WO2024027113A1 - 一种灵芝提取物纳米乳及其制备方法和应用 - Google Patents

一种灵芝提取物纳米乳及其制备方法和应用 Download PDF

Info

Publication number
WO2024027113A1
WO2024027113A1 PCT/CN2023/072380 CN2023072380W WO2024027113A1 WO 2024027113 A1 WO2024027113 A1 WO 2024027113A1 CN 2023072380 W CN2023072380 W CN 2023072380W WO 2024027113 A1 WO2024027113 A1 WO 2024027113A1
Authority
WO
WIPO (PCT)
Prior art keywords
ganoderma lucidum
nanoemulsion
surfactant
lucidum extract
extract nanoemulsion
Prior art date
Application number
PCT/CN2023/072380
Other languages
English (en)
French (fr)
Inventor
彭灿
张静
鲁嘉
吴梦飞
方灿灿
段效晖
桂双英
彭代银
Original Assignee
安徽中医药大学
安徽百草精研医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 安徽中医药大学, 安徽百草精研医药科技有限公司 filed Critical 安徽中医药大学
Publication of WO2024027113A1 publication Critical patent/WO2024027113A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/06—Emulsions
    • A61K8/062—Oil-in-water emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/345—Alcohols containing more than one hydroxy group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/41—Particular ingredients further characterized by their size
    • A61K2800/413—Nanosized, i.e. having sizes below 100 nm
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/596—Mixtures of surface active compounds

Definitions

  • the invention relates to the technical field of cosmetics, and in particular to a Ganoderma lucidum extract nanoemulsion and its preparation method and application.
  • Ganoderma lucidum is the dried fruiting body of Ganoderma lucid-um or Ganoderma sinense (Ganoderma sinense Zhao, Xu et Zhang) of the Polyporaceae family. According to records, Ganoderma lucidum has the effects of nourishing and strengthening, prolonging life, and uprightness and spirituality. Modern medical research and experiments have further proved that Ganoderma lucidum has antioxidant and anti-aging functions. Research shows that Ganoderma lucidum polysaccharides and Ganoderma lucidum triterpenes are the main active ingredients of Ganoderma lucidum and are closely related to the above-mentioned medicinal functions.
  • Nanoemulsion is a stable system formed spontaneously from oil, water, surfactant (SA) and co-surfactant (CoSA).
  • SA surfactant
  • CoSA co-surfactant
  • the particle size ranges from 1 to 100nm. It is clear and transparent and can produce the Tyndall effect. There are also some systems that will Glowing blue opalescence.
  • nanoemulsion can increase the solubility of drugs, extend the drug's action time, and increase its bioavailability by virtue of its own characteristics.
  • the primary purpose of the present invention is to provide a Ganoderma lucidum extract nanoemulsion, which is spherical in shape, evenly distributed, and has good stability.
  • a kind of Ganoderma lucidum extract nanoemulsion consisting of Ganoderma lucidum triterpene, Ganoderma lucidum polysaccharide, surfactant, co-surfactant, oil phase and water, wherein the mass ratio of the surfactant to the co-surfactant is 1:2 ⁇ 2:1, the mass ratio of the total mass of surfactant and co-surfactant to the oil phase is 4:6 ⁇ 10:1.
  • the surfactant is one or more of polysorbate-80 (Tween 80), castor oil polyoxyethylene ether 40 (EL 40) or polyoxyethylene castor oil 35 (EL 35), and more Polyoxyethylene castor oil 35 (EL 35) is preferred.
  • the co-surfactant is one or more of anhydrous ethanol, glycerol or propylene glycol, more preferably glycerol.
  • the oil phase is one or a mixture of two of medium chain triglyceride (MCT) or isopropyl myristate (IPM), and more preferably medium chain triglyceride (MCT).
  • MCT medium chain triglyceride
  • IPM isopropyl myristate
  • MCT medium chain triglyceride
  • the Ganoderma triterpenes and Ganoderma polysaccharides described in the present invention can be obtained commercially, or can be prepared by conventional methods in this field.
  • the average particle size of the Ganoderma lucidum extract nanoemulsion prepared by the present invention is 28.01nm, the PDI is 0.105, and the average Zeta potential is -4.05mV.
  • the maximum drug loading capacity of Ganoderma lucidum polysaccharide in the Ganoderma lucidum extract nanoemulsion is 36.35mg/mL.
  • the maximum drug loading capacity of triterpenes is 5.99 mg/mL.
  • the present invention also provides a preparation method of the above-mentioned Ganoderma lucidum extract nanoemulsion, which includes the following steps:
  • steps (a)-(c) are performed in a water bath at 50-60°C and a magnetic stirring speed of 1000-1500 r/min.
  • the third aspect of the present invention provides the application of the above Ganoderma lucidum extract nanoemulsion in external skin care preparations.
  • the present invention combines Ganoderma lucidum extract and nanoemulsion to prepare an external skin care preparation with anti-aging and antioxidant effects. It can increase the solubility of the active ingredients of Ganoderma lucidum, improve the utilization value of Ganoderma lucidum, and promote the transdermal absorption of its active ingredients to achieve beauty and anti-aging effects due to the unique advantages of nanoemulsion preparations.
  • the external skin care preparation is essence, lotion, cream, facial mask, lotion or spray.
  • the present invention has the following beneficial effects:
  • the present invention prepares an O/W type Ganoderma lucidum extract nanoemulsion by screening the types and amounts of each component in the formula.
  • the nanoemulsion is spherical, stable in nature, evenly distributed, and relatively fluid. good;
  • the Ganoderma lucidum extract nanoemulsion prepared by the present invention significantly improves the solubility of Ganoderma lucidum triterpenes and Ganoderma lucidum polysaccharides, which helps to increase the drug loading capacity of the system and the utilization rate of its active ingredients;
  • the present invention combines Ganoderma lucidum extract with nanoemulsion, which is suitable for application in the field of cosmetics. It can produce an external skin care preparation with anti-aging and antioxidant effects, which can not only increase the solubility of Ganoderma lucidum's active ingredients, but also improve the efficacy of Ganoderma lucidum. Utilization value, and because of the unique advantages of nanoemulsion preparations, it can promote the transdermal absorption of its active ingredients to achieve beauty and anti-aging effects, which provides a certain experimental basis for the subsequent process production and theoretical research of Ganoderma lucidum extract nanoemulsion cosmetics.
  • Figure 1 shows the pseudo-ternary phase diagram of different surfactants SA
  • Figure 2 shows the pseudo-ternary phase diagram of different km values of surfactant SA and co-surfactant
  • Figure 3 is a pseudo-ternary phase diagram of different oil phases
  • Figure 4 is the standard curve of Ganoderma lucidum polysaccharide
  • Figure 5 is the standard curve of Ganoderma lucidum triterpenes
  • Figure 6 shows the appearance inspection of Ganoderma lucidum extract nanoemulsion
  • Figure 7 shows the Tyndall effect of Ganoderma lucidum extract nanoemulsion
  • Figure 8 shows the type identification of Ganoderma lucidum extract nanoemulsion
  • Figure 9 shows the morphological distribution of Ganoderma lucidum extract nanoemulsion under a transmission electron microscope.
  • the material-liquid ratio is 1:30 (W/V), and the total polysaccharide solution of Ganoderma lucidum fruiting bodies can be obtained by extracting for 2 hours at 70°C. Concentrate by rotary evaporation, add ethanol to the extract until the alcohol content is 80%, stir evenly, place at 4°C for 24 hours, centrifuge and pour off the supernatant, and evaporate the precipitated ethanol to obtain crude polysaccharide solids of the fruiting body.
  • HLB HLB A *A%+HLB B *B%
  • the present invention preselects Tween80, RH 40, EL40, and EL35 as candidate SAs, with HLB values in the range of 13-16, and glycerol is selected as CoSA.
  • Tween80, RH 40, EL40, EL 35 and glycerin at 1:1, 1:2, and 2:1 respectively, and perform a centrifugation experiment. Set the speed to 5000r/min and the time to 15min to record the phenomenon.
  • the mixed solvent of RH40 and glycerin is milky white and has a high viscosity under different conditions.
  • the mixed solvents of Tween80, EL 40, EL 35 and glycerin are mostly clear and transparent, with low viscosity and high viscosity under various conditions. There is no stratification, so Tween80, EL 40, and EL 35 are selected as candidate SAs for the next experimental operation.
  • Tween80 and EL40 are not easy to emulsion in the high oil phase area, and the emulsion area area from large to small is: EL35>EL40>Tween80, EL 35 has the largest emulsion area, so EL 35 is preferably nanometer Milk SA.
  • CoSAs in nanoemulsions include anhydrous ethanol, glycerin, propylene glycol, etc.
  • the fat-soluble Ganoderma triterpene compound used in the present invention is extracted from anhydrous ethanol.
  • anhydrous ethanol has a certain irritation to the skin, it should not be used as In external skin care products, glycerin is harmless to the skin, is compatible with many substances, and has water-absorbing, moisturizing, and antioxidant effects. Therefore, it can be used as a solvent, emollient, and moisturizer in various skin care products.
  • glycerol was selected as CoSA in this experiment.
  • mix SA and CoSA according to Km 1:1, 1:2, 2:1, and then mix S mix and oil phase at a mass ratio of 1 :9 ⁇ 9:1 ratio, add the water phase drop by drop under stirring, observe and record the critical point when the emulsion changes from turbid to clear or from clear to turbid, and draw a pseudo-ternary phase diagram ( Figure 2).
  • MCT medium chain triglyceride
  • IPM isopropyl myristate
  • olive oil is a vegetable oil, which is more common in external cosmetics and has natural beauty and skin care effects.
  • the present invention preselects the above three oil phases as candidate oil phases. Since Ganoderma lucidum triterpenes are fat-soluble compounds, Ganoderma lucidum triterpenoid extracts were dissolved in each oil phase to examine the solubility and stability of Ganoderma lucidum triterpenes in each oil phase. Excess Ganoderma lucidum triterpenes were added to the above three oils respectively, and then each mixture was vortexed using a vortexer to accelerate the uniform mixing of the medicine and various solvents. Vortex for 30 seconds every 5 minutes and observe the dissolution within 30 minutes until the drug is no longer dissolved and some solids precipitate after standing.
  • the optimal formula for this nanoemulsion is to select EL 35 as SA, glycerin as CoSA, km value as 2:1, MCT as the oil phase, and distilled water as the water phase. And through the emulsion-forming area of the pseudo-ternary phase diagram, the most stable nanoemulsion formula ratio is: EL 35: 30%, glycerol: 14.4%, MCT: 11.1%, water: 44.5%.
  • Example 5 Quality evaluation of Ganoderma lucidum extract nanoemulsion (Example 1)
  • the prepared Ganoderma lucidum extract nanoemulsion was dissolved in methanol for demulsification, and the absorbance values of Ganoderma lucidum polysaccharide and triterpene solution in the nanoemulsion were measured at 626nm and 546nm using UV spectrophotometry, and compared with the standard curve, the nanoemulsion was obtained.
  • Drug-loaded content of Ganoderma lucidum polysaccharides and triterpenes in milk were obtained.
  • the results show that the solubility of Ganoderma triterpenes in the oil phase is 1.70 mg/ml, the solubility of Ganoderma polysaccharides in water is 21.72 mg/ml, and the solubilities of Ganoderma triterpenes and Ganoderma polysaccharides in nanoemulsion are 5.99 mg/ml and 36.35, respectively. mg/ml.
  • Experimental results show that nanoemulsion significantly improves the solubility of Ganoderma lucidum triterpenes and Ganoderma lucidum polysaccharides, helping to increase the drug loading capacity of the system and the utilization rate of its active ingredients.
  • Ganoderma extract nanoemulsion with a polysaccharide content of 6.00 mg/ml and a triterpene content of 5.99 mg/ml was prepared for subsequent experiments.
  • the prepared blank nanoemulsion and Ganoderma lucidum extract nanoemulsion were placed in containers respectively, as shown in Figure 6.
  • the appearance of the prepared blank nanoemulsion was a clear and transparent emulsion with light blue opalescence (A ); the nanoemulsion after adding Ganoderma lucidum triterpene is a light yellow clear emulsion (B); the solution after adding Ganoderma lucidum triterpene and Ganoderma lucidum polysaccharide is a dark brown clear emulsion (C).
  • the Tyndall effect refers to the phenomenon that colloidal emulsions with particle sizes ranging from 1 to 100 nm produce a visible "light band" under concentrated light irradiation. Therefore, when the laser is irradiated from the side, the above-mentioned nanoemulsions produce a relatively obvious Tyndall effect, which proves that the nanoemulsions prepared in this experiment meet the nanometer-level particle size requirements. As shown in Figure 7.
  • the identification methods of nanoemulsion types include dilution method, staining method and conductivity method.
  • the dyeing method was chosen for this experiment because of its simple operation and obvious phenomena.
  • the identification reagent is a methylene blue solution, because the methylene blue solution is water-soluble and will diffuse rapidly in O/W emulsions. There are obvious phenomena that are easy to observe, but it cannot be observed in W/O emulsions. Take the nanoemulsion prepared in this experiment and put it into the EP tube. Drop an appropriate amount of methylene blue solution into the EP tube and observe and record the phenomenon (Figure 8).
  • the average particle size of the nanoemulsion is 28.01nm, and the PDI is 0.105. That is, the overall particle size of the prepared nanoemulsion is uniform, the distribution is uniform, the system is stable, and it conforms to the standard particle size range of nanoemulsion.
  • the zeta potential is -4.05mV. Zeta potential refers to the potential difference between the continuous phase and the fluid stable layer attached to the dispersed particles. The greater the absolute value, the more stable the system. From the zeta potential results of the nanoemulsion obtained in this experiment, it can be seen that the electrostatic repulsive force of the nanoemulsion particles is relatively large, that is, the entire nanoemulsion system is relatively stable.
  • the dilution stability of the nanoemulsion prepared in this experiment was investigated. After the Ganoderma lucidum extract nanoemulsion was diluted 50 times, 100 times, 500 times, and 1000 times with pure water, it was observed that there was no stratification, demulsification, flocculation, etc. Since the condition of infinite dilution can be achieved by diluting the nanoemulsion 500 times, diluting the nanoemulsion prepared in this experiment will not affect its stability and can be diluted infinitely. At the same time, it was further proved that the structural type of Ganoderma lucidum extract nanoemulsion is O/W type nanoemulsion.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Emergency Medicine (AREA)
  • Mycology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Dispersion Chemistry (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Dermatology (AREA)
  • Cosmetics (AREA)

Abstract

一种灵芝提取物纳米乳及其制备方法和应用，纳米乳由灵芝三萜、灵芝多糖、表面活性剂、助表面活性剂、油相和水组成，其中，表面活性剂与助表面活性剂的质量比为1:2-2:1，表面活性剂和助表面活性剂的总质量与油相的质量比为4:6-10:1。通过组方中各组分的种类及用量的筛选，制备得到一种O/W型灵芝提取物纳米乳，显著提高灵芝三萜、灵芝多糖的溶解度，适用于化妆品领域。

Description

一种灵芝提取物纳米乳及其制备方法和应用 技术领域
本发明涉及化妆品技术领域，具体涉及一种灵芝提取物纳米乳及其制备方法和应用。
背景技术
灵芝为多孔菌科真菌赤芝(Ganoderma lucid-um)或紫芝(Ganoderma sinense Zhao，Xu et Zhang)的干燥子实体，据记载灵芝具有滋补强壮、延年益寿、正气修神等功效。现代医学研究与实验也进一步佐证了灵芝具有抗氧化、抗衰老的功能。研究表明，灵芝多糖与灵芝三萜为灵芝的主要有效成分，并且与上述药效功能密切相关。
纳米乳是由油、水、表面活性剂(SA)与助表面活性剂(CoSA)等自发形成，粒径在1～100nm范围内的稳定体系，澄清透明能产生丁达尔效应，也有一些体系会泛着蓝色乳光。纳米乳作为一种新型的载药系统，凭借其自身特点，可以增加药物的溶解度，延长药物作用时间，也可以增大其生物利用度。
目前对于灵芝提取物的研究多在于灵芝提取物口服制剂的制备，功效主要为免疫调节及治疗、预防肿瘤等，尚未发现将灵芝提取物制备成外用型纳米乳的文献报道。
发明内容
鉴于此，本发明的首要目的在于提供一种灵芝提取物纳米乳，该灵芝提取物纳米乳呈类球型，分布均匀，具有较好的稳定性。
本发明是通过以下技术方案实现的：
一种灵芝提取物纳米乳，由灵芝三萜、灵芝多糖、表面活性剂、助表面活性剂、油相和水组成，其中，所述表面活性剂与助表面活性剂的质量比为1:2～2:1，表面活性剂和助表面活性剂的总质量与油相的质量比为4:6～10:1。
优选地，所述表面活性剂为聚山梨酯-80(Tween 80)、蓖麻油聚氧乙烯醚40(EL 40)或聚氧乙烯蓖麻油35(EL 35)中的一种或几种，更优选为聚氧乙烯蓖麻油35(EL 35)。
优选地，所述助表面活性剂为无水乙醇、甘油或丙二醇中的一种或几种，更优选为甘油。
优选地，所述油相为中链甘油三酯(MCT)或肉豆蔻酸异丙酯(IPM)中的一种或两种的混合，更优选为中链甘油三酯(MCT)。
本发明所述的灵芝三萜和灵芝多糖可通过市购方式获得，也可以通过本领域常规方法制备得到。
本发明制备得到的灵芝提取物纳米乳的平均粒径为28.01nm，PDI为0.105，平均Zeta电位为-4.05mV，灵芝提取物纳米乳中灵芝多糖的最大载药量为36.35mg/mL，灵芝三萜的最大载药量为5.99mg/mL。
本发明另一方面还提供了上述灵芝提取物纳米乳的制备方法，包括以下步骤：
a、将表面活性剂和助表面活性剂混合均匀，得到混表面活性剂，将混表面活性剂和油相混合均匀，得到混合体系；
b、在搅拌状态下，在混合体系中滴加水，搅拌至澄清即得空白纳米乳；
c、将灵芝三萜加入空白纳米乳中，静置，离心，得上清液，将灵芝多糖溶解于上清液，充分搅拌均匀至完全溶解，即得灵芝提取物纳米乳。
优选地，步骤(a)-(c)在50-60℃的水浴、磁力搅拌转速为1000-1500r/min条件下进行。
本发明第三方面提供了上述灵芝提取物纳米乳在外用护肤制剂中的应用，本发明将灵芝提取物与纳米乳结合，可制得一种具有抗衰老、抗氧化作用的外用护肤制剂，既能增加灵芝有效成分的溶解度，提高灵芝的利用价值，又能因为纳米乳制剂的独特优点而促进其有效成分的透皮吸收达到美容抗衰的效果。
优选地，所述外用护肤制剂为精华、乳液、面霜、面膜、化妆水或喷雾。
本发明与现有技术相比，具有如下有益效果：
(1)本发明通过组方中各组分的种类及用量的筛选，制备得到一种O/W型灵芝提取物纳米乳，该纳米乳呈类球型、性质稳定、分布均匀、流动性较好；
(2)本发明制备得到的灵芝提取物纳米乳显著提高了灵芝三萜、灵芝多糖的溶解度，有助于提高体系的载药量及其有效成分利用率；
(3)本发明将灵芝提取物与纳米乳结合，适用于应用于化妆品领域，可制得一种具有抗衰老、抗氧化作用的外用护肤制剂，既能增加灵芝有效成分的溶解度，提高灵芝的利用价值，又能因为纳米乳制剂的独特优点而促进其有效成分的透皮吸收达到美容抗衰的效果，为后续灵芝提取物纳米乳化妆品的工艺生产及理论研究提供了一定的实验基础。
附图说明
图1为不同表面活性剂SA的伪三元相图；
图2为表面活性剂SA与助表面活性剂的不同km值的伪三元相图；
图3为不同油相的伪三元相图；
图4为灵芝多糖标准曲线；
图5为灵芝三萜标准曲线；
图6为灵芝提取物纳米乳的外观考察；
图7为灵芝提取物纳米乳的丁达尔效应；
图8为灵芝提取物纳米乳的类型鉴别；
图9为透射电镜下灵芝提取物纳米乳形态分布。
具体实施方式
下面通过具体实施方式来进一步说明本发明，以下实施例为本发明较佳的实施方式，但本发明的实施方式并不受下述实施例的限制。
【灵芝多糖的制备】
采用水浴浸提法，料液比为1:30(W/V)，在70℃条件下，提取2h，可得灵芝子实体的总多糖溶液。旋蒸浓缩，提取液加乙醇至含醇量为80％，搅拌均匀，4℃放置24h，离心倾去上清液，挥干沉淀中的乙醇，即得到子实体粗多糖固体。
【灵芝三萜的制备】
采用乙醇浸提法，精密称取灵芝子实体粗粉100g，至于圆底烧瓶中，按照料液比1：20(W/V)加入无水乙醇，70℃加热回流提取2次，每次1.5h。用抽滤法收集萃取液，摇匀。将得到的萃取液在55℃下旋蒸浓缩至浸膏状，保存备用。
实施例1：
(1)将EL 35 2.7g和甘油1.3g混合均匀，得到混表面活性剂(Smix)，再将MCT 1.0g加入Smix中，置于55℃的转速为1000-1500r/min的磁力搅拌水浴锅中混合均匀，得到混合体系；
(2)在搅拌状态下，逐滴滴加4.0g的水于混合体系中，搅拌至澄清即得空白纳米乳；
(3)少量多次的将过量灵芝三萜加入空白纳米乳中，静置24h后，通过低速离心法(2000rpm)将沉淀与载药后的纳米乳剂分离，保留上清液；少量多次的将灵芝多糖粉末溶解于上清液中充分搅拌均匀至完全溶解，即得棕黄色澄清透明的灵芝提取物纳米乳液。
实施例2：
(1)将EL 35 3.75g和甘油3.75g混合均匀，得到混表面活性剂(Smix)，再将MCT 1.25g加入Smix中，置于50℃的转速为1000-1500r/min的磁力搅拌水浴锅中混合均匀，得到混合体系；
(2)在搅拌状态下，逐滴滴加1.25g的水于混合体系中，搅拌至澄清即得空白纳米乳；
(3)少量多次的将过量灵芝三萜加入空白纳米乳中，静置24h后，通过低速离心法(2000rpm)将沉淀与载药后的纳米乳剂分离，保留上清液；少量多次的将灵芝多糖粉末溶解于上清液中充分搅拌均匀至完全溶解，即得棕黄色澄清透明的灵芝提取物纳米乳液。
实施例3：
(1)将EL35 4.2g和甘油2.1g混合均匀，得到混表面活性剂(Smix)，再将IPM 2.5g加入Smix中，置于60℃的转速为1000-1500r/min的磁力搅拌水浴锅中混合均匀，得到混合体系；
(2)在搅拌状态下，逐滴滴加1.25g的水于混合体系中，搅拌至澄清即得空白纳米乳；
(3)少量多次的将过量灵芝三萜加入空白纳米乳中，静置24h后，通过低速离心法(2000rpm)将沉淀与载药后的纳米乳剂分离，保留上清液；少量多次的将灵芝多糖粉末溶解于上清液中充分搅拌均匀至完全溶解，即得棕黄色澄清透明的灵芝提取物纳米乳液。
实施例4：纳米乳的处方筛选
4.1表面活性剂(SA)的选择
4.1.1表面活性剂稳定性实验
当HLB值在3-6时为W/O型乳化剂，当HLB值在8-18时为O/W型乳化剂，根据CoSA的HLB值计算公式：HLB＝HLBA*A％+HLBB*B％，本发明预选用Tween80、RH 40、EL40、EL35作为备选SA，其HLB值在13-16范围之间，选用甘油作为CoSA。将Tween80、RH 40、EL40、EL 35与甘油分别按照1:1、1:2、2:1混匀，进行离心实验，设置转速为5000r/min，时间为15min，记录现象。按照上述操作将试剂混匀，分别置于4℃、室温(25℃)、55℃条件下，10min后观察其颜色、澄清度、黏度及是否分层等情况。从中选取混合后澄清、透明、不分层、黏度小的SA作为纳米乳的备选SA。结果见下表(表3)。
表3不同混合表面活性剂的配伍稳定性
由结果可知，RH40与甘油的混合溶剂在不同条件下呈乳白色且黏度较大，Tween80、EL 40、EL 35与甘油的混合溶剂在多种条件下的状态大都表现为澄清透明、黏度较小并且不分层，故选用Tween80、EL 40、EL 35为备选SA进行下一步的实验操作。
4.1.2最佳表面活性剂的选择
将Tween80、EL40、EL35在55℃水浴、磁力搅拌转速为1000-1500r/min的条件下，分别与甘油按照质量比km＝2:1进行混合均匀，再将Smix与油相以质量比1:9-9:1比例混匀，然后逐滴加入水相，观察并记录乳液由浊到清或由清到浊时的临界点，绘制伪三元相图(如图1)。
由图1可知，Tween80和EL40在高油相区不易成乳，且成乳区面积由大到小依次顺序为：EL35>EL40>Tween80,EL 35成乳区面积最大，故优选EL 35为纳米乳的SA。
4.2助表面活性剂(CoSA)及km值的确定
纳米乳常用的CoSA有无水乙醇、甘油、丙二醇等，本发明所用的脂溶性的灵芝三萜化合物为无水乙醇提取制得，但由于无水乙醇对皮肤具有一定的刺激性，不宜用作外用护肤品中，而甘油对皮肤无害、可以和许多物质相溶，且具有吸水保湿性、抗氧化的功效，因此可在各种护肤品中用作溶剂、润肤剂、保湿剂等。
综上所述，本实验选用甘油作为CoSA。在55℃水浴、磁力搅拌转速为1000-1500r/min的条件下，将SA与CoSA按Km＝1:1、1:2、2:1混匀，再将Smix与油相以质量比1:9～9:1的比例混匀，搅拌状态下逐滴加入水相，观察记录乳液由浊到清或由清到浊时的临界点，绘制伪三元相图(如图2)。
由图2可知，当km＝2:1时的伪三元相图成乳区面积最大，因此本发明配方SA与CoSA的最佳km值为2:1。
4.3油相的选择
4.3.1油相的溶解度实验
MCT(中链甘油三酯)、IPM(肉豆蔻酸异丙酯)、橄榄油均为化妆品中比较常用的油相，MCT润滑性好、黏度小与其他溶剂能很好的互溶；IPM的延展性好、清爽、有补水保湿的作用；橄榄油为植物油，在外用化妆品中较为常见，有天然的美容护肤作用。
本发明预选用上述三种油相为待选油相。由于灵芝三萜为脂溶性化合物，因此将灵芝三萜提取物溶解于各油相中，考察灵芝三萜在各油相中的溶解度及稳定性。将过量的灵芝三萜分别加入到上述三种油相当中，然后，使用涡旋仪对各混合物进行涡旋，以加速药物与各种溶剂混合均匀。每隔5min涡旋30s，观察30min内的溶解情况，直至药物不再溶解且静置后有部分固体沉淀。随后将上述充分混合后的每个离心管置于25±2℃水浴恒温振荡器中持续震荡24h，观察溶解情况。若依旧有固体沉淀，说明溶液处于饱和状态，即得灵芝三萜饱和油溶液。将饱和溶液以12000r/min离心10min，去除掉过量的药物。取母液，用紫外分光光度法测量母液中灵芝三萜的含量。从而筛选溶解度较好的SA备选试剂。由实验可知，灵芝三萜在MCT中溶解度为1.7mg/ml，在IPM中溶解度为2.8mg/ml，而在橄榄油中溶解度较差，且离心后会出现明显的分层现象，故本发明选择MCT和IPM为待选油相。
4.3.2最佳油相的选择
将EL35在55℃水浴、磁力搅拌转速为1000-1500r/min的条件下与甘油按照质量比km＝2:1进行混合均匀，再将Smix与待选油相MCT、IPM分别以质量比1:9、2:8、3:7、4:6、5:5、6:4、7:3、8:2、9:1比例混匀，搅拌状态下逐滴加入水相，观察记录乳液由浊到清或由清到浊转变时的临界点，绘制伪三元相图(如图3)。
由图3可知，成乳区面积MCT>IPM，因此选用MCT为此纳米乳配方的最佳油相。
综上所述，此纳米乳的最优处方选定EL 35为SA，选定甘油为CoSA，选定km值为2:1，选定MCT为油相，选定蒸馏水为水相。并且通过伪三元相图的成乳区域筛选出最稳定的纳米乳的配方比例为：EL 35：30％，甘油：14.4％，MCT：11.1％，水：44.5％。
实施例5灵芝提取物纳米乳(实施例1)的质量评价
5.1纳米乳载药量测定
将已经制得的灵芝提取物纳米乳溶解于甲醇进行破乳，使用紫外分光光度法分别在626nm、546nm处测量纳米乳中灵芝多糖和三萜溶液的吸光度值，对照标准曲线，从而得出纳米乳中灵芝多糖和三萜的载药含量。
5.1.1分光光度法测定灵芝多糖含量
精密称取D-(+)-葡萄糖2.5mg，加纯水定容至25mL,制备得0.1mg/ml葡萄糖标准溶液。精密称取蒽酮25mg，加浓硫酸定容至25mL，制备得硫酸-蒽酮溶液。精密量取0.2、0.4、0.6、0.8、1.0mL的葡萄糖标准品溶液，加纯水至1.0mL，分别加入3.0mL的硫酸-蒽酮溶液，立即混合均匀，室温下反应5min。在626nm波长处，在空白对照下，测定吸光度值。以标准葡萄糖含量为X轴，吸光度值为Y轴，即得标准曲线和回归方程。结果如图4所示。
精密量取灵芝提取物纳米乳1.0mL,用甲醇稀释100倍，超声破乳10min，混合均匀，从上述溶液中精密量取0.17mL甲醇稀释破乳后的多糖溶液至另一试管中，加甲醇至1.0mL，再加入3.0mL的硫酸-蒽酮溶液，立即混合均匀，室温下反应5min。在626nm波长处，在空白对照下，测定吸光度值。平行测定3次，取其平均值。代入已经得到的灵芝多糖浓度的回归方程，测得本纳米乳中灵芝多糖的含量为36.35mg/ml。
5.1.2分光光度法测定灵芝三萜含量
将齐墩果酸加甲醇配制成含量为0.2mg/ml的标准溶液。取上述溶液0.1，0.2，0.3，0.4，0.5ml，分别置10.0ml具塞试管中，挥干(55℃)，放冷，加入新配制的5％香草醛冰醋酸溶液0.2ml，高氯酸0.8ml，混匀后水浴(70℃)15min，随后立即冰浴5min，取出后尽快加入乙酸乙酯4mL，混合均匀。空白对照下，在波长为546nm处，测定吸光度值，以质量浓度为X轴，吸光度为Y轴，即得标准曲线和回归方程，结果图5所示。
精准量取灵芝提取物纳米乳1.0mL,用甲醇稀释100倍，超声破乳10min，混合均匀,取待测样品0.2ml，置10ml具塞试管中，按上述步骤，自“挥干”起，同法操作，测定吸光度，每个样品平行测定3次，取平均值并记录。代入已经得到的灵芝三萜浓度的回归方程中，即得本纳米乳中灵芝三萜的含量为5.99mg/ml。
综上结果表明，灵芝三萜在油相中溶解度为1.70mg/ml，灵芝多糖在水中溶解度为21.72mg/ml，而灵芝三萜和灵芝多糖在纳米乳中的溶解度分别5.99mg/ml和36.35mg/ml。实验结果表明，纳米乳显著提高了灵芝三萜、灵芝多糖的溶解度，有助于提高体系的载药量及其有效成分利用率。
制备多糖含量为6.00mg/ml、三萜含量为5.99mg/ml的灵芝提取物纳米乳以供后续实验使用。
5.2纳米乳的外观考察
将所制得的空白纳米乳和灵芝提取物纳米乳分别置于容器中，如图6所示，所制得的空白纳米乳的外观为澄清透明乳液，并泛有淡蓝色乳光(A)；加入灵芝三萜后的纳米乳则为淡黄色澄清乳液(B)；加入灵芝三萜和灵芝多糖后的溶液为深棕色澄清乳液(C)。
丁达尔效应是指，粒径为1～100nm范围的胶体乳液在汇聚光照射下会产生一条可见“光带”的现象。因此，当激光从侧面照射时，上述纳米乳均产生较为明显的丁达尔效应，由此可证明，本实验制得的纳米乳液符合纳米级的粒径要求。如图7所示。
5.3纳米乳的类型鉴定
纳米乳类型的鉴定方法有稀释法、染色法及电导率法等。本实验选用染色法，因为其操作简单、现象明显。鉴定试剂选择亚甲基蓝溶液，因为亚甲基蓝溶液是水溶性的，在O/W型乳液中会迅速扩散，有明显的现象易被观察，而在W/O型乳液中则不能。取本实验所制得的纳米乳于EP管中，向EP管中滴加适量亚甲基蓝溶液，观察并记录现象(图8)，由实验结果可知，当亚甲基蓝溶液滴加到纳米乳中后，蓝色迅速扩散，因此本实验制得的灵芝提取物纳米乳为O/W型纳米乳。
5.4粒径分布和Zeta电位
取灵芝提取物纳米乳1ml,用纯水稀释20倍，取适量分别置于各自的样品池中，用激光粒度仪测定其平均粒径和Zeta电位，结果如表所示(表4)。
纳米乳平均粒径为28.01nm，PDI为0.105，即所制得的纳米乳整体粒径大小均一、分布均匀、体系稳定，符合纳米乳剂的标准粒径范围，Zeta电位为-4.05mV。Zeta电位指的是连续相与附着在分散粒子上的流体稳定层之间的电势差值，其绝对值越大，体系则越稳定。由本实验所制得的纳米乳Zeta电位结果可知，纳米乳粒静电排斥力较大，即整个纳米乳体系较为稳定。
表4灵芝提取物纳米乳平均粒径、PDI、Zeta电位
5.5形态学考察
取灵芝提取物纳米乳适量稀释至适宜浓度，滴一滴于铜网上，浸润约1min，用滤纸吸干多余液体，使其自然晾干；随后滴加1滴3％磷钨酸溶液，染色约5min，滤纸吸干多余溶液后，使其自然晾干，用透射电镜观察制备好的铜网。结果(如图9)表明，本实验所制得的灵芝提取物纳米乳呈类球型,分布均匀。
5.6离心稳定性
本实验采用高速离心法对灵芝提取物纳米乳进行离心稳定性的考察，观察其是否会出现静置分层、分布不均、聚集沉降等现象。将样品在转速为9000r/min的条件下离心15min，结果显示，经过高速离心后的纳米乳并未出现分层、沉淀等现象。因此，本实验制得的纳米乳具有较好的稳定性。
5.7稀释稳定性
对本实验制得的纳米乳进行稀释稳定性考察，将灵芝提取物纳米乳用纯水分别稀释50倍、100倍、500倍、1000倍后，观察发现均没有分层、破乳、絮凝等现象产生，由于将纳米乳稀释500倍即达到无限稀释的条件，因此稀释本实验制得的纳米乳不会影响其稳定性，可被无限稀释。同时，也进一步证明了灵芝提取物纳米乳的结构类型为O/W型纳米乳。

Claims (9)

  1. 一种灵芝提取物纳米乳，其特征在于，由灵芝三萜、灵芝多糖、表面活性剂、助表面活性剂、油相和水组成，其中，所述表面活性剂与助表面活性剂的质量比为1:2-2:1，表面活性剂和助表面活性剂的总质量与油相的质量比为4:6-10:1。
  2. 根据权利要求1所述的灵芝提取物纳米乳，其特征在于，所述表面活性剂为聚山梨酯-80、蓖麻油聚氧乙烯醚40或聚氧乙烯蓖麻油35中的一种或几种，优选为聚氧乙烯蓖麻油35。
  3. 根据权利要求1所述的灵芝提取物纳米乳，其特征在于，所述助表面活性剂为无水乙醇、甘油或丙二醇中的一种或几种，优选为甘油。
  4. 根据权利要求1所述的灵芝提取物纳米乳，其特征在于，所述油相为中链甘油三酯或肉豆蔻酸异丙酯中的一种或两种的混合，优选为中链甘油三酯。
  5. 根据权利要求1所述的灵芝提取物纳米乳，其特征在于，所述灵芝提取物纳米乳的平均粒径为28.01nm，PDI为0.105，平均Zeta电位为-4.05mV，灵芝提取物纳米乳中灵芝多糖的最大载药量为36.35mg/mL，灵芝三萜的最大载药量为5.99mg/mL。
  6. 权利要求1-5任一项所述的灵芝提取物纳米乳的制备方法，其特征在于，包括以下步骤：
    a、将表面活性剂和助表面活性剂混合均匀，得到混表面活性剂，将混表面活性剂和油相混合均匀，得到混合体系；
    b、在搅拌状态下，在混合体系中滴加水，搅拌至澄清即得空白纳米乳；
    c、将灵芝三萜加入空白纳米乳中，静置，离心，得上清液，将灵芝多糖溶解于上清液，充分搅拌均匀至完全溶解，即得灵芝提取物纳米乳。
  7. 根据权利要求6所述的灵芝提取物纳米乳的制备方法，其特征在于，步骤(a)-(c)在50-60℃的水浴、磁力搅拌转速为1000-1500r/min条件下进行。
  8. 权利要求1-5任一项所述的灵芝提取物纳米乳在外用护肤制剂中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述外用护肤制剂为精华、乳液、面霜、面膜、化妆水或喷雾。
PCT/CN2023/072380 2022-08-02 2023-01-16 一种灵芝提取物纳米乳及其制备方法和应用 WO2024027113A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210919490.XA CN115282089A (zh) 2022-08-02 2022-08-02 一种灵芝提取物纳米乳及其制备方法和应用
CN202210919490.X 2022-08-02

Publications (1)

Publication Number Publication Date
WO2024027113A1 true WO2024027113A1 (zh) 2024-02-08

Family

ID=83827148

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/072380 WO2024027113A1 (zh) 2022-08-02 2023-01-16 一种灵芝提取物纳米乳及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN115282089A (zh)
WO (1) WO2024027113A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115282089A (zh) * 2022-08-02 2022-11-04 安徽中医药大学 一种灵芝提取物纳米乳及其制备方法和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101601695A (zh) * 2009-06-26 2009-12-16 江苏省中医药研究院 灵芝三萜提取物的自微乳化纳米组合物及其制备方法
CN102058530A (zh) * 2010-12-14 2011-05-18 于荣敏 一种灵芝多糖口服纳米乳剂及其制备方法
CN109568354A (zh) * 2019-02-15 2019-04-05 中国科学院上海药物研究所 一种包含灵芝孢子油和灵芝多糖的组合物及其用途
CN109646399A (zh) * 2019-02-15 2019-04-19 中国科学院上海药物研究所 一种包含灵芝孢子油和灵芝多糖的口服乳剂及其制备方法和用途
CN109876023A (zh) * 2019-03-08 2019-06-14 暨南大学 一种灵芝孢子油纳米乳及其制备方法与应用
CN110613643A (zh) * 2019-09-21 2019-12-27 云南辰锦威生物科技有限公司 一种修复除皱霜及其制备方法
CN115282089A (zh) * 2022-08-02 2022-11-04 安徽中医药大学 一种灵芝提取物纳米乳及其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101601695A (zh) * 2009-06-26 2009-12-16 江苏省中医药研究院 灵芝三萜提取物的自微乳化纳米组合物及其制备方法
CN102058530A (zh) * 2010-12-14 2011-05-18 于荣敏 一种灵芝多糖口服纳米乳剂及其制备方法
CN109568354A (zh) * 2019-02-15 2019-04-05 中国科学院上海药物研究所 一种包含灵芝孢子油和灵芝多糖的组合物及其用途
CN109646399A (zh) * 2019-02-15 2019-04-19 中国科学院上海药物研究所 一种包含灵芝孢子油和灵芝多糖的口服乳剂及其制备方法和用途
CN109876023A (zh) * 2019-03-08 2019-06-14 暨南大学 一种灵芝孢子油纳米乳及其制备方法与应用
CN110613643A (zh) * 2019-09-21 2019-12-27 云南辰锦威生物科技有限公司 一种修复除皱霜及其制备方法
CN115282089A (zh) * 2022-08-02 2022-11-04 安徽中医药大学 一种灵芝提取物纳米乳及其制备方法和应用

Also Published As

Publication number Publication date
CN115282089A (zh) 2022-11-04

Similar Documents

Publication Publication Date Title
Rani et al. Self-emulsifying oral lipid drug delivery systems: advances and challenges
EP2891486B1 (en) Polymeric finasteride and minoxidil nanoparticles, method for preparing same, aqueous suspension containing same, pharmaceutical composition and use thereof
WO2024027113A1 (zh) 一种灵芝提取物纳米乳及其制备方法和应用
Shi et al. Development, optimization and evaluation of emodin loaded nanoemulsion prepared by ultrasonic emulsification
CN103462846A (zh) 人参美白抗衰纳米乳精华液及其制备方法
RU2662069C2 (ru) Липидные композиции рацекадотрила
RU2632441C2 (ru) Липидные композиции рацекадотрила
US11752450B2 (en) Selective extraction method for natural substances
Mishra et al. Liranaftate loaded Xanthan gum based hydrogel for topical delivery: Physical properties and ex-vivo permeability
Shaikh et al. Formulation and evaluation of nanoemulsion for topical application
CN112386507A (zh) 一种囊泡型载体的制备方法及其在化妆品中的应用
Nanjwade et al. Development and evaluation of nanoemulsion of Repaglinide
Sutthanut et al. Solid lipid nanoparticles for topical administration of Kaempferia parviflora extracts
Zhang et al. Improved stability and skin penetration through glycethosomes loaded with glycyrrhetinic acid
US20110250239A1 (en) Pharmaceutical and/or cosmetic composition for treating the skin
Kurul et al. Skin permeation of two different benzophenone derivatives from various vehicles
CN111419900A (zh) 一种基于冠心宁改进的纳米混悬冻干制剂及其制备方法
CN106511272A (zh) 一种抗肝炎的黄岑苷o/w型口服纳米乳及其制备方法
CN113117091A (zh) 一种姜黄素水溶性制剂及其制备方法
CN112870161B (zh) 一种雪松醇纳米乳及其优化制备方法
David et al. Development and evaluation of liquid crystal systems of combination of 5-fluorouracil and curcumin for cervical cancer cell line
CN112263542A (zh) 地奈德纳米乳凝胶组合物及其制备方法
WO2009007622A2 (fr) Composition dermatologique comprenant des vésicules lipidiques de calcitriol, son procédé de préparation et son utilisation
KR101827611B1 (ko) 피코시아닌을 포함하는 유중수 마이크로 또는 나노에멀젼
TWI832471B (zh) 具清除自由基及美白功效的粉團蓼微膠囊載體及其製備方法與用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23848828

Country of ref document: EP

Kind code of ref document: A1