CN116440080A - Safflower yellow nanometer flexible liposome and preparation method thereof - Google Patents
Safflower yellow nanometer flexible liposome and preparation method thereof Download PDFInfo
- Publication number
- CN116440080A CN116440080A CN202310159311.1A CN202310159311A CN116440080A CN 116440080 A CN116440080 A CN 116440080A CN 202310159311 A CN202310159311 A CN 202310159311A CN 116440080 A CN116440080 A CN 116440080A
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- China
- Prior art keywords
- safflower yellow
- flexible liposome
- liposome
- mucous membrane
- safflower
- Prior art date
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Links
- 239000002502 liposome Substances 0.000 title claims abstract description 101
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- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
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- IAVUBSCVWHLRGE-UXEKTNMQSA-N (6e)-2,5-dihydroxy-6-[(e)-1-hydroxy-3-(4-hydroxyphenyl)prop-2-enylidene]-2,4-bis[(2s,3r,4r,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]cyclohex-4-ene-1,3-dione Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C(C(C(O)([C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C1=O)=O)=C(O)\C1=C(/O)\C=C\C1=CC=C(O)C=C1 IAVUBSCVWHLRGE-UXEKTNMQSA-N 0.000 description 1
- XIAYFENBYCWHGY-UHFFFAOYSA-N 2-[2,7-bis[[bis(carboxymethyl)amino]methyl]-3-hydroxy-6-oxoxanthen-9-yl]benzoic acid Chemical compound C=12C=C(CN(CC(O)=O)CC(O)=O)C(=O)C=C2OC=2C=C(O)C(CN(CC(O)=O)CC(=O)O)=CC=2C=1C1=CC=CC=C1C(O)=O XIAYFENBYCWHGY-UHFFFAOYSA-N 0.000 description 1
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- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Physiology (AREA)
- Vascular Medicine (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Nutrition Science (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a safflower yellow nanometer flexible liposome and a preparation method thereof, wherein the preparation of the safflower yellow nanometer flexible liposome is carried out based on a film hydration-probe ultrasonic method, and the obtained safflower yellow nanometer flexible liposome has affinity and absorption activity on the skin and/or mucous membrane of an organic organism and is used for replacing an injection administration route to realize synergistic attenuation administration of the safflower yellow. The invention provides a brand new technical idea and a technical path, based on anatomical and physiological characteristics of human mucous membrane, especially sublingual mucous membrane, and deep prolongation of pharmacological technical means, the traditional Chinese medicine infusion administration is converted into nano flexible liposome so as to ensure the safety of the nano flexible liposome while the high efficiency of the nano flexible liposome is maintained, and the successful research and development of a safflower yellow nano flexible liposome preparation process system is realized.
Description
Technical Field
The invention relates to the technical field of medicine improvement and preparation, in particular to a novel safflower yellow nano flexible liposome preparation with high affinity and absorption activity to skin mucosa of an organic organism and a preparation process thereof.
Background
The traditional Chinese medicines for preventing and treating cardiovascular diseases are numerous in number and different in administration form. Among them, the Chinese medicine injection preparation is widely used because of rapid onset of drug action, high bioavailability, definite curative effect, etc. However, with the expansion of the application range, adverse reactions/events of the traditional Chinese medicine injection preparation are gradually increased, and the traditional Chinese medicine injection preparation has attracted great attention in various social circles. The national drug adverse reaction monitoring annual report (2018) shows that: adverse reaction/event is counted according to medicine category, the traditional Chinese medicine ratio is 14.6%, wherein the highest dosage mode is 48.7% by intravenous injection, and the number and the ratio of the related reports of cardiovascular system are in an ascending trend. Adverse reactions/events frequently occur, which not only seriously threatens the health and safety of patients; but also greatly preventing the traditional Chinese medicine variety with definite curative effect from exerting the effect of preventing and treating diseases; in addition, as clinical use is gradually limited, manufacturers of Chinese medicinal injection preparations face disastrous consequences.
How to avoid adverse reaction/event, fully exerting the reliable disease prevention and treatment effect of the traditional Chinese medicine injection preparation, firstly, defining the cause of the adverse reaction/event, and analyzing according to literature, wherein non-drug factors and drug factors are main reasons, wherein the non-drug factors comprise: age, sex and special constitution of the patient; improper solvent selection or incorrect operation such as higher instillation speed during infusion; unreasonable combination or overdose is not relevant to the drug itself. The drug factor means that substances contained in the preparation can cause anaphylactic reaction or anaphylactic reaction-like components lack of being selectively trapped by biological membranes of organisms (natural defense barriers evolved by human beings) during infusion, and can be quickly and directly introduced into blood to cause adverse reactions. Enhancing the administration guidelines and normative practices for non-pharmaceutical factors may reduce some adverse events, but the risk cannot be completely eliminated due to the dosage form of the injectable formulation itself and the route of administration. In regard to the latter, a great deal of experiments are currently carried out by the scholars on the cause of the anaphylactic reaction and the sensitized substance, and the theory is that the allergic reaction and the sensitized substance can be related to the infusion speed, the dose, auxiliary materials in the preparation and even effective components. However, there are few studies on allergic reactions, and at present, all the allergic components cannot be confirmed. Therefore, it is obviously time-consuming and laborious and impractical to solve the problem by finding the substance that causes the adverse reaction-! In addition, enterprises adopt multi-step refining means to obtain active ingredients with higher purity for injection, but adverse events are not completely eliminated yet. Based on the above, a new way for improving the safety of the traditional Chinese medicine injection preparation and ensuring the effectiveness of the traditional Chinese medicine injection preparation is explored, the life safety of patients is concerned, the inherited development of traditional Chinese medicine is concerned, the death and the death of medicine enterprises are concerned, and the method is certainly a complex and important scientific problem to be solved urgently.
Among a plurality of Chinese medicinal injection preparations, the safflower injection is used as a first-line medicament for preventing and treating cardiovascular and cerebrovascular diseases, and plays an important role in preventing and treating serious diseases such as coronary heart disease, vasculitis, occlusive cerebrovascular diseases and the like. The safflower yellow is an effective part extracted from safflower, and the Hydroxy Safflower Yellow A (HSYA) is the flavonoid compound with the highest content, and the extract is brown yellow powder, and is easy to dissolve in water. A plurality of researches show that the hydroxysafflor yellow A has the functions of resisting oxidization, resisting inflammation, promoting blood circulation, removing blood stasis, resisting thrombosis, resisting platelet aggregation and the like.
The safflower injection or the safflower yellow for injection is commonly used clinically for preventing and treating cardiovascular system diseases such as vasculitis, and with the gradual expansion of clinical application, adverse reaction events are gradually increased, the use of the safflower injection is severely limited by increasingly severe safety problems, and the original province (Shanxi province) of 8 production enterprises is reserved at present only for 4. In order to examine the cause of adverse reaction, the subject group studied the sensitization substances in safflower injection earlier, found that protein substances with smaller molecular weight (40 KD, 26 KD) can cause allergic reaction, however, the cause of the residual allergic substances can not be clarified by comparing the production process of enterprises (the finished product is filtered by using a filter membrane with the cutoff molecular weight of 10000), but it can be presumed that if the pore diameter of the ultrafiltration membrane is continuously reduced, other possibly effective components are lost, further the clinical curative effect is reduced or lost, and the hidden danger of adverse reaction is still unavoidable. At present, another safflower injection preparation which is widely applied to clinic, namely safflower yellow for injection (only 2 production enterprises in China: zhejiang Yongning and Shanxi Yuan Tang at present), is an effective part with higher purity (the content of hydroxy safflower yellow A is more than 60 percent) which is continuously refined by a multi-step column separation technology, and compared with safflower injection, adverse reactions are reduced, but still occur, so that the problem of safety of the safflower injection preparation is difficult to be fundamentally solved by a continuous purification way.
In summary, developing a new idea and a new technical path for solving the safety problem of traditional Chinese medicines is a key and difficult problem in the industry at present.
Disclosure of Invention
The invention aims to solve the technical problems that: based on a brand new technical idea and a brand new technical path, the infusion administration is converted into nano flexible liposome by fully applying a pharmacy means so as to improve the safety of the nano flexible liposome and keep the high efficiency of the nano flexible liposome, and the safflower yellow nano flexible liposome and the preparation method thereof are provided.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
The safflower yellow nanometer flexible liposome has affinity and absorption activity to the skin and/or mucous membrane of organic organism, and is used to replace injection administration route to realize synergistic attenuation administration of safflower yellow.
As a preferable technical scheme of the invention, the mucous membrane is an animal body or human mucous membrane with a three-layer structure of an epithelial layer, an intrinsic layer and a basal layer, wherein the epithelial layer is formed by orderly arranging flat compact cells to form a main barrier for drug absorption.
As a preferred technical scheme of the invention, the mucous membrane is sublingual mucous membrane.
As a preferable technical scheme of the invention, the core process flow is as follows: firstly, a compound organic composition containing carthamin yellow is subjected to rotary evaporation to prepare a film, then an aqueous phase solution containing a softening agent and small glass beads are added into the obtained film for rotary hydration, and the obtained solution is subjected to ultrasonic treatment by a specific probe to obtain the carthamin yellow nano flexible liposome.
As a preferable technical scheme of the invention, the core process parameters are as follows: the compound organic composition is prepared by compounding carthamin, egg yolk lecithin, cholesterol and an organic solvent, wherein the compound dosage of the carthamin, the egg yolk lecithin and the cholesterol is set according to the following standard: the mass ratio of the medicine to the fat is 1:20-1:150, and the mass ratio of the gall to the fat is 1:2-1:40; the softening agent in the water phase solution containing the softening agent adopts 1, 2-propylene glycol, and the mass fraction of the softening agent in the water phase solution is 10% -30%; the ultrasonic time of the probe in the ultrasonic treatment process is 4-6 min, and the rotating speed is 7000-9000 r/min.
As a preferable technical scheme of the invention, the mass ratio of the medicine to the fat is 1:30-1:100, and the mass ratio of the bile to the fat is 1:10-1:30.
As a preferable technical scheme of the invention, the mass ratio of the medicine to the fat is 1:50, and the mass ratio of the bile to the fat is 1:20.
As a preferable technical scheme of the invention, the organic solvent is absolute ethanol or methylene dichloride-methanol, and the mass ratio of the organic solvent to the carthamin yellow is (20-40) 1; the temperature of the rotary evaporation film-making process is controlled to be 45-50 ℃, and the rotation speed is controlled to be 65-70 r/min.
As a preferable technical scheme of the invention, the dosage of the small glass beads is 1 small glass bead for each 3-5mL of aqueous phase solution; the duration of the rotary hydration process is controlled to be 0.5h-1.5h.
As a preferable technical scheme of the invention, the probe ultrasonic treatment process has the probe ultrasonic time of 5min and the rotating speed of 8000r/min.
As a preferable technical scheme of the invention, 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection are accurately weighed, the three are placed in an eggplant type bottle, and 50mL of absolute ethyl alcohol is added for full dissolution, so as to obtain an oil phase; adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and mixing uniformly to obtain a water phase; removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, pouring 15mL of water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, ultrasonically treating with a probe of 8000r/min for 5min, passing through a polypropylene filter membrane of 0.22 mu m, adding the residual water phase, and measuring the encapsulation rate of liposome to complete the preparation.
A safflower yellow nano-flexible liposome having affinity and absorption activity to skin and/or mucous membrane of an organic organism, characterized in that: the preparation method is used for replacing injection administration route to realize synergistic attenuation administration of the carthamin yellow.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
in the whole, the invention provides a brand-new technical idea and a technical path, based on anatomical and physiological characteristics of human mucous membrane, especially sublingual mucous membrane, and deep prolongation of pharmacological technical means, the traditional Chinese medicine infusion administration is converted into nano flexible liposome so as to ensure the safety of the nano flexible liposome while the high efficiency of the nano flexible liposome is maintained, the successful research and development of a safflower yellow nano flexible liposome preparation process system is realized, and the brand-new technical idea, technical path and technical method are provided for the synergism and attenuation of the traditional Chinese medicine injection preparation.
The invention finally adopts a nano liposome technical route, and points the research direction to sublingual mucosa, which is based on technical consideration of various angles. From the effect, the invention forms a brand new effective, safe, convenient and economic mode of administration for treating cardiovascular disease or even an ideal mode of administration for daily prevention and slowing down the disease course by sublingual administration of the traditional Chinese medicine injection preparation. From the technical point of view, the liposome is a single-layer or multi-layer spherical vesicle structure formed by dispersing amphiphilic substances (phospholipids) and other amphoteric compounds (cholesterol and the like) in an aqueous phase, wherein water is compatible with water-soluble compounds, and a lipid bilayer accommodates the fat-soluble compounds; the liposome has a structure similar to that of a vesicle of a biological membrane, can directly enter cells, can be adsorbed on the outer layer of a target cell, can be metabolized and degraded by biological self-catabolic enzymes, has less adverse reaction, and has the advantages of good biocompatibility, targeting property and the like; the sublingual has abundant venous plexus capillaries, and the medicine can quickly and directly reach the heart through the internal jugular vein after being absorbed by the capillaries, so that the first pass effect of the liver and the adverse effect of the gastrointestinal tract on the medicine absorption can be effectively avoided, and the sublingual administration can obtain the curative effect equivalent to the intravenous administration. Sublingual administration avoids most of the non-pharmaceutical factors that produce adverse effects/events compared to injection administration: such as improper solvent, too fast dripping, unreasonable combined medication, and the like, and reduces the blood inflow speed and the quantity of harmful components due to the selective absorption of sublingual mucosa, thereby being expected to further improve the safety. Meanwhile, sublingual administration is simple and convenient to operate, and patient compliance is high; the sublingual formulation is less costly to prepare and use than injectable formulations.
Drawings
FIG. 1 is a dialysis balancing curve. The method comprises the following specific steps: taking 3 parts of 10mL self-made liposome solution in three dialysis bags respectively, placing the three bags in 3 100mL distilled water respectively, and sampling the liquid inlet every 1h until the HSYA content is nearly constant. The HSYA content in the dialysis external solution is basically constant for about 48 hours.
FIG. 2 is a graph showing the particle size distribution of nano flexible liposomes. The method comprises the following specific steps: appropriate amounts of SY-FNL and SY-CL were diluted with distilled water and injected into a Markov particle size potentiometric analyzer to determine the particle size of the liposome particles, 3 times per sample.
FIG. 3 is a graph showing potential profiles of nano-flexible liposomes. The method comprises the following specific steps: appropriate amounts of SY-FNL and SY-CL were diluted with distilled water and injected into a Markov particle size potentiometric apparatus to determine the potential of the liposome particles, 3 times per sample.
Fig. 4 shows the cumulative amount of drug substance solution and liposome accumulated on the mucous membrane. See embodiment 7 for specific steps.
FIG. 5 shows a liposome transmission electron micrograph (about 100000) and a scanning electron micrograph (about 5000). The method comprises the following specific steps: putting a proper amount of SY-FNL on a copper-carrying net, drying in the air, performing negative dyeing with phosphotungstic acid (1.5%), and observing by adopting a Transmission Electron Microscope (TEM); after fixing SY-FNL with a conductive adhesive, the metal is sprayed and observed by a Scanning Electron Microscope (SEM). It can be seen that SY-FNL is spherical or ellipsoidal with a layered structure.
FIG. 6 is a laser confocal microscopy image of fluorescent probes in liposomes (10X 100). In the figure, both the water-soluble probe (right) and the fat-soluble probe (left) can be uniformly distributed in the space of the whole flexible liposome.
Detailed Description
The following examples illustrate the invention in detail. The raw materials and the equipment used by the invention are conventional commercial products, and can be directly obtained through market purchase. As used in this specification and the appended claims, the term "if" may be interpreted as "when..once" or "in response to a determination" or "in response to detection" depending on the context. Similarly, the phrase "if a determination" or "if a [ described condition or event ] is detected" may be interpreted in the context of meaning "upon determination" or "in response to determination" or "upon detection of a [ described condition or event ]" or "in response to detection of a [ described condition or event ]". In addition, in the description of the present application and the appended claims, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and are not to be construed as indicating or implying relative importance.
Reference in the specification to "one embodiment" or "some embodiments" or the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," and the like in the specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless expressly specified otherwise.
Example 1 sublingual mucosa
Based on modern medicine, the structure of the sublingual mucosa is divided into three layers: the mucosal epithelium layer, lamina propria and basal layer, wherein the mucosal epithelium layer is composed of ordered arrangement of flat and dense cells, with a thickness of about 150 μm, is a major obstacle to drug absorption. As a lipid barrier membrane, drugs with low lipid solubility or large molecular weight are not easy to be absorbed through sublingual mucosa, and most of the active ingredients contained in the traditional Chinese medicine injection preparation are water-soluble substances. Therefore, the efficient promotion of drug passage through the mucosal upper epidermis layer is the key to successful construction of sublingual delivery thereof.
EXAMPLE 2 phospholipid-associated development
Category 1: the safflower yellow nanometer flexible liposome is prepared by lecithin. Accurately weighing 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection, placing the three raw material medicines into an eggplant type bottle, and adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase; and adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and uniformly mixing to obtain a water phase. Removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, drying for 1h, pouring 15mL of water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, performing ultrasonic treatment for 5min by a probe, passing through a 0.22 mu m polypropylene filter film, adding the rest water phase, and measuring the encapsulation rate of the liposome.
Category 2: adopts soybean phosphatide (for injection) to prepare safflower yellow nanometer flexible liposome. Accurately weighing 1.44g soybean lecithin (for injection), 24mg cholesterol and 20mg safflower yellow raw material medicine for injection, placing the three medicines into an eggplant type bottle, adding 50mL absolute ethyl alcohol for full dissolution, and obtaining an oil phase; and adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and uniformly mixing to obtain a water phase. Removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, drying for 1h, pouring 15mL of water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, performing ultrasonic treatment for 5min by a probe, passing through a 0.22 mu m polypropylene filter film, adding the rest water phase, and measuring the encapsulation rate of the liposome.
Category 3: adopts soybean phosphatide (for oral administration) to prepare safflower yellow nanometer flexible liposome. Accurately weighing 1.44g soybean lecithin (for oral administration), 24mg cholesterol, 20mg safflower yellow raw material for injection, placing the three materials into a eggplant type bottle, adding 50mL absolute ethyl alcohol, and fully dissolving to obtain an oil phase; and adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and uniformly mixing to obtain a water phase. Removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, drying for 1h, pouring 15mL of water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, performing ultrasonic treatment for 5min by a probe, passing through a 0.22 mu m polypropylene filter film, adding the rest water phase, and measuring the encapsulation rate of the liposome.
The encapsulation efficiency results of safflower yellow nano flexible liposome prepared by three phospholipids are shown in the following table.
| Phospholipid species | Encapsulation efficiency |
| Category 1 | 55.79％ |
| Class 2 | 30.35％ |
| Class 3 | 45.54％ |
The egg yolk lecithin adopted by the invention has obvious technical advantages.
EXAMPLE 3 development of drug-to-lipid ratio
Precisely weighing 24mg of cholesterol and 20mg of safflower yellow raw material medicines for injection, precisely weighing a certain amount of egg yolk lecithin according to the medicine-to-fat ratio of 1:10, 1:20, 1:50, 1:100 and 1:150 respectively, placing the three medicines into an eggplant type bottle, and adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase; 3mL of 1, 2-propylene glycol is added into 16mL of distilled water and mixed uniformly to obtain a water phase. Removing absolute ethanol from the oil phase by rotary evaporation under reduced pressure at 50deg.C to obtain dry film, pouring the water phase preheated at 50deg.C into film medium, adding 3-5 small glass beads for rotary hydration for 1h, ultrasound with probe for 5min, passing through 0.22 μm polypropylene filter membrane, adding the rest water phase, and determining liposome encapsulation rate by protamine aggregation-HPLC method. The encapsulation efficiency results of the safflower yellow nanometer flexible liposome prepared by different medicine-fat ratios are shown in the following table.
| Ratio of medicine to fat | Encapsulation efficiency |
| 1:10 | 15.74％ |
| 1:20 | 55.91％ |
| 1:50 | 71.37％ |
| 1:100 | 60.36％ |
| 1:150 | 58.75％ |
According to the above results, the encapsulation efficiency of safflower yellow nano flexible liposome increases with the increase of the amount of egg yolk lecithin, but decreases to some extent, possibly due to the following reasons: the liposome is a thermodynamically unstable system, and when the egg yolk lecithin is increased to a certain degree, the collision probability among liposome small particles is increased, so that fusion and precipitation are easy to occur.
EXAMPLE 4 development of bile lipid ratio
Precisely weighing 24mg of cholesterol and 20mg of safflower yellow raw material medicines for injection, precisely weighing a certain amount of egg yolk lecithin according to the cholesterol ratio of 1:2, 1:5, 1:10, 1:20 and 1:50 respectively, placing the three medicines into an eggplant type bottle, and adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase; 3mL of 1, 2-propylene glycol is added into 16mL of distilled water and mixed uniformly to obtain a water phase. Removing absolute ethanol from the oil phase by rotary evaporation under reduced pressure at 50deg.C to obtain dry film, pouring the water phase preheated at 50deg.C into film medium, adding 3-5 small glass beads for rotary hydration for 1h, ultrasound with probe for 5min, passing through 0.22 μm polypropylene filter membrane, adding the rest water phase, and determining liposome encapsulation rate by protamine aggregation-HPLC method. The encapsulation efficiency results of safflower yellow nano flexible liposome prepared by different bile lipid ratios are shown in the following table.
| Ratio of bile to lipid | Encapsulation efficiency |
| 1:50 | 12.47％ |
| 1:20 | 71.46％ |
| 1:10 | 68.10％ |
| 1:5 | 57.10％ |
| 1:2 | 57.67％ |
According to the above results, the increase of the cholesterol amount allows more cholesterol to be incorporated into the phospholipid molecules, and the cholesterol is arranged at intervals with the phospholipid molecules, so that the volume of the aqueous phase in the liposome is increased, and the encapsulation efficiency of the water-soluble component is increased; meanwhile, cholesterol can increase the surface viscosity of phospholipid to a certain extent, reduce the permeability of a phospholipid membrane to water-soluble drugs, reduce aggregation and fusion of liposome and increase the stability of the liposome.
EXAMPLE 5 1, 2-propanediol related development
Accurately weighing 50mg of cholesterol, 20mg of safflower yellow raw material medicine for injection and 1g of lecithin, placing the three into an eggplant type bottle, and adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase; respectively preparing 20mL of 1, 2-propylene glycol aqueous solution with mass fractions of 10%, 15%, 20%, 25% and 30%, and uniformly mixing to obtain a water phase. Removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, drying for 1h, pouring the water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, performing ultrasonic treatment for 5min by a probe, passing through a 0.22 mu m polypropylene filter membrane, adding the rest water phase, and determining the encapsulation rate of the liposome by using a protamine coacervation method-HPLC method.
Encapsulation efficiency results of safflower yellow nanoflexible liposomes prepared with different mass fractions of propylene glycol are shown in the following table.
According to the above results, the phospholipid bilayer of propylene glycol incorporated into the liposome increases its water solubility, so that part of water-soluble molecules enter the internal aqueous phase of the liposome to increase its encapsulation efficiency, when the mass fraction of propylene glycol increases to a certain extent, liposomes with smaller particle size can be formed due to excessive softness of the phospholipid membrane, and under the same phospholipid dosage, the small particle size lipid can reduce the volume of the internal aqueous phase to reduce its encapsulation efficiency; too much propylene glycol can also reduce the stability of liposome membrane materials, so that partial liposome is destroyed to cause leakage of liposome drugs, and the encapsulation efficiency is reduced.
Example 6, comparative test
The safflower yellow nanometer flexible liposome is prepared by adopting the preparation method (method 1) and four other preparation methods (method 2-method 5) respectively.
Method 1: the safflower yellow nanometer flexible liposome is prepared by adopting a film hydration-probe ultrasonic method. Accurately weighing 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection, placing the three raw material medicines into an eggplant type bottle, and adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase; and adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and uniformly mixing to obtain a water phase. Removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, pouring 15mL of the water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, ultrasonically treating with a probe of 8000r/min for 5min, passing through a polypropylene filter membrane of 0.22 mu m, adding the residual water phase, and measuring the encapsulation rate of the liposome.
Method 2: the safflower yellow nanometer flexible liposome is prepared by adopting an ethanol injection method. Accurately weighing 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection, placing the lecithin and the cholesterol into a eggplant type bottle, adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase, and removing an organic solvent by rotary evaporation under reduced pressure at 50 ℃; and adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and uniformly mixing to obtain a water phase. Dissolving carthamin 15mL of water phase, placing on a magnetic stirrer, slowly injecting the oil phase into the water phase, stirring for 5min, ultrasonic treating with 8000r/min probe for 5min, filtering with 0.22 μm polypropylene filter membrane, adding the rest water phase, and measuring liposome encapsulation efficiency.
Method 3: the safflower yellow nanometer flexible liposome is prepared by adopting a reverse evaporation method. Accurately weighing 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection, placing the lecithin and the cholesterol into a eggplant type bottle, and adding 50mL of dichloromethane-methanol (5:2) mixed solvent for dissolution to obtain an oil phase; and adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and uniformly mixing to obtain a water phase. Dissolving carthamus tinctorius yellow colour in 15mL of water phase, adding into the oil phase, performing water bath ultrasonic treatment for 5min, and standing for 1min each time for 1min for two times to obtain emulsion. The organic solvent was removed by rotary evaporation under reduced pressure at 50℃to gel state, shaken and homogenized at high speed for 10min, passed through a 0.22 μm polypropylene filter, and the liposome entrapment was determined after adding the remaining aqueous phase.
Method 4: the safflower yellow nanometer flexible liposome is measured by adopting a multiple emulsion method. Accurately weighing 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection, placing the lecithin and the cholesterol into a eggplant type bottle, and adding 50mL of dichloromethane-methanol (5:2) mixed solvent for dissolution to obtain an oil phase; adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and mixing uniformly to obtain a water phase; dissolving safflower yellow in 15mL of water phase, injecting the water phase into an oil phase, performing ultrasonic treatment to form emulsion, obtaining W/O type emulsion subjected to primary emulsification treatment, adding 10mL of ultrapure water, performing ultrasonic treatment under the same power to obtain W/O/W type emulsion subjected to secondary emulsification treatment, transferring the emulsion into a rotary evaporation bottle, performing reduced pressure rotary evaporation at 50 ℃ to remove organic solvent, passing through a 0.22 mu m polypropylene filter membrane, and adding the rest water phase to determine liposome encapsulation efficiency.
Method 5: the safflower yellow nanometer flexible liposome is prepared by adopting a pH gradient method. 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection are precisely weighed, lipid is dissolved in 50mL of dichloromethane, organic solvent is removed by rotary evaporation, a uniform film is formed on the bottom of a bottle by liposome mixture, 15mL of citric acid buffer solution with pH value of 4 is added (21 g of citric acid is added into 1000mL for standby, 71.63g of sodium dihydrogen phosphate is added into 1000mL of water for standby, 38.55mL of new preparation is mixed with 61.45mL of standby liquid for shaking, and the mixture is fully hydrated for 1h, so that the blank liposome is obtained. And mixing 7.2mL of 1, 2-propylene glycol in 38mL of distilled water uniformly to obtain a water phase, fully dissolving the safflower yellow with the prescription amount by using 15mL of the water phase, adding the water phase into blank liposome, incubating in a water bath for 30min, passing through a 0.22 mu m polypropylene filter membrane, adding the rest of the water phase, and measuring the liposome encapsulation efficiency.
The encapsulation efficiency results of the safflower yellow nano flexible liposome prepared by the five methods are shown in the following table.
| Preparation method | Encapsulation efficiency |
| Method 1 | 57.03％ |
| Method 2 | 13.81％ |
| Method 3 | 10.50％ |
| Method 4 | 11.38％ |
| Method 5 | 8.07％ |
The film hydration-probe ultrasonic method has obvious technical advantages and good application value.
Example 7 absorption test and confocal microscopy test
The experimental mucous membrane of the absorption test is selected from the sublingual mucous membrane of the pig, the sublingual mucous membrane of the pig with proper size is carefully peeled off, the mucous membrane is washed clean by normal saline, and then the mucous membrane is placed on a vertical double-chamber diffusion cell, and the mucous membrane side faces a supply cell. The double-chamber diffusion cell was fixed on a temperature-controlled magnetic stirrer, and the receiving liquid was stirred at a constant speed, maintaining a constant temperature of (37.+ -. 0.5) °c. 1mL of each of the crude drug solution and the liposome solution is added into a supply tank, 2mL of each of the crude drug solution and the liposome solution is sampled at 0.5, 1,2, 3 and 4 hours, 2mL of physiological saline preheated at 37 ℃ is injected into each of the two solutions, the extracted samples are dissolved by 200 mu L of purified water after being dried by nitrogen, the solution is subjected to centrifugal treatment at 14800r/min for 30min, the supernatant is taken for sample injection to determine the content of HSYA, and the cumulative permeation amounts of the two solutions are calculated and compared. The experimental results are shown in figure 4. In the whole transmucosal administration process, the accumulated permeation quantity of the safflower yellow nano flexible liposome (SY-FNL) is always higher than that of the safflower yellow common liposome (SY-CL) and the Safflower Yellow (SY) bulk drug solution, and when the time is 4 hours, the accumulated permeation quantity of HY in the SY-FNL, SY-CL and the SY bulk drug solution is (100.79 +/-4.35) mug/cm respectively 2 、(72.98±3.33)μg/cm 2 And (60.99.+ -. 5.37) μg/cm 2 . SY-FNL was shown to penetrate more easily the sublingual mucosal structure.
And (3) performing laser confocal microscopy, respectively adding a fat-soluble fluorescent probe Nile Red (Nile Red) and a water-soluble fluorescent probe Calcein (Calcein) into an oil phase and a water phase to prepare the carthamin nano flexible liposome, setting the Nile Red excitation wavelength to be 561nm and the Calcein excitation wavelength to be 488nm by using a laser confocal microscope (CLSM), observing the fluorescence distribution condition, and photographing and recording. The results are shown in FIG. 6, which shows that both the water-soluble probe (green) and the lipid-soluble probe (red) can be uniformly distributed throughout the space of the flexible liposome.
Example 8 development related to encapsulation Rate determination
Method 1: the entrapment rate of the safflower Huang Suse nanometer flexible liposome is measured by a dialysis-HPLC method. Precisely sucking 10mL self-made carthamin yellow liposome into a dialysis bag with a cut-off molecular weight of 14000, clamping two sides of the dialysis bag by a dialysis clamp, placing the dialysis bag into a 250mL beaker, adding 100mL 1, 2-propanediol as a dialysis medium, sucking 2mL of dialysis external liquid and supplementing 1,2 propanediol with the same volume respectively in 0.25, 0.5, 1,2, 4, 8, 10, 12, 24, 48 and 72 hours, allowing the dialysis external liquid in different time periods to pass through a 0.45 mu m filter membrane, sampling to measure the content of hydroxy A in the dialysis external liquid, drawing a dialysis balance curve, and measuring the encapsulation rate of the liposome.
Method 2: the liposome encapsulation efficiency of safflower Huang Suse was measured by protamine coacervation-HPLC. Precisely sucking 0.1mL of carthamin yellow liposome into a 5mL centrifuge tube, adding 0.1mL of 10mg/mL protamine solution, standing for 3min, adding 3mL of physiological saline, placing into a centrifuge, centrifuging for 30min at 14800r/min, discarding supernatant, adding 12% Triton X-100 into precipitate to demulsify, passing through a 0.45 μm filter membrane, sampling to determine the content of hydroxy A therein, and calculating liposome encapsulation efficiency.
The encapsulation efficiency of safflower yellow nano flexible liposome measured by the two methods is shown in the table below.
| Encapsulation efficiency measuring method | Encapsulation efficiency |
| Method 1 | 63.03％ |
| Method 2 | 61.37％ |
According to the results, the liposome encapsulation efficiency measured by the two methods is relatively close, but the dialysis equilibrium time in the dialysis method is 48 hours and takes a long time, so that the encapsulation efficiency measuring method is selected as a protamine coacervation method-HPLC method.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
Claims (10)
1. The preparation method of the safflower yellow nanometer flexible liposome is characterized by comprising the following steps: the safflower yellow nanometer flexible liposome has affinity and absorption activity to the skin and/or mucous membrane of organic organism, and is used to replace injection administration route to realize synergistic attenuation administration of safflower yellow.
2. The method for preparing safflower yellow nano flexible liposome according to claim 1, wherein: the mucous membrane is an animal body or human body mucous membrane with a three-layer structure of an upper epidermis layer, an inherent layer and a basal layer, and the upper epidermis layer is formed by orderly arranging flat compact cells to form a main barrier for drug absorption.
3. The method for preparing safflower yellow nano flexible liposome according to claim 1, wherein: the mucous membrane is sublingual mucous membrane.
4. The method for preparing safflower yellow nano flexible liposome according to claim 1, wherein:
the core process flow is as follows: firstly, a compound organic composition containing carthamin yellow is subjected to rotary evaporation to prepare a film, then an aqueous phase solution containing a softening agent and small glass beads are added into the obtained film for rotary hydration, and the obtained solution is subjected to ultrasonic treatment by a specific probe to obtain carthamin yellow nano flexible liposome;
the core technological parameters are as follows: the compound organic composition is prepared by compounding carthamin, egg yolk lecithin, cholesterol and an organic solvent, wherein the compound dosage of the carthamin, the egg yolk lecithin and the cholesterol is set according to the following standard: the mass ratio of the medicine to the fat is 1:20-1:150, and the mass ratio of the gall to the fat is 1:2-1:40; the softening agent in the water phase solution containing the softening agent adopts 1, 2-propylene glycol, and the mass fraction of the softening agent in the water phase solution is 10% -30%; the ultrasonic time of the probe in the ultrasonic treatment process is 4-6 min, and the rotating speed is 7000-9000 r/min.
5. The method for preparing safflower yellow nano flexible liposome according to claim 4, wherein: the mass ratio of the medicine to the lipid is 1:30-1:100, and the mass ratio of the bile to the lipid is 1:10-1:30.
6. The method for preparing safflower yellow nano flexible liposome according to claim 4, wherein: the mass ratio of the medicine to the lipid is 1:50, and the mass ratio of the bile to the lipid is 1:20.
7. The method for preparing safflower yellow nano flexible liposome according to claim 4, wherein: the organic solvent is absolute ethanol or dichloromethane-methanol, and the mass ratio of the organic solvent to safflower yellow is (20-40) 1; the temperature of the rotary evaporation film-making process is controlled to be 45-50 ℃, and the rotation speed is controlled to be 65-70 r/min.
8. The method for preparing safflower yellow nano flexible liposome according to claim 4, wherein: the dosage of the small glass beads is 1 small glass bead for each 3-5mL of aqueous phase solution; the duration of the rotary hydration process is controlled to be 0.5h-1.5h; in the probe ultrasonic treatment process, the probe ultrasonic time is 5min, and the rotating speed is 8000r/min.
9. The method for preparing safflower yellow nano flexible liposome according to claim 4, wherein: accurately weighing 1.44g of egg yolk lecithin, 24mg of cholesterol and 20mg of safflower yellow raw material medicine for injection, placing the three raw material medicines into an eggplant type bottle, and adding 50mL of absolute ethyl alcohol for full dissolution to obtain an oil phase; adding 7.2mL of 1, 2-propylene glycol into 38mL of distilled water, and mixing uniformly to obtain a water phase; removing absolute ethyl alcohol from the oil phase by rotary evaporation under reduced pressure at 50 ℃ to obtain a dry film, pouring 15mL of water phase preheated at 50 ℃ into a film medium, adding 3-5 small glass beads for rotary hydration for 1h, ultrasonically treating with a probe of 8000r/min for 5min, passing through a polypropylene filter membrane of 0.22 mu m, adding the residual water phase, and measuring the encapsulation rate of liposome to complete the preparation.
10. A safflower yellow nano-flexible liposome having affinity and absorption activity to skin and/or mucous membrane of an organic organism, characterized in that: a method of manufacture according to any one of claims 1 to 9 for achieving synergistic attenuation of safflor yellow by alternative routes of administration by injection.
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