最稳定。Zeta potential can also directly reflect the stability of tea tree oil nano-liposomes, so it is also the main reference index. The larger the absolute value of Zeta potential of liposomes, the more stable the liposomes are. The absolute value of Zeta potential is between 0 and 30 It is unstable within the range, and the liposome is more stable when it is greater than 30. As shown in Table 1, three kinds of tea tree oil nano-liposomes are all negatively charged, and the Zeta potential of single-layer tea tree oil nano-liposome is-23.7mV, and its absolute value is less than 30. The liposome is unstable, and the double-layer tea tree oil nano-liposome The Zeta potential of the liposome is -32.1mV, and its absolute value is greater than 30. The liposome is more stable. The Zeta potential of the multilayer tea tree oil nano liposome is -44.2mV, and its absolute value is the largest, and the liposome is the most stable.
实施例4多层茶树油纳米脂质体的荧光显微镜观察The fluorescence microscope observation of embodiment 4 multilayer tea tree oil nano liposomes
1实验材料1 Experimental materials
多层茶树油纳米脂质体。Multilamellar tea tree oil nanoliposomes.
2实验方法2 Experimental methods
用德国徕卡仪器公司生产的型号为TCS-SP5的荧光显微镜，直接将待测脂质体样品放入荧光显微镜中观察。The liposome sample to be tested was directly put into the fluorescence microscope for observation with a model TCS-SP5 fluorescence microscope produced by Germany Leica Instruments.
荧光显微镜样品前处理方法：Fluorescence microscope sample pretreatment method:
（1）多层茶树油纳米脂质体样品的制备（A液）：取1mL茶树油脂质体，0.5mL甲醇和0.5mL氯仿混合。(1) Preparation of multilayer tea tree oil nanoliposome sample (solution A): Take 1mL tea tree oil liposome, mix with 0.5mL methanol and 0.5mL chloroform.
（2）荧光染料DIL的制备（B液）：0.1mL的DIL溶于0.1mL的二氯甲烷中。(2) Preparation of fluorescent dye DIL (solution B): 0.1 mL of DIL was dissolved in 0.1 mL of dichloromethane.
（3）取A液0.5mL和B液50μL放入小离心管混合，震荡均匀。(3) Take 0.5mL of solution A and 50μL of solution B into a small centrifuge tube to mix and shake evenly.
（4）将上述混合液体放入真空干燥箱，干燥一夜。(4) Put the above mixed liquid into a vacuum drying oven and dry overnight.
（5）取已干燥的离心管，加入0.5mL超纯水，在振荡器上震荡30min。(5) Take the dried centrifuge tube, add 0.5mL ultrapure water, and shake on the shaker for 30min.
（6）室温放置3h。(6) Place at room temperature for 3 hours.
（7）滴在载玻片上进行观察。(7) Drop on a glass slide for observation.
3多层茶树油纳米脂质体的荧光显微镜观察3 Fluorescence microscope observation of multilayered tea tree oil nanoliposomes
由以上荧光显微镜拍摄到的显微照片可以看出，脂质体染色后，呈现圆形，分散较均匀。It can be seen from the microphotographs taken by the above fluorescent microscope that after staining, the liposomes are round and dispersed evenly.
实施例5多层茶树油纳米脂质体的原子力显微镜观察The atomic force microscope observation of embodiment 5 multilayer tea tree oil nano liposomes
1实验材料1 Experimental materials
多层茶树油纳米脂质体。Multilamellar tea tree oil nanoliposomes.
2实验方法2 Experimental methods
用美国安捷伦科技公司生产的型号为Agilent5500的原子力显微镜，直接将待测脂质体样品放入原子力显微镜中观察。原子力前处理方法是取植物精油脂质体样品10μL滴在云母片上10min。然后用移液枪吸除表面的液体，再滴上10μL超纯水30s，重复清洗3次，通风处静置3h，放置于原子力显微镜下观察。With the atomic force microscope model Agilent5500 produced by American Agilent Technologies, directly put the liposome sample to be tested into the atomic force microscope for observation. The method of atomic force pretreatment is to take 10 μL of plant essential oil liposome sample and drop it on the mica sheet for 10 minutes. Then use a pipette to suck off the liquid on the surface, then drip 10 μL of ultrapure water for 30 seconds, repeat the cleaning 3 times, let it stand in a ventilated place for 3 hours, and place it under an atomic force microscope for observation.
3多层茶树油纳米脂质体的原子力显微镜观察3 Atomic Force Microscopy Observation of Multilayered Tea Tree Oil Nanoliposomes
由以上原子力显微镜拍摄到的显微照片可以看出，脂质体呈现圆形，分散较均匀。It can be seen from the microphotographs taken by the above atomic force microscope that the liposomes are round and dispersed evenly.
实施例6多层茶树油纳米脂质体的抗菌性能The antibacterial property of embodiment 6 multilayer tea tree oil nano liposomes
1实验材料1 Experimental materials
① 单层茶树油纳米脂质体（保存7天、30天、60天、90天）。① Monolayer tea tree oil nanoliposomes (storage for 7 days, 30 days, 60 days, 90 days).
② 双层茶树油纳米脂质体（保存7天、30天、60天、90天）。② Double-layer tea tree oil nanoliposomes (storage for 7 days, 30 days, 60 days, 90 days).
③ 多层茶树油纳米脂质体（保存7天、30天、60天、90天）。③ Multi-layer tea tree oil nano-liposomes (storage for 7 days, 30 days, 60 days, 90 days).
2实验方法2 Experimental methods
采用平板菌落计数法，以大肠杆菌(Escherichiacoli)和金黄色葡萄球菌(Staphylococcusaureus)为模式菌测定茶树油纳米脂质体的残存菌数，将大肠杆菌和金黄色葡萄球菌接种到液体培养基中，分别置于气浴摇床中在37℃、150rpm条件下震荡培养24~48h，获得对数生长期的细菌，取适量处于对数期的大肠杆菌和金黄色葡萄球菌分别加入含有一定量无菌磷酸缓冲液的试管中(菌浓度约为105~106cfu/mL），然后再向试管中加入浓度为10%的各种茶树油纳米脂质体，同时另取两个分别含有以上两种菌的试管并向其中加入等量无菌水(不加茶树油纳米脂质体)作为对照，将各试管均置于气浴摇床中在37℃、150rpm条件下震荡反应24h，分别于不同时间点的取适量培养液进行十倍梯度稀释到合适的浓度，然后移取100μL稀释液滴到无菌固体平板培养基上，涂布均匀，之后放入37℃恒温恒湿培养箱中倒置培养，24~48h后进行平板菌落计数，从而对评价各茶树油纳米脂质体的抗菌活性，做三次重复，结果取平均值。Adopt plate colony counting method, take Escherichia coli (Escherichiacoli ) and Staphylococcus aureus (Staphylococcus aureus) as model bacteria to measure the residual bacterial count of tea tree oil nanoliposome, Escherichia coli and Staphylococcus aureus are inoculated in the liquid medium, Place them in an air-bath shaker at 37°C and 150rpm for 24-48 hours of shaking culture to obtain bacteria in the logarithmic growth phase. Take appropriate amounts of Escherichia coli and Staphylococcus aureus in the logarithmic phase and add a certain amount of sterile phosphate buffer solution (bacterial concentration is about 105 ~106 cfu/mL), and then add various tea tree oil nanoliposomes with a concentration of 10% to the test tube, and take another two containing the above two Inoculate the test tube and add the same amount of sterile water (without adding tea tree oil nano liposomes) therein as a control, place each test tube in an air bath shaker at 37°C and 150rpm for a shaking reaction for 24h, respectively Take an appropriate amount of culture solution at different time points for ten-fold gradient dilution to a suitable concentration, then pipette 100 μL of the diluted solution onto a sterile solid plate medium, spread evenly, and then place it upside down in a constant temperature and humidity incubator at 37°C Cultivate, carry out plate colony count after 24～48h, thereby to evaluate the antibacterial activity of each tea tree oil nano liposome, do three repetitions, the result takes the average value.
3多层茶树油纳米脂质体的抗菌性能3 Antibacterial properties of multilayered tea tree oil nanoliposomes
不同保存期的茶树油纳米脂质体的抗菌活性的变化也可以间接反映脂质体的稳定性，因此对保存7天、30天、60天、90天的各种茶树油纳米脂质体进行了抗菌性能评价，结果如图5、图6所示；保存时间为7天时，单层茶树油纳米脂质体、双层茶树油纳米脂质体、多层茶树油纳米脂质体的抗菌活性均相同，对大肠杆菌和金黄色葡萄球菌均显示了良好的抗菌效果；保存时间为30天时，单层茶树油纳米脂质体的抗菌效果明显降低、而双层茶树油纳米脂质体和多层茶树油纳米脂质体均显示良好的抗菌效果；保存时间为60天和90天时，单层茶树油纳米脂质体没有显示抗菌效果，双层茶树油纳米脂质体的抗菌效果明显降低，而多层茶树油纳米脂质体一直保持良好的抗菌效果。The change of the antibacterial activity of the tea tree oil nano-liposomes of different storage periods can also reflect the stability of liposomes indirectly, so the various tea tree oil nano-liposomes stored for 7 days, 30 days, 60 days, and 90 days were tested. Antibacterial performance evaluation, the results as shown in Figure 5 and Figure 6; when the storage time was 7 days, the antibacterial activity of single-layer tea tree oil nanoliposomes, double-layer tea tree oil nanoliposomes, and multilayer tea tree oil nanoliposomes Both showed good antibacterial effect on Escherichia coli and Staphylococcus aureus; when the storage time was 30 days, the antibacterial effect of single-layer tea tree oil nanoliposomes was significantly reduced, while double-layer tea tree oil nanoliposomes and multilayer Layered tea tree oil nanoliposomes all showed good antibacterial effect; when the storage time was 60 days and 90 days, single layer tea tree oil nanoliposomes did not show antibacterial effect, and the antibacterial effect of double layer tea tree oil nanoliposomes was significantly reduced. The multilayered tea tree oil nanoliposomes have always maintained a good antibacterial effect.
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