WO2021088983A1 - 一种高生物利用度的7,8-二羟基黄酮复合纳米生物材料及其制备方法和用途 - Google Patents

一种高生物利用度的7,8-二羟基黄酮复合纳米生物材料及其制备方法和用途 Download PDF

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WO2021088983A1
WO2021088983A1 PCT/CN2020/127104 CN2020127104W WO2021088983A1 WO 2021088983 A1 WO2021088983 A1 WO 2021088983A1 CN 2020127104 W CN2020127104 W CN 2020127104W WO 2021088983 A1 WO2021088983 A1 WO 2021088983A1
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dhf
zein
composite nano
nanoparticles
glycosylated
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PCT/CN2020/127104
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French (fr)
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张英
陈玉峰
韩建欣
黄骆镰
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杭州尤美特科技有限公司
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    • 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
    • A61K31/352—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 condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the fields of medicines, natural products, functional foods and dietary supplements. More specifically, it relates to 7,8-dihydroxyflavonoid composite nano biomaterials with high bioavailability, and preparation methods and uses thereof.
  • 7,8-Dihydroxyflavone (7,8-DHF) is a flavonoid aglycone with low frequency and abundance that exists in nature. It was first discovered in a vegetable used in salads by Westerners. So far it has only been reported. It has been detected in the plants Godmania aesculifolia, Tridax procumbens, Primula and Malus hupehensis in the central and southern United States. A large number of studies have shown that 7,8-DHF can penetrate the blood-brain barrier, effectively mimic brain-derived neurotrophic factor (BDNF), and specifically activate TrkB receptors, thereby inducing dimerization and autophosphorylation of TrkB.
  • BDNF brain-derived neurotrophic factor
  • the team has obtained two potential new drugs by chemical structure modification: (1) the original drug A ring 7,
  • the oral bioavailability of the simulated drug R13 (known as the prodrug) after the two OHs at the 8-position are protected is close to 18% (increased by 3.75 times compared to the original drug), and the treatment of Alzheimer's disease has been completed in mainland China and Australia respectively.
  • the pre-clinical study of Schimheimer’s disease has now been approved by the US FDA for clinical trials.
  • the oral bioavailability of the pure chemical drug (CF3-CN) after further structural modification on the B ring of the original drug increased by 136%.
  • the purpose of the present invention is to provide a 7,8-dihydroxyflavone composite nano biomaterial with the advantages of high bioavailability, high brain targeting, excellent digestive tract stability, good storage stability, and convenient long-term storage. Preparation method and use.
  • a composite nano-biological material which includes the following components:
  • a drug carrier comprising zein and lactoferrin
  • the drug carrier encapsulates the drug.
  • the drug carrier has a core-shell structure, zein is the core, and lactoferrin is the shell.
  • the molecular weight of the zein is 25-45Kda, preferably 25-35Kda, more preferably 25-29Kda, preferably 25Kda.
  • the lactoferrin is an iron-binding protein derived from human milk, cow milk or goat milk.
  • the molecular weight of the lactoferrin is 60-200Kda, preferably 65-150Kda, more preferably 70-120Kda, most preferably 75-100Kda, preferably 80Kda.
  • the lactoferrin is glycosylated lactoferrin.
  • the glycosylated lactoferrin is dextran glycosylated lactoferrin.
  • the molecular weight of the dextran is 5-100Kda, preferably 10-80Kda, more preferably 20-60Kda, most preferably 30-50Kda, preferably 40Kda.
  • the dextran glycosylated lactoferrin is processed by Maillard reaction, such as at a temperature of 40-80°C (preferably 50-70°C) and a relative humidity of 60-90% (preferably, the graft product is obtained under the conditions of 70-85%) and reaction time of 20-60h (preferably 30-55h).
  • the drug further comprises a bioflavonoid selected from the group consisting of: bamboo leaf carboside flavonoids, hesperetin, naringenin, EGCG, baicalein, leucoxanthin, kaempferol, chickpein A. Quercetin, myricetin, genistein or a combination thereof.
  • a bioflavonoid selected from the group consisting of: bamboo leaf carboside flavonoids, hesperetin, naringenin, EGCG, baicalein, leucoxanthin, kaempferol, chickpein A. Quercetin, myricetin, genistein or a combination thereof.
  • the bamboo leaf carbon glycoside flavonoids are selected from the group consisting of orientin, isoorientin, vitexin, isovitexin, or a combination thereof.
  • the medicine is 7,8-dihydroxyflavonoids and 70% pure flavonoids.
  • the drug carrier encapsulates the drug, and the encapsulation rate is> 60% (preferably> 70%, preferably> 80%, preferably> 90%, preferably> 92%, more preferably >95%).
  • the mass ratio of the zein to the lactoferrin is 0.8-1.5 (preferably 0.9-1.2, more preferably 0.95-1.1).
  • the mass content of the drug is 3-10 wt% (preferably 4-8 wt%, more preferably 5-7 wt%) based on the total freeze-dried weight of the composite nano biomaterial.
  • the mass ratio of 7,8-dihydroxyflavone to zein in the composite nano biomaterial is 1:5-15, preferably 1:8-12.
  • the mass ratio of 7,8-dihydroxyflavonoids, bamboo flavonoids and zein is 1:1:5-15, preferably 1: 1: 8-12.
  • the average particle size of the composite nano-biological material is 50-150nm (preferably 60-140nm, more preferably 70-130nm, most preferably 80-120nm);
  • the dispersion coefficient of the composite nano-biological material is 0.2-0.5 (preferably 0.25-0.4);
  • the composite nano biomaterial is amorphous.
  • the second aspect of the present invention provides a method for preparing the composite nano biomaterial according to the first aspect of the present invention, including the steps:
  • the first mixed solution includes a first solvent, a drug, and zein;
  • the second mixed liquid includes a second solvent and lactoferrin
  • the first solvent is selected from the following group: ethanol, water, or a combination thereof.
  • the first solvent is an ethanol-water solution, preferably the ethanol concentration is 70-95%, preferably 75-90%, more preferably 80-85%.
  • the mass ratio of the drug to the zein in the first mixed solution is 1:4-15, preferably 1:5-12, more preferably 1. :7-11, preferably 1:10.
  • the second solvent is water.
  • step 2) the volume ratio of the first mixed liquid and the second mixed liquid is 1:2-5, preferably 1:2.5-4.
  • the mass ratio of the zein to the lactoferrin in the third mixed solution is 0.3-20, preferably 0.5-15, more preferably 0.8-10 , The best is 0.9-2.
  • the third aspect of the present invention provides a use of the composite nano biomaterial according to the first aspect of the present invention for use selected from the following group:
  • the form of the product is selected from the group consisting of solid beverages, formula milk powder, compressed candies, tablets, granules, capsules, freeze-dried powder injections, or combinations thereof.
  • Figure 2 is the chemical characterization of glycosylated LF:
  • A is the SDS-PAGE chart of LF and LF-dextran grafts, strip 0: LF, strip 1: LF 10K , strip 2: LF 40K and strip Band 3: LF 70K ;
  • B is the grafting efficiency and browning degree of the LF-dextran graft;
  • C is the zeta potential electromotive force of LF and LF-dextran graft;
  • D is the circular dichrograph;
  • E It is the Fourier infrared spectrogram.
  • Figure 3 is the TEM image of different nanoparticles; A is DHF-zein nanoparticles, B is DHF-zein/LF nanoparticles, C is DHF-zein/LF 10K nanoparticles, D is DHF-zein/LF 40K nanoparticles, E is DHF-zein/LF 70K nanoparticles, 50000 ⁇ magnification.
  • Figure 4 is the FE-SEM image of different nanoparticles; A is zein nanoparticles, B is DHF-zein nanoparticles, C is DHF-zein/LF nanoparticles, D is DHF-zein/LF 10K nanoparticles, E is DHF -zein/LF 40K nanoparticles, F is DHF-zein/LF 70K nanoparticles, 50000 ⁇ magnification.
  • Figure 5 shows the thermal behavior of 7,8-DHF and different nanoparticles.
  • Figure 6 shows the X-ray diffraction patterns of different samples.
  • Fig. 7 is the Fourier infrared spectrogram of no-load and loaded nanoparticles; A is no-load, and B is a load.
  • Figure 8 shows the physical and chemical stability of the nanocarrier; A is the effect of different pH conditions on the average particle size of different nanosystems, B is the effect of different ionic strength and pH on the average particle size of zein, and C is the effect of different ionic strength and pH values.
  • D is the influence of different ionic strength and pH value on the average particle size of zein/LF 10K
  • E is the influence of different ionic strength and pH value on the average particle size of zein/LF 40K
  • F is The effect of different ionic strength and pH value on the average particle size of zein/LF 70K
  • G is the effect of storage time on the average particle size of different nanoparticles
  • H is the effect of heat treatment on the average particle size of different nanoparticles.
  • Figure 9 shows the effect of in vitro digestion simulation on the average particle size of loaded nanoparticles and the in vitro bioavailability; A is the average particle size, and B is the in vitro bioavailability.
  • Figure 10 is FE-SEM images of different nanoparticles before and after in vitro digestion; 15000 ⁇ magnification.
  • Figure 11 is the concentration and time curve of 7,8-DHF after oral administration of free 7,8-DHF and loading 7,8-DHF composite nanoparticles.
  • the present inventors unexpectedly prepared a 7,8-dihydroxyflavonoid composite nano-biomaterial, the material is 7,8-dihydroxyflavonoid alone or a complex composed of other bioflavonoids.
  • the core material uses Zein (Zein) and (glycosylated) lactoferrin (LF) as packaging materials.
  • LF lactoferrin
  • the coating load of the core material by the packaging material makes the obtained material have the following advantages: high bioavailability, high brain targeting, excellent digestive tract stability, good storage stability, and convenient long-term storage Wait. On this basis, the inventor completed the present invention.
  • core material refers to 7,8-DHF alone or in combination with other bioflavonoids.
  • the core material is a composite composed of 7,8-DHF and 70% accuracy of bamboo leaf carboside flavonoids at a mass ratio of 1:1.
  • the term "packaging material” refers to the use of two edible proteins, Zein (Zein) and Lactoferrin (LF), preferably composed of Zein and glycosylated LF.
  • glycosylated LF is obtained by Maillard reaction (for example, at a temperature of 60°C, a relative humidity of 79%, and a reaction of 48h) with dextran of different molecular weights (such as 10, 40, and 70kDa).
  • the grafted product (denoted as LF 10K , LF 40K and LF 70K ), more preferably, refers to the grafted product of LF and 40 kDa glucan (denoted as LF 40K ).
  • nanotransmitter refers to a composite nanocarrier composed of Zein and LF, preferably a composite nanocarrier composed of Zein and glycosylated LF. More preferably, it is a composite nanotransmitter constructed by Zein and LF 40K.
  • the term "composite nanoparticle” refers to a nanotransmitter composed of Zein and LF or glycosylated LF, wrapped in 7,8-DHF or a complex composed of other plant flavonoids, and then prepared as a composite bio-nanoparticle material.
  • it refers to a nanocarrier composed of Zein and glycosylated LF (LF 10K , LF 40K and LF 70K ), using 7,8-DHF as the core material to make composite nanoparticles.
  • it refers to a composite bio-nano material prepared by using a carrier package composed of Zein and LF 40K with 7,8-DHF and bamboo leaf carbon glycoside flavone composition as the core material.
  • in vitro anti-digestion performance refers to the varying degrees of average particle size and particle structure of different composite nanoparticles carrying 7,8-DHF after being digested in the in vitro simulated gastrointestinal tract.
  • bioavailability refers to the 7,8-DHF content level in the mixed micelles after digestion of gastric juice and intestinal juice in the process of simulated digestion in vitro, and the level of 7,8-DHF in the undigested initial sample. , The ratio of 8-DHF content.
  • oral bioavailability refers to the “relative bioavailability", that is, the difference between different composite nano-formulations carrying 7,8-DHF and the original substance (7,8-DHF).
  • the bioavailability value obtained by comparing the degree of absorption and the rate of absorption with each other.
  • drug targeting refers to the level of 7,8-DHF actually detected in the main target organ (ie brain tissue) after the test animals take different 7,8-DHF composite nano-biomaterials. Compared to oral non-nanoized raw materials, the degree of improvement.
  • 7,8-Dihydroxyflavone (7,8-DHF) is one of the naturally occurring flavonoid family members.
  • BDNF brain-derived neurotrophic factor
  • TrkB receptors bind to TrkB receptors, thereby inducing dimerization and autophosphorylation of TrkB, and further activation of receptors.
  • MAPK/ERK MAPK/ERK
  • PI3K/Akt PI3K/Akt
  • 7,8-DHF is used in various BDNF/TrkB signal related diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Rett syndrome, depression, obesity, etc.) in the prevention and treatment research, and A series of exciting results have been achieved.
  • the inventor's recent cell, animal and human experimental studies have shown that 7,8-DHF has a very prominent effect in the prevention and treatment of women's menopausal (also known as perimenopausal) syndrome and its osteoporosis.
  • the 7,8-DHF composite nano biomaterial with high bioavailability provided by the present invention refers to: using zein and (glycosylated) lactoferrin as packaging materials, using 7,8-DHF or other
  • the composite nano-particles composed of bioflavonoids that is, the former is the monarch drug and the latter is the minister drug
  • the composite nanoparticles are prepared by the anti-solvent precipitation method.
  • the nanomaterial has much higher bioavailability and brain targeting than free 7,8-DHF. It has excellent stability in a wide pH range (pH 3-9), different ionic strength (0-500mmol/L NaCl) and high temperature (95°C), as well as good storage stability.
  • the average particle size of the nano particles is between 50 nm and 150 nm, and the encapsulation rate of the target object (that is, the core material) is above 90%, and can be stored for a long time after lyophilization, and has good rehydration.
  • the results of the in vitro simulated digestion test show that after digestion of gastric juice and intestinal juice, the nanomaterial can still maintain a complete structure to reach the absorption site of the small intestine.
  • the results of the in vivo bioavailability test showed that compared with the free state 7,8-DHF, the oral bioavailability was increased by 3 to 8 times, and the effective concentration in the brain tissue was also significantly increased, showing good blood-brain barrier targeting. This is closely related to the presence of lactoferrin on the surface of nanoparticles.
  • 7,8-DHF and bamboo leaf carbon glycoside flavonoids are beneficial to improve The absorption of intestinal mucosal cells significantly improves its oral bioavailability.
  • the lactoferrin used in the present invention is an iron-binding protein with a molecular weight of about 80Kda derived from human milk, cow milk or goat milk. It has certain resistance to pepsin and trypsin, and is found in human intestinal epithelial cells and blood-brain barrier. All have corresponding receptors. Lactoferrin is compounded with glucans of different molecular weights (10, 40 and 70kDa) at a mass ratio of 1:1. The glycosylated lactoferrin prepared by Maillard reaction has more powerful functions and more stable structure.
  • the preparation method of the nanomaterial is roughly as follows: dissolve zein in a high-concentration ethanol solution (such as an 80% ethanol aqueous solution), and add lactoferrin or glycosylated lactoferrin, zein alcohol in a certain volume ratio
  • the mass ratio of soluble protein to lactoferrin/glycosylated lactoferrin is approximately 1:1.
  • the mass percentage of dry powder is about 5 to 7%).
  • the nano material of the present invention greatly improves the bioavailability and brain targeting of 7,8-DHF, and its biological effect is more powerful. It can be widely used in the fields of functional (health) foods and new medicines to prevent and treat Alzheimer's. Chronic diseases such as Hymer's disease, Parkinson's syndrome, depression, obesity, osteoporosis and menopausal syndrome.
  • the preparations can appear in various forms such as solid beverages, formula milk powders, compressed candies, tablets, granules, capsules, freeze-dried powder injections and so on.
  • the present invention provides a high bioavailability 7,8-DHF composite nano biomaterial, which uses Zein (Zein) and (glycosylated) lactoferrin (LF) as carriers, Using 7,8-DHF or a compound with other plant flavonoids (the former is a monarch drug, the latter is a minister drug) as the core material, the composite nanoparticles prepared by the anti-solvent precipitation method have much higher than the original material ( 7,8-DHF) oral bioavailability and brain targeting, at the same time has a high pH, ionic strength, high temperature, storage stability and digestion resistance, the average particle size is in the range of 60 ⁇ 150nm, the carrier to the target The encapsulation rate of the material (ie the core material) is above 90%, and the nano-suspension can be stored for a long time after freeze-drying, and the freeze-dried powder has good rehydration.
  • Zein Zein
  • LF lactoferrin
  • the composite nano-particles refer to composite nano-biological materials constructed with Zein and LF as packaging materials and 7,8-DHF as core materials.
  • it refers to a composite nano biomaterial prepared by wrapping 7,8-DHF with a binary transmitter composed of Zein and glycosylated LF.
  • glycosylated LF is the grafting of LF and glucans of different molecular weights (such as 10, 40 and 70kDa) through Maillard reaction (such as at a temperature of 60°C, a relative humidity of 79%, and a reaction time of 48 hours).
  • the products are denoted as LF 10K , LF 40K and LF 70K . .
  • the composite nanoparticle refers to a composite nanotransmitter composed of Zein and LF 40K (ie packaging material), loaded with 7,8-DHF or a combination with bamboo leaf flavonoids (ie core material) The composite nano-biological material prepared afterwards.
  • the core material is a combination of 7,8-DHF and 70% precision bamboo leaf flavonoids (BLF 70 ).
  • the four carbon glycoside flavonoids (orientin, isoorientin) in BLF 70 Glucoside, vitexin and isovitexin) together account for 65-75% (w/w) of the total mass of the preparation.
  • the present invention also provides a method for preparing the above-mentioned composite nano biological material: the core material and Zein are respectively dissolved in 80% ethanol-water solution with different mass ratios as the solvent phase, and LF or glycosylated LF is dissolved in distilled water
  • the solvent phase was quickly added to the anti-solvent phase at a volume ratio of 1:3 (v/v), stirred for 30 minutes, and the mass ratio of Zein to LF or glycosylated LF in the mixed solvent system was controlled at 20: 1 ⁇ 1:3; then, use a rotary evaporator to remove the excess ethanol solution under reduced pressure (40°C, -0.1Mpa), and finally get an average diameter of 60 ⁇ 150nm and a core material encapsulation ratio of 66 ⁇ A suspension of 99.5% composite nanoparticles; this suspension was pre-frozen at -80°C for 24h and then vacuum freeze-dried (-50°C, -0.1Mpa) for 36h to obtain
  • 7,8-DHF:Zein is between 1:5 and 1:15.
  • the mass ratio of 7,8-DHF to Zein is 1:10 (w/w).
  • the mass ratio of 7,8-DHF:BLF 70 :Zein is 1:1:10 (w/w).
  • the anti-solvent system is an aqueous solution of LF or glycosylated LF.
  • the anti-solvent system is an aqueous solution of glycosylated LF.
  • the mass ratio of Zein to LF or glycosylated LF can be 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3 (w /w).
  • the mass ratio of Zein to LF is 1:1 (w/w).
  • the mass ratio of Zein to glycosylated LF is 1:1 (w/w).
  • the encapsulation rate of the composite transmitter to the core material in the present invention when the mass ratio of 7,8-DHF to Zein is between 1:5 and 1:15, and the mass ratio of Zein to LF or glycosylated LF is 1 :1, the encapsulation rate of 66 to 99.5% can be achieved.
  • the encapsulation ratio of the composite transmitter to the core material when the mass ratio of 7,8-DHF and Zein is 1:10, and the mass ratio of Zein to LF or glycosylated LF is 1:1, A 98-99.5 percent encapsulation rate can be achieved.
  • the optimized preparation method of the composite nano-biological material of the present invention is as follows: the core material (7,8-DHF or a combination with other plant flavonoids) and Zein are dissolved in an 80% ethanol-water solution at a mass ratio of 1:10. Solvent phase, dissolve LF or glycosylated LF in distilled water as the anti-solvent phase, quickly add the solvent phase to the anti-solvent phase at a volume ratio of 1:3 and stir for 30 minutes (stirring strength 800rmp/min). Zein The mass ratio to LF (or glycosylated LF) is 1:1.
  • a more preferred preparation method is as follows: Dissolve 7,8-DHF:BLF 70 :Zein in a mass ratio of 1:1:10 in an 80% ethanol-water solution as the solvent phase, and dissolve the glycosylated LF Distilled water was used as the anti-solvent phase.
  • the solvent phase was quickly added to the anti-solvent phase at a volume ratio of 1:3, stirred for 30 minutes (stirring intensity 800rmp/min), and then used a rotary evaporator to reduce pressure (40°C, -0.1Mpa)
  • Excess ethanol was removed under conditions to obtain a suspension of composite bio-nanoparticles with an average particle size between 70 and 100 nm and an encapsulation efficiency of 96.21% and 92.13% for 7,8-DHF and BLF, respectively.
  • the suspension was placed at -80°C. After being pre-frozen for 24 hours and then subjected to vacuum freeze-drying (-50°C, -0.1Mpa, 36h), 7,8-DHF and bamboo leaf flavonoids composite nano-particle freeze-dried powder can be obtained.
  • the present invention also provides the structural characterization of the composite nano biomaterial after loading 7,8-DHF (and its bamboo leaf flavonoids), which meets all or any of the following conditions:
  • the composite nanomaterials are spherical in appearance, with an average particle size in the range of 60-150nm; and, due to the presence of LF or glycosylated LF, the adsorption of LF significantly changes the properties of ordinary Zein nanoparticles. Surface structure. As shown in Figure 3 and Figure 4.
  • the thermal properties of composite nanomaterials The composite transmitter composed of glycosylated LF and Zein significantly improves the thermal stability of the nanomaterials; at the same time, as the carbon chain of glucan used during branching grows (that is, the molecular weight increases) ), the thermal stability of the composite nanotransmitter also increased (melting temperature increased from 68.57°C to 75.74, 80.89 and 85.60°C respectively). As shown in Figure 5.
  • 7,8-DHF is encapsulated in the composite transmitter through hydrogen bonding, hydrophobic force and electrostatic interaction, and at the same time LF or glycosylated LF and Zein are formed by hydrogen bonding and hydrophobic interaction
  • the special structure of the transmitter surface (similar to the core-shell structure where Zein is the core, LF or glycosylated LF is the shell). As shown in Figure 7.
  • the present invention also provides the physical and chemical stability of the composite nanotransmitter and the nanoparticle loaded with the target substance and the in vitro simulated digestion resistance:
  • the composite nanotransmitter of Zein and LF can remain stable in the range of pH 3-9, and can withstand a wide range of ionic strength (0-500mmol/L NaCl) under neutral and alkaline conditions.
  • Good storage stability nanonosuspension can be kept stable for 30 days
  • thermal stability heating at 95°C for 60 minutes).
  • the physical and chemical stability of the nanotransmitter is that the composite transmitter constructed by Zein and glycosylated LF (LF 10K , LF 40K, and LF 70K ) can remain stable in the range of pH 3-9, at pH Under 3-9 conditions, it can tolerate a wide range of ionic strength (0-500mmol/L NaCl), and has good storage stability (nanosuspension can be kept stable for 30 days) and thermal stability (heating at 95°C for 60 minutes).
  • Zein and glycosylated LF LF 10K , LF 40K, and LF 70K
  • the physical and chemical stability of the nanotransmitter is that the composite nanotransmitter constructed by Zein and LF 40K can remain stable in the range of pH 3-9, and can withstand a wide range under the condition of pH 3-9
  • the ionic strength (0 ⁇ 500mmol/L NaCl), has good storage stability (nanosuspension can be kept stable for 30d) and thermal stability (heating at 95°C for 60min).
  • the in vitro simulated digestion resistance of composite nanoparticles loaded with 7,8-DHF is that Zein/LF composite nanoparticles loaded with 7,8-DHF undergo digestion in the gastrointestinal tract, and the average particle size increases and precipitation occurs.
  • the carrier structure The change is obvious. Compared with free 7,8-DHF, the in vitro bioavailability increased by 3.52 times.
  • the in vitro simulated digestion resistance of the composite nanoparticles loaded with 7,8-DHF is Zein/glycosylated LF (LF 10K , LF 40K and LF 70K ) composite nanoparticles loaded with 7,8-DHF
  • LF 10K , LF 40K and LF 70K Zein/glycosylated LF
  • the in vitro simulated digestion resistance of the 7,8-DHF-loaded composite nanoparticles is that the 7,8-DHF-loaded Zein/glycosylated LF (LF 40K ) composite nanoparticles undergo gastrointestinal digestion After that, the average particle size is stable without precipitation, and the support structure is still spherical. Compared with free 7,8-DHF, the in vitro bioavailability increased by 4.65 times.
  • the present invention also provides data on the improvement of the bioavailability of the 7,8-DHF composite nano-biological material by oral administration in mice and the substantial increase of the 7,8-DHF level in the main target organ (brain homogenate).
  • the same mass of 7,8-DHF (ie DHF-Zein/LF 40K ) encapsulated in Zein/LF 40K composite nanoparticles has the highest relative oral bioavailability An increase of 8.46 times.
  • the composite of 7,8-DHF and BLF 70 ie DHF-BLF 70 -Zein/LF encapsulated in Zein/LF 40K composite nanoparticles 40K ) has the highest relative oral bioavailability, increasing by 10.12 times.
  • the level of 7,8-DHF in the brain homogenate (main target organ) of experimental animals is determined by liquid mass spectrometry: the oral dose of mice is 25mg/kg.bw, and different samples are converted into the same 7,8-DHF Absolute quality.
  • Chromatographic conditions Shimadzu Nexerra UPLC system, analytical column AcquityHSS T3 1.8 ⁇ m 2.1 ⁇ 50mm; mobile phase flow rate: 0.7mL/min, mobile phase A is 0.1% ammonium formate solution, mobile phase B is acetonitrile (containing 0.1% formic acid, v/ v).
  • Mass spectrometry conditions AB Sciex Qtrap 6500, ion source ESI (+) and (-), scan mode is MRM.
  • the composite nanoparticle DHF-Zein/LF 40K (the absolute mass of 7,8-DHF is 25mg/kg.bw)
  • the absolute mass of 7,8-DHF is 25mg/kg.bw
  • the actual content of 7,8-DHF was 70.1ng/g, and 42.5ng/g at 2h; compared with the control group that took the original substance of 7,8-DHF (dissolved in 5% sodium carboxymethylcellulose aqueous solution) orally,
  • the actual content of 7,8-DHF in mouse brain homogenate was increased by 2.73 times at 0.5h, and by 3.10 times at 2h.
  • the mouse oral administration of composite nanoparticles DHF-BLF 70 -Zein/LF 40K converted to the absolute mass of 7,8-DHF is 25mg/kg.bw
  • the mouse is measured at 0.5h
  • the actual content of 7,8-DHF in brain homogenate is 93.2ng/g and 36.8ng/g at 2h; compared with the original substance of 7,8-DHF (dissolved in 5% sodium carboxymethylcellulose aqueous solution)
  • the actual content of 7,8-DHF in mouse brain homogenate measured at 0.5h increased by 3.63 times, and at 2h it increased by 2.67 times.
  • a kind of complexes with Zein (Zein) and (glycosylated) lactoferrin (LF) as carriers, with 7,8-DHF or other plant flavonoids (the former is a prime drug, the latter is a minister drug) ) Is a core material and is prepared by using an anti-solvent precipitation method to prepare composite nano biological particles, and a preparation method and application thereof.
  • the composite nano biological particles have an average particle size of 60-150 nm, and the encapsulation rate of the carrier to the target (ie core material) is above 90%.
  • the nano-suspension can be stored for a long time after freeze-drying, and the freeze-dried powder has good rehydration It has much higher oral bioavailability and brain targeting than the original substance (7,8-DHF), and has good storage stability and anti-digestive properties.
  • the 7,8-DHF composite nano-biological material is characterized in that: the composite nano-biological material is a composite nano-particle prepared by using a composite nano-carrier composed of Zein and LF and using 7,8-DHF as a core material.
  • a composite nano biomaterial prepared by wrapping 7,8-DHF with a binary transmitter composed of Zein and glycosylated LF.
  • glycosylated LF is the grafting of LF and glucans of different molecular weights (such as 10, 40 and 70kDa) through Maillard reaction (such as at a temperature of 60°C, a relative humidity of 79%, and a reaction for 48 hours).
  • the products are denoted as LF 10K , LF 40K and LF 70K .
  • the 7,8-DHF composite nano-biological material is characterized in that: the composite nano-biological material is a composite nanotransmitter (ie packaging material) composed of Zein and LF 40K, loaded with 7,8-DHF or with bamboo leaves Composite nano biomaterials prepared by flavonoid composition (ie core material).
  • the composite nano-biological material is a composite nanotransmitter (ie packaging material) composed of Zein and LF 40K, loaded with 7,8-DHF or with bamboo leaves Composite nano biomaterials prepared by flavonoid composition (ie core material).
  • BLF 70 flavone C-glycosides Orientin, Isoorientin, vitexin and isovitexin
  • the anti-solvent precipitation method for preparing 7,8-DHF composite nano biomaterials the core material and Zein are dissolved in 80% ethanol-water solution with different mass ratios as the solvent phase, and LF or glycosylated LF is dissolved in Distilled water is used as the anti-solvent phase.
  • the solvent phase is quickly added to the anti-solvent phase at a volume ratio of 1:3 (v/v), stirred for 30 minutes, and the mass ratio of Zein to LF or glycosylated LF in the mixed solvent system is controlled within Between 20:1 and 1:3; subsequently, use a rotary evaporator to remove excess ethanol solution under reduced pressure (40°C, -0.1Mpa), and finally obtain an average diameter of 60-150nm and a core material encapsulation rate 66 ⁇ 99.5% of composite nanoparticle suspension; this suspension is pre-frozen at -80°C for 24h and then vacuum freeze-dried (-50°C, -0.1Mpa) for 36h to obtain 7,8-DHF composite nano-organism Freeze-dried powder of materials.
  • the preparation method of 7,8-DHF composite nano biomaterial is characterized in that: in the mass ratio of the key core material to the wall material, 7,8-DHF:Zein is between 1:5 and 1:15.
  • 7,8-DHF:Zein in the mass ratio of the key core material to the wall material, 7,8-DHF:Zein is between 1:5 and 1:15.
  • the mass ratio of 7,8-DHF to Zein is 1:10 (w/w)
  • the mass ratio of zein to LF or glycosylated LF is 1:1 (w/w)
  • 7,8-DHF The mass ratio of :BLF 70 :Zein is 1:1:10 (w/w).
  • the composite nano biological material has good stability and storage.
  • the composite transmitter constructed by Zein and glycosylated LF can remain stable in the range of pH 3-9, and can tolerate a wide range of ionic strength (0-500mmol/L NaCl) under the condition of pH 3-9, and has a good
  • the composite nano biological material has good digestion resistance. After the Zein/LF composite nanoparticles loaded with 7,8-DHF undergo digestion in the gastrointestinal tract, the average particle size increases and precipitation occurs, the carrier structure changes significantly, and the in vitro bioavailability increases compared to free 7,8-DHF 3.52 times; the in vitro simulated digestion resistance of composite nanoparticles loaded with 7,8-DHF is that Zein/glycosylated LF (LF 40K ) composite nanoparticles loaded with 7,8-DHF undergo gastrointestinal digestion. The average particle size is stable without precipitation, and the carrier structure is still spherical. Compared with free 7,8-DHF, the in vitro bioavailability increased by 4.65 times.
  • the composite nano biomaterial significantly improves the bioavailability improvement data of the 7,8-DHF composite nano biomaterial and the level of 7,8-DHF in the main target organ (brain homogenate):
  • 7,8-DHF ie DHF-Zein/LF 40K
  • Zein/LF 40K composite nanoparticles has the highest relative oral bioavailability Increased by 8.46 times;
  • the 7,8-DHF and BLF 70 complex ie DHF-BLF 70 -Zein/LF 40K encapsulated in Zein/LF 40K composite nanoparticles
  • the relative oral bioavailability of increased by 10.12 times.
  • the composite nano-biological material significantly increases the effective concentration level of 7,8-DHF in the main target organ (brain homogenate):
  • the high-bioavailability 7,8-DHF composite nano-biological material provided by the present invention has more powerful biological effects and is suitable for the possible applications of 7,8-DHF original material
  • Most uses such as oral drugs, health foods, functional foods, foods for special medical purposes, and daily chemicals or personal care products, can prevent and treat Alzheimer's disease, Parkinson's disease, depression, and obesity It has a variety of effects such as disease, osteoporosis and menopausal syndrome, improving sleep and skin.
  • the end product can be in various forms such as capsules, tablets, granules, powders, foods, drinks, candies, gels, etc.
  • the invention discloses a 7,8-DHF composite nano biomaterial with high bioavailability. It uses Zein and/or glycosylated lactoferrin (LF) as a carrier and uses 7,8 -DHF or a composite composed of bamboo leaf flavonoids as the core material, composite nanoparticles prepared by the anti-solvent precipitation method, the encapsulation efficiency is above 90%, the average particle size is about 100nm, and has a high degree of physical and chemical stability and Anti-digestive performance, freeze-dried powder can be stored stably for a long time, and has good rehydration. Compared with the original material of 7,8-DHF, the in vitro bioavailability of the composite nanomaterial is 3 to 5 times.
  • LF Zein and/or glycosylated lactoferrin
  • the composite nano-biological material of the present invention except that 7,8-DHF can be a natural product or a chemical synthesis, other raw and auxiliary materials are all derived from food or new food raw materials; the preparation process does not contain any other than ethanol (edible alcohol). Other organic solvents or chemical catalysts, the process is green and environmentally friendly.
  • the 7,8-DHF composite nano-biological material of the present invention is suitable for most uses where the original 7,8-DHF material may be used, such as oral medicine, health food, functional food, food for special medical purposes, and daily chemicals Or personal care products, etc., play various roles in preventing and treating Alzheimer's disease, Parkinson's disease, depression, obesity, osteoporosis and menopausal syndrome, improving sleep and skin.
  • the end product can be in various forms such as capsules, tablets, granules, powders, foods, drinks, candies, gels, etc.
  • the present invention has the following main advantages:
  • the raw and auxiliary materials are edible, degradable in vivo, safe, and the preparation method is convenient and green; for 7,8-DHF, encapsulation in the composite nanotransmitter of Zein-LF is a physical process (Adsorption and embedding), there is no substantial chemical structure change.
  • the composite nano biomaterials (DHF-Zein/LF, DHF-Zein/LF 40K and DHF-BLF 70 -Zein/LF 40K ) have an average particle size ranging from 60 to 150 nm, and the target (ie core material)
  • the encapsulation rate is above 90%. It can be stored for a long time after lyophilization, and has good rehydration. It has good system stability in a wide range of pH value (3-9) and ionic strength (0-500mmol/L NaCl), high temperature (95°C) and 30d storage process, and it is not easy to precipitate.
  • the composite nanoparticles (DHF-Zein/LF 40K ) can still maintain a complete structure to reach the absorption site of the small intestine of the experimental animal.
  • the composite nano-biological material of the present invention greatly improves the bioavailability and brain targeting of 7,8-DHF, which is due to the specific construction method of the composite nano-material of the present invention.
  • the main advantages are: 1Increase of absorption: Due to the high dispersion coefficient of nanoparticles and large surface area, the contact area of the biofilm with the absorption site is greatly increased, which improves the bioavailability; 2Increase of membrane permeability: Nano particles enter Cells are mainly endocytosed, which is conducive to the improvement of drug absorption and the performance of intracellular effects; 3Increase of targeting: LF and glycosylated LF in nanocarriers have a high degree of targeting and can identify intestinal and blood The LF receptor on the cell membrane of the brain barrier is mediated and targeted to bind to the brain through the LF receptor; 4Slow release is increased: the release efficiency of the nano drug delivery system can adjust the release of 7,8-DHF and enhance Its retention time in the body.
  • the biological efficacy of the composite nano-biological material of the present invention is stronger than that of 7,8-DHF, and can be widely used in the fields of functional (health) food and new drugs, and can prevent and treat Alzheimer’s disease, Parkinson’s syndrome, and depression.
  • Chronic diseases such as disease, obesity, osteoporosis and menopausal syndrome. It can appear in various forms such as solid beverages, formula milk powder, compressed candies, tablets, granules, capsules, freeze-dried powder injections, etc.
  • Zein mono- and binary (Zein/LF) nanotransmitters were prepared by anti-solvent precipitation method. Accurately weigh the zein powder, place it in 80% ethanol-water, and ultrasonically in a water bath for 5 minutes to prepare a storage solution (1%, w/v). Subsequently, the storage solution was quickly added to distilled water (ie, anti-solvent) at a volume ratio of 1:3, and continuously stirred (800 rpm) for 30 min at room temperature. Use a rotary evaporator to remove excess ethanol solution under reduced pressure (-0.1Mpa) at 40°C. Finally, the zein monotransmitter was obtained, and the final concentration of zein in the system was 2.5 mg/mL.
  • the average particle size and PDI of the zein primary and zein/LF binary transmitters were measured with a nanoparticle size analyzer at 25°C. The measurement angle was 90°, and the refractive index of water was 1.45.
  • the average particle size and dispersion coefficient of Zein/LF binary nanotransmitter under different mass ratios of zein and LF are shown in Figure 1.
  • the average particle size of the Zein monotransmitter is 169.56 nm.
  • the mass ratio of LF to Zein gradually increases, the average particle size of the binary transmitter first decreases and then increases.
  • the mass ratio of Zein to LF is 1:1, the average particle size is the smallest (74.63nm).
  • the PDI value also shows a trend of first rising and then decreasing.
  • the mass ratio of Zein:LF is 5:1, the PDI value is the highest.
  • the mass ratio is in the range of 2:1 to 1:3, the PDI value is at a low value ( ⁇ 0.200), indicating that a stable and uniform size nanocolloid system is formed.
  • the grafted products of LF-dextran ie glycosylated LF
  • LF 10K ie glycosylated LF
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): using 5% concentrated gel and 8% separating gel. Take 5 ⁇ L of sample solution (2mg/mL) and mix with 20 ⁇ L of protein loading buffer, place it in boiling water and heat for 5min, and quickly cool. Take 10 ⁇ L of the mixed sample and add it to each groove of the gel electrophoresis with a voltage of 80-120 mV. After electrophoresis, stain with 0.25% ((w/v) Coomassie Brilliant Blue R-250 solution, and decolorize with decolorizing solution (10% acetic acid, 50% methanol and 40% distilled water, v/v) overnight.
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the OPA working solution is configured as follows: 40mg OPA (dissolved in 1mL methanol), 2.5mL 20% SDS solution, 25mL 0.1mol/L sodium tetraborate buffer and 100 ⁇ L ⁇ -mercaptoethanol solution, mix well, and dilute to 50mL with distilled water. Mix 4.0 mL OPA working solution with 200 ⁇ L lactoferrin-dextran conjugate (2 mg/mL), react for 3 minutes at room temperature, and measure the absorbance at 340 nm. The standard curve of amino content was established with different concentrations of L-leucine (0.25 ⁇ 2.5mmol/L). The grafting efficiency is calculated by the following formula (4-5):
  • Degree of browning In order to evaluate the degree of browning caused by Maillard reaction, the graft was dissolved in distilled water (1.0mg/mL), and the LF-dextran graft was measured at 420nm with an ultraviolet-visible spectrophotometer The absorbance.
  • Zeta electromotive potential Use dynamic light scattering (DLS) to test Zeta electromotive potential at 25°C.
  • DLS dynamic light scattering
  • Circular Dichroism Scan at room temperature and continuous nitrogen flow, the scanning speed is 50nm/min, the bandwidth is 1.0nm, and the path length is 0.1cm.
  • the LF-dextran graft was dissolved in distilled water, the concentration of the sample was 0.2 mg/mL when the secondary structure was measured, and the scanning was performed in the wavelength range of 190 to 260 nm. And analyze the data obtained.
  • Infrared spectroscopy mix 1% LF-dextran grafted product with KBr and place it in an agate mortar and grind it evenly. Use FTIR to scan the sample. The scanning range is 500 ⁇ 4000cm -1 , and the resolution is 4cm -1 .
  • OMNIC software version 8.0 was used for analysis.
  • SDS-PAGE was used to determine the molecular weight of LF and glycosylated LF.
  • the main zone corresponding to LF is about 80kDa (band 0).
  • band 0 the bands of the three LF-dextran grafts at 80kDa decrease and the color becomes lighter, while the color of the band at high molecular weight becomes darker, resulting in high mobility, indicating that the two pass through the United States.
  • the Rader reaction produces high molecular weight glycosylated proteins (bands 1 to 3).
  • assessing the amino group content is an important indicator for analyzing the degree of polysaccharide binding.
  • the Maillard reaction occurs, the free amino group of the protein and the carbonyl group of the reducing sugar are covalently linked to form a Schiff base.
  • the OPA method was used to determine the number of free amino groups.
  • the grafting efficiencies of LF and dextran with molecular weights of 10, 40, and 70kDa were 24.96, 16.54, and 11.39%, respectively, indicating that the molecular weight of dextran has a significant effect on the grafting efficiency of LF.
  • the color depth of the Maillard reaction product can intuitively reflect the degree of the Maillard reaction, and the absorbance value at the wavelength of 420nm is usually used as an indicator of the amount of browning product.
  • the A 420 was 0.081, 0.041, and 0.030, respectively.
  • the zeta potential electromotive force of the LF-dextran grafted product is significantly lower than that of the mixture of LF and dextran (p ⁇ 0.05) ( Figure 3C), which is due to the participation of the positively charged -NH 2 group on LF The formation of Schiff base leads to the reduction of zeta potential electromotive force.
  • CD can reflect the secondary structure information of the protein.
  • the effect of glycosylation on the secondary structure of LF was studied, and the results are shown in Figure 4.12D.
  • the CD scan of the far-ultraviolet region of LF showed a negative minimum at 208nm and 215nm, and a maximum at 190-195nm, which is a typical protein spectrum with more ⁇ -helix and ⁇ -sheet structures.
  • the inset in Figure 3D shows that after glycosylation modification, the ⁇ -helix and ⁇ -sheet of LF are reduced, and the disordered structure increases, indicating that the secondary structure of glycosylated LF has changed to a certain extent.
  • FTIR spectroscopy data shows that the four LF samples have significant differences near 3300 ⁇ 3600cm -1 and 950 ⁇ 1150cm -1 (Figure 3E).
  • the absorption peaks of 3300 ⁇ 3600cm -1 are considered to be NH tensile vibration and OH tensile vibration.
  • the absorption peak of natural LF is mainly at 3291 cm -1 (amide A band, which represents NH stretched and combined hydrogen bond).
  • the absorption peak of glycosylated LF at 3351 cm -1 is significantly blue-shifted, and LF 10K , LF 40K and LF 70K increase by 54, 66 and 72 cm -1 respectively , indicating the -NH 2 group of LF Participated in Maillard reaction.
  • LF has two typical peaks in the 1500-1700 cm -1 band, which are 1651 cm -1 (amide I band) and 1538 cm -1 (amide II band).
  • the amide II band is mainly related to the CN stretching vibration.
  • Encapsulation efficiency (EE) measurement remove the free 7,8-DHF in the nanosuspension by high-speed centrifugation (4°C, 10000 ⁇ g, 10min). Take the supernatant (containing the nanoparticles loaded with 7,8-DHF), demulsify and dilute it with methanol 5 times, and dilute the same amount of the initial nanosuspension 5 times with methanol. 7,8-DHF is measured by UPLC method.
  • EE and LC calculation formulas (4-1 and 4-2) are as follows:
  • Table 1 summarizes the EE, average particle size and PDI values of 7,8-DHF loaded by different nano-delivery systems.
  • the mass ratio of Zein to 7,8-DHF is 5:1
  • the EE of DHF-Zein, DHF-Zein/LF DHF-Zein/LF 10K , DHF-Zein/LF 40K and DHF-Zein/LF 70K nanoparticles are respectively They were 37.27, 66.10, 72.41, 84.75 and 83.61%.
  • the encapsulation efficiency of LF, LF 10K , LF 40K and LF 70K combined with Zein has increased by about 1.7, 1.9, 2.3 and 2.2 times.
  • the EE of DHF-Zein, DHF-Zein/LF DHF-Zein/LF 10K , DHF-Zein/LF 40K and DHF-Zein/LF 70K nanoparticles are respectively It is 46.38, 98.31, 98.66, 99.41, 99.21%. It shows that LF and glycosylated LF stabilize Zein nanoparticles and form a stable binary transmitter system through non-covalent interactions (hydrogen bonding and hydrophobic force), thereby enhancing the effect of encapsulating 7,8-DHF, especially LF 40K combined with Zein as the carrier has the best encapsulation effect.
  • the average particle size of the Zein unitary system is 169.56nm, while the average particle size of the Zein/glycosylated LF binary system is reduced to 78.63 ⁇ 87.24nm.
  • the average particle size of Zein/LF 10K , Zein/LF 40K and Zein/LF 70K is slightly larger, which is related to the sugar chains bound on LF.
  • PDI also shows a trend similar to the average particle size.
  • Zein/LF and Zein/glycosylated LF binary nanoparticles loaded with 7,8-DHF show good water resolubility, and the reconstituted EE has decreased, but it is still higher than 90%.
  • the average particle size is In the range of 103.6-110.3nm
  • PDI is in the range of 0.265-0.295, showing a stable system with low particle size and uniform dispersion.
  • DHF-Zein/LF 40K nanoparticles have the best effect.
  • TEM transmission electron microscope
  • a differential scanning calorimeter was used to analyze the thermal characteristics of the freeze-dried samples. Accurately weigh 6-10 mg of sample into an aluminum crucible and seal it. Use an empty crucible with the same conditions as a reference. A N 2 atmosphere was used to raise the temperature from 20°C to 300°C at a heating rate of 10°C/min for scanning calorimetry analysis.
  • X-ray diffraction was performed for powdered 7,8-DHF, dry unloaded or loaded nanoparticles (lyophilized powder) XRD analysis.
  • the copper anode of the instrument generates copper K ⁇ radiation, the acceleration voltage is 40kV, and the tube current is 40mA.
  • the Soller slit is set to 2.5°, and the divergence slit is set to 0.5°.
  • the 2 ⁇ angle range is 5° ⁇ 70°, the step length is 0.02°, and the step length is 0.2s.
  • FTIR Fourier infrared spectroscopy
  • TEM shows the shape, size, uniformity and integrity of colloidal transmitters. It can be seen from Figure 3 that the average particle size of DHF-Zein (Zein nanoparticles loaded with 7,8-DHF) nanoparticles is above 100 nm3. In addition, the DHF-Zein nanoparticles are in a state of interconnection, which may be due to the aggregation of the Zein particles due to the hydrophobic interaction between the Zein particles after the Zein colloidal system is diluted 10 times.
  • DHF-Zein/glycosylated LF nanoparticles all showed a spherical shape in the range of 70-100nm (C, D and E in Figure 3), and the dispersion was uniform, indicating that the glycosylated emulsion
  • the adsorption of ferritin can increase electrostatic repulsion and steric repulsion effects.
  • the aggregation of DHF-Zein nanoparticles can be prevented.
  • the average particle size of DHF-Zein/glycosylated LF nanoparticles is relatively larger (Figure 3B).
  • X-ray diffraction was used to determine the crystal diffraction pattern of each sample within the range of 2 ⁇ values of 5°-90°.
  • LF 10K , LF 40K, and LF 70K also showed peak patterns similar to LF, indicating that these grafted proteins all exist in an amorphous form.
  • 7,8-DHF DHF-Zein/LF, DHF-Zein/LF 10K , DHF-Zein/LF 40K and DHF-Zein/LF 70K
  • 7,8 The characteristic diffraction peak of DHF crystalline morphology indicates that it becomes amorphous after being effectively encapsulated. This phenomenon is consistent with the results of thermal analysis.
  • Figure 7 shows the absorption peaks of different samples in the wavenumber range of 4000-500cm -1.
  • Zein's OH group stretching characteristic peak is 3306cm -1 ( Figure 7A).
  • the hydrogen bond characteristic peak The transfer from 3306 cm -1 to 3406, 3404 and 3417 cm -1 , respectively, indicates that the binding of Zein and glycosylated LF is involved in hydrogen bonding, and compared with Zein/LF nanoparticles, the hydrogen bonding ability is stronger.
  • the stretching vibration peak of the CH group of Zein hydrophobic at 2953 cm -1 is considered.
  • Example 3 The physical and chemical stability of nanotransmitters and the gastrointestinal stability of Zein/LF and Zein/glycosylated LF composite nanoparticles loaded with 7,8-DHF
  • the effect of storage time adjust the pH of the freshly prepared nanosuspension to 3.0-9.0, and store in light for 30 days at 25°C.
  • SGF simulated gastric juice
  • SIF simulated intestinal fluid
  • the final digestion solution was centrifuged at a centrifugal force of 20000 ⁇ g for 1 hour, and the supernatant (ie mixed micellar phase containing dissolved 7,8-DHF) was collected, and its 7,8-DHF content was determined by UPLC method.
  • the bioavailability is calculated as follows:
  • the final digestion solution was made into freeze-dried powder, which was characterized by FE-SEM, and its microscopic surface morphology was observed.
  • Figure 8 B, C, D, E, and F show the effect of ionic strength on the stability of the nanocarrier under different pH conditions.
  • the Zein unitary system is highly sensitive to ionic strength (Figure 8B).
  • Figure 8C Although the binary system is improved after adding LF, it is still unstable under low pH (3 ⁇ 5) conditions ( Figure 8C), while after LF glycosylation It exerts different degrees of stabilization effect.
  • the average particle size of nanoparticles increases with the increase of ionic strength. Under the conditions of high concentration of NaCl (500mmol/L) and low pH (3 and 4), its The average particle size exceeds 350 nm (Figure 8D).
  • the average particle size of Zein/LF 40K ( Figure 8E) and Zein/LF 70K ( Figure 8F) is in a wide range of pH (3-9) and ionic strength (0-500mmol/L NaCl) are less than 250nm and 200nm respectively, Zein/LF 70K shows the best stabilizing effect. It shows that as the molecular weight of dextran increases, its chain length increases correspondingly, resulting in greater steric hindrance and preventing the agglomeration effect of nanoparticles. At the same time, the interface layer formed by glycosylated LF on the surface of the particles also plays a role. To shield the external electric charge.
  • LF indicated by the nanoparticles has been grafted, which not only provides a strong spatial repulsion to overcome the mutual attraction between particles (hydrophobic interaction and van der Waals force), but also indicates that a new interface is formed on the particles to shield The degradation of acid, alkali and enzymes.
  • the FE-SEM image shows that the in vitro digestion simulation has an important influence on the surface morphology of the loaded nanoparticles.
  • the morphology of DHF-Zein and DHF-Zein/LF particles have undergone significant changes, especially DHF-Zein (the original particles are spherical and have a block structure after digestion).
  • DHF-Zein/LF binary particles After the DHF-Zein/LF binary particles are digested in the gastrointestinal tract, they change from a spherical shape to a square shape, which is caused by a large amount of DHF-Zein/LF binary particles agglomerated during the SIF digestion process.
  • the 7,8-DHF (made into 0.5% CMC suspension), DHF-Zein, DHF-Zein/LF and DHF-Zein/LF 40K samples were intragastrically administered respectively, and the intragastric dose was 50mg/kg (all at 7 , 8-DHF absolute content calculation).
  • blood 200 ⁇ L was taken from the orbit and placed in the K2-EDTA pretreatment anticoagulant tube. The collected blood samples were centrifuged at 4000 ⁇ g for 10 minutes and then the upper plasma samples were collected. Store the blood sample at -80°C for testing.
  • UPLC test conditions Chromatographic column: C 18 , 1.7 ⁇ m, 2.1mm ⁇ 50mm.
  • the mobile phase is methanol (solvent A) and 0.05% trifluoroacetic acid (solvent B).
  • the gradient elution procedure is as follows: 20% solvent A (0 ⁇ 1min), 20 ⁇ 80% solvent A (1 ⁇ 5min), 80 ⁇ 100% solvent A (5 ⁇ 7min), 100 ⁇ 20% solvent A (7 ⁇ 8min) ) And 20% solvent A (8min).
  • the flow rate is 0.2mL/min
  • the temperature is 37°C
  • the PDA detection wavelength is 330nm.
  • the calibration curve is obtained through three experiments.
  • the quantitative range of 7,8-DHF by UPLC method is 10 ⁇ 10000ng/mL.
  • the plasma concentration-time curve of the pharmacokinetic test is shown in Figure 11. After the control sample (free form 7,8-DHF) rapid oral absorption, peak concentration 127.36ng / mL of (C max) at the time of 0.25h (T max). Elimination half-life (t 1/2 ), elimination rate constant (K e ), mean retention time (MRT), area under the curve (AUC (0-t) ) are 2.23h, 0.20h -1 , 2.67h, 278.98ng, respectively ⁇ H/mL (Table 2).
  • 7,8-DHF bamboo Leaf Flavonoids (BLF): Zein was dissolved in 80% ethanol-water solution at a mass ratio of 1:1:10 as the solvent system, and glycosylated LF was dissolved in distilled water as the anti-solvent system, The solvent system was quickly added to the anti-solvent system at a volume ratio of 1:3 and stirred for 30 minutes. The mass ratio of Zein to glycosylated LF in the mixed solvent system was 1:1, and then the rotary evaporator was used at 40°C under reduced pressure (-0.1 Under the condition of Mpa), the excess ethanol solution was removed to obtain 7,8-DHF composite nano-biological material (DHF-BLF-Zein/LF 40K ). At the same time, the encapsulation efficiency, average particle size and oral bioavailability were determined (Table 3).
  • the encapsulation rate of 7,8-DHF is 96.21%, which is compared with DHF-Zein/LF and DHF-Zein/LF 40K have declined, but still maintain a high encapsulation rate.
  • the encapsulation rate of BLF has reached 92.13%.
  • 7,8-DHF composite nano-biomaterials have an increase in average particle size to 97.8nm.
  • the relative oral bioavailability of 7,8-DHF composite nano-biomaterials reached 1012.41%, which also increased compared to DHF-Zein/LF and DHF-Zein/LF 40K.
  • mice 40 C57BL/6 mice aged 1.5-2 months, half male and half male, weighing 20-30g.
  • 7,8-DHF made into 0.5% CMC suspension
  • DHF-Zein/LF DHF-Zein/LF 40K
  • DHF-BLF 70 -Zein/LF 40K total 4 groups, divided into 0.5h and 2h
  • the samples were intragastrically administered at a dose of 25 mg/kg (all based on the absolute content of 7,8-DHF).
  • the mice were sacrificed to take brain tissue, and the content of 7,8-DHF in the brain homogenate was determined.
  • Quantitative analysis of 7,8-DHF content in brain tissue and sample preparation method Weigh 0.1g brain tissue, add 3 times volume (v/w) of PBS (1X, pH 7.4), homogenize, and take 50 ⁇ L brain homogenate , Add 200 ⁇ L of acetonitrile solution, vortex for 1min, centrifuge at 4°C, 3000rpm for 10min, take 100 ⁇ L of supernatant and transfer to a new test tube, add 100 ⁇ L of ultrapure water, shake and mix for later use.
  • the brain tissues of DHF-Zein/LF, DHF-Zein/LF 40K, and DHF-BLF-Zein/LF 40K all increased compared with the free 7,8-DHF group, and increased respectively by 0.5 hours 1.75, 2.76 and 3.63 times; at 2 hours, although the level of 7,8-DHF was degraded, the content was still higher than that of the 7,8-DHF control group, and the content was 25.5, 42.5 and 368ng/g, respectively.
  • the DHF-BLF 70- Zein/LF 40K group has the highest content of 7,8-DHF in the brain, showing a high degree of brain targeting and a strong ability to penetrate the blood-brain barrier.

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Abstract

一种高生物利用度的7,8-二羟基黄酮复合纳米生物材料及其制备方法和用途。具体地，所述材料以玉米醇溶蛋白和乳铁蛋白为包材，以7,8-二羟基黄酮为芯材。所述材料具有高生物利用度、高脑靶向性、优异稳定性、良好贮藏稳定性、便于长期储存等优点。还公开了所述材料的制备和应用。

Description

一种高生物利用度的7,8-二羟基黄酮复合纳米生物材料及其制备方法和用途 技术领域
本发明涉及药物、天然产物、功能食品和膳食补充剂等领域。更具体地涉及高生物利用度的7,8-二羟基黄酮复合纳米生物材料及其制备方法和用途。
背景技术
7,8-二羟基黄酮(7,8-DHF)是自然界存在的一种出现频率和丰度都很低的黄酮苷元，最早发现于西方人用作沙拉的一种蔬菜中，迄今报道仅在美国中南部植物Godmania aesculifolia、长柄菊(Tridax procumbens)、报春花(Primula)及湖北海棠(Malus hupehensis)中被检出。大量的研究表明，7,8-DHF能透过血脑屏障，有效模拟脑源性神经营养因子(BDNF)，专一性地激活TrkB受体，从而诱导TrkB发生二聚化及自磷酸化，并进一步激活其下游的MAPK/ERK、PI3K/Akt和PC3K三条神经信号通路。目前，7,8-DHF已被广泛应用于各类BDNF/TrkB信号相关疾病及其肥胖、糖尿病等代谢综合征的防治，并取得了一系列令人振奋的研究成果。
然而，动物试验证实，它易在体内被糖醛酸化、硫酸化和甲酯化，实测天然来源或化学合成的7,8-DHF(称为原药)在试验动物(代码为C57BL/6的阿尔氏海默症模式小鼠)中的口服生物利用度仅为4.8％。美国Emory大学医学院的叶克强教授是7,8-DHF防治大脑退行性疾病研究领域的领军科学家，目前该团队采用化学结构修饰得到了两个有潜力的新药：(1)原药A环7,8-位上的两个OH被保护后的仿真药物R13(称为前药)的口服生物利用度接近18％(相比原药提高了3.75倍)，分别在中国大陆和澳大利亚完成了治疗阿尔氏海默症的临床前研究，目前已获得美国FDA的临床试验批件。(2)进一步在原药B环上结构改造后的纯化学药物(CF3-CN)的口服生物利用度又提高了136％。
7,8-DHF、R13和CF3-CN的化学结构示意如下：
Figure PCTCN2020127104-appb-000001
上述均为通过化学结构改造提升7,8-DHF生物利用度的研究，目前尚未见有采用物理改性提升7,8-DHF生物利用度的报道。
发明内容
本发明的目的在于提供一种具有高生物利用度、高脑靶向性、优异消化道稳定性、良好贮藏稳定性、便于长期储存等优点的7,8-二羟基黄酮复合纳米生物材料及其制备方法和用途。
本发明的第一方面，提供了一种复合纳米生物材料，包含如下组分：
1)药物载体，所述药物载体包含玉米醇溶蛋白和乳铁蛋白；和
2)药物，所述药物为7,8-二羟基黄酮；
所述药物载体包封所述药物。
在另一优选例中，所述药物载体具有核壳结构，玉米醇溶蛋白为核，乳铁蛋白为壳。
在另一优选例中，所述玉米醇溶蛋白的分子量为25-45Kda，较佳地25-35Kda，更佳地25-29Kda，优选25Kda。
在另一优选例中，所述乳铁蛋白为来源于人乳、牛乳或羊乳中的铁结合蛋白。
在另一优选例中，所述乳铁蛋白的分子量为60-200Kda，较佳地65-150Kda，更佳地70-120Kda，最佳地75-100Kda，优选80Kda。
在另一优选例中，所述乳铁蛋白是糖基化的乳铁蛋白。
在另一优选例中，所述糖基化的乳铁蛋白为葡聚糖糖基化的乳铁蛋白。
在另一优选例中，所述葡聚糖的分子量为5-100Kda，较佳地10-80Kda，更佳地20-60Kda，最佳地30-50Kda，优选为40Kda。
在另一优选例中，所述葡聚糖糖基化的乳铁蛋白是通过美拉德反应，如在温度40-80℃(较佳地50-70℃)、相对湿度60-90％(较佳地70-85％)、反应时间20-60h(较佳地30-55h)的条件下得到的接枝产物。
在另一优选例中，所述药物还包含选自下组的生物黄酮：竹叶碳苷黄酮、橙皮素、柚皮素、EGCG、黄芩素、漆黄素、山奈酚、鹰嘴豆芽素A、槲皮素、杨梅素、染料木素或其组合。
在另一优选例中，所述竹叶碳苷黄酮选自下组：荭草苷、异荭草苷、牡荆苷、异牡荆苷、或其组合。
在另一优选例中，所述药物为7,8-二羟基黄酮与70％纯度的竹叶碳苷黄酮。
在另一优选例中，所述药物载体包封所述药物，且包封率＞60％(较佳地＞70％， 较佳地＞80％，较佳地＞90％，较佳地＞92％，更佳地＞95％)。
在另一优选例中，所述药物载体中，所述玉米醇溶蛋白和所述乳铁蛋白的质量比为0.8-1.5(较佳地0.9-1.2，更佳地0.95-1.1)。
在另一优选例中，以所述复合纳米生物材料的冻干总重计，所述药物的质量含量为3-10wt％(较佳地4-8wt％，更佳地5-7wt％)。
在另一优选例中，所述的复合纳米生物材料中，7,8-二羟基黄酮与玉米醇溶蛋白的质量比为1：5-15，较佳地为1：8-12。
在另一优选例中，所述的复合纳米生物材料中，7,8-二羟基黄酮、竹叶碳苷黄酮与玉米醇溶蛋白的质量比为1：1：5-15，较佳地为1：1：8-12。
在另一优选例中，具有选自下组的一个或多个特征：
1)所述复合纳米生物材料的平均粒径为50-150nm(较佳地为60-140nm，更佳地为70-130nm，最佳地为80-120nm)；
2)所述复合纳米生物材料的分散系数为0.2-0.5(较佳地为0.25-0.4)；
3)所述复合纳米生物材料为非晶态。
本发明的第二方面，提供了一种本发明第一方面所述的复合纳米生物材料的制备方法，包括步骤：
1)提供第一混合液和第二混合液；
所述第一混合液包含第一溶剂、药物和玉米醇溶蛋白；
所述第二混合液包含第二溶剂和乳铁蛋白；
2)将所述第一混合液加入所述第二混合液中，搅拌得到第三混合液；
3)旋转蒸发所述第三混合液，得到所述的复合纳米生物材料。
在另一优选例中，所述第一溶剂选自下组：乙醇、水、或其组合。
在另一优选例中，所述第一溶剂为乙醇-水溶液，优选地乙醇浓度为70-95％，较佳地为75-90％，更佳地为80-85％。
在另一优选例中，所述第一混合液中，所述药物和所述玉米醇溶蛋白的质量比为1：4-15，较佳地为1：5-12，更佳地为1：7-11，优选1：10。
在另一优选例中，所述第二溶剂为水。
在另一优选例中，步骤2)中，所述第一混合液和所述第二混合液的体积比为1：2-5，较佳地为1：2.5-4。
在另一优选例中，所述第三混合液中，所述玉米醇溶蛋白和所述乳铁蛋白的质量比为0.3-20，较佳地为0.5-15，更佳地为0.8-10，最佳地为0.9-2。
本发明的第三方面，提供了一种本发明第一方面所述的复合纳米生物材料的用途，用于选自下组的用途：
1)用于制备预防和/或治疗选自下组的疾病的药物制剂或功能食品：阿尔茨海默症、帕金森综合症、亨廷顿舞蹈症、Rett综合症、抑郁症、肥胖症、糖尿病、骨质疏松症、更年期综合征；
2)用于制备预防和/或治疗BDNF/TrkB信号相关疾病的药物。
在另一优选例中，所述产品的形态选自下组：固体饮料、配方奶粉、压片糖果、片剂、颗粒剂、胶囊剂、冻干粉针剂、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为不同zein和LF质量比条件下zein/LF二元纳米递质的平均粒径和分散系数；A为原始pH，B为调节pH＝6。
图2为糖基化LF化学性质表征：A为LF和LF-葡聚糖接枝物的SDS-PAGE图，条带0：LF、条带1：LF 10K、条带2：LF 40K和条带3：LF 70K；B为LF-葡聚糖接枝物的接枝效率和褐变程度；C为LF和LF-葡聚糖接枝物的zeta电位电动势；D为圆二色谱图；E为傅里叶红外光谱图。
图3为不同纳米粒子的TEM图像；A为DHF-zein纳米粒子，B为DHF-zein/LF纳米粒子，C为DHF-zein/LF 10K纳米粒子，D为DHF-zein/LF 40K纳米粒子，E为DHF-zein/LF 70K纳米粒子，50000×放大倍数。
图4为不同纳米粒子的FE-SEM图像；A为zein纳米粒子，B为DHF-zein纳米粒子，C为DHF-zein/LF纳米粒子，D为DHF-zein/LF 10K纳米粒子，E为DHF-zein/LF 40K纳米粒子，F为DHF-zein/LF 70K纳米粒子，50000×放大倍数。
图5为7,8-DHF和不同纳米粒子的热行为。
图6为不同试样的X-射线衍射图。
图7为空载和负载纳米粒子的傅里叶红外光谱图；A为空载，B为负载。
图8为纳米载体的理化稳定性；A为不同pH条件对不同纳米体系平均粒径 的影响，B为不同离子强度和pH值对zein平均粒径的影响，C为为不同离子强度和pH值对zein/LF平均粒径的影响，D为不同离子强度和pH值对zein/LF 10K平均粒径的影响，E为不同离子强度和pH值对zein/LF 40K平均粒径的影响，F为不同离子强度和pH值对zein/LF 70K平均粒径的影响，G为贮藏时间对对不同纳米粒子平均粒径的影响，H为热处理对对不同纳米粒子平均粒径的影响。
图9为体外消化模拟对负载纳米粒子平均粒径和体外生物可及度的影响；A为平均粒径，B为体外生物可及度。
图10为不同纳米粒子的体外消化前与后的FE-SEM图；15000×放大倍数。
图11为口服游离7,8-DHF和负载7,8-DHF复合纳米粒子后的7,8-DHF浓度与时间曲线。
具体实施方式
本发明人经过长期而深入的研究，意外地制备了一种7,8-二羟基黄酮复合纳米生物材料，所述材料以7,8-二羟基黄酮单独或与其他生物黄酮组成的复合物为芯材，以玉米醇溶蛋白(Zein)和(糖基化)乳铁蛋白(LF)为包材。具体地，通过所述包材对所述芯材的包覆负载使得所得材料具有如下优点：具有高生物利用度、高脑靶向性、优异消化道稳定性、良好贮藏稳定性、便于长期储存等。在此基础上，发明人完成了本发明。
术语
本发明中，“芯材”是指7,8-DHF单独或与其他生物黄酮的组合物。优选地，所述芯材为7,8-DHF与70％精度的竹叶碳苷黄酮按1:1质量比组成的复合物。
本发明中，术语“包材”是指采用玉米醇溶蛋白(Zein)和乳铁蛋白(LF)两种食用蛋白，优选地，是由Zein与糖基化的LF共同组成。其中，糖基化LF是LF分别与不同分子量的葡聚糖(如10、40和70kDa)通过美拉德反应(如在温度60℃、相对湿度79％、反应48h的条件下)后得到的接枝产物(记为LF 10K、LF 40K和LF 70K)，更为优选地，是指LF与40kDa葡聚糖的接枝物(记为LF 40K)。
本发明中，术语“纳米递质”是指以Zein和LF组成的复合纳米载体，优选以Zein与糖基化LF组成的复合纳米载体。更为优选地，是Zein与LF 40K共同构建的复合纳米递质。
本发明中，术语“复合纳米粒子”是指以Zein与LF或糖基化LF组成的纳米递 质，包裹7,8-DHF或与其他植物黄酮组成的复合物后制备而成的复合生物纳米材料。优选地，是指以Zein和糖基化LF(LF 10K、LF 40K和LF 70K)组成的纳米载体，以7,8-DHF为芯材制成复合纳米粒子。更为优选地，是指以Zein与LF 40K组成的载包裹7,8-DHF与竹叶碳苷黄酮组合物为芯材制备而成的复合生物纳米材料。
本发明中，术语“体外抗消化性能”是指载有7,8-DHF的不同复合纳米粒子经过体外模拟的胃肠道消化后，其平均粒径和粒子结构呈现出的不同变化程度。
本发明中，术语“生物可及度”是指在体外模拟消化过程中，经过胃液和肠液依次消化后的混合胶束中的7,8-DHF含量水平与未经消化的初始试样中7,8-DHF含量的比值。
本发明中，术语“口服生物利用度”指的是“相对生物利用度”，即为载有7,8-DHF的不同复合纳米制剂之间及其与原物质(7,8-DHF)之间相互比较吸收程度与吸收速度后而得出的生物利用度值。
本发明中，术语“药物靶向性”是指试验动物口服不同的7,8-DHF复合纳米生物材料后，在主要靶器官(即脑组织)中实际检测到的7,8-DHF含量水平相较口服非纳米化原物质的提升程度。
复合纳米生物材料
7,8-二羟基黄酮(7,8-DHF)是天然存在的黄酮类化合物家族成员之一。大量的研究表明，它能够穿过血脑屏障，有效模拟脑源性神经营养因子(BDNF)，专一性地结合TrkB受体，从而诱导TrkB发生二聚化及自磷酸化，并进一步激活受体下游的MAPK/ERK、PI3K/Akt以及PCγ1三条信号通路。目前，7,8-DHF被应用于各类BDNF/TrkB信号相关疾病(如阿尔兹海默症、帕金森症、亨廷顿舞蹈症、Rett综合症、抑郁症及肥胖等)中的防治研究，并取得一系列令人振奋的成果。发明人近期的细胞、动物和人体试验研究均表明，7,8-DHF在妇女更年期(又称围绝经期)综合征及其骨质疏松症的防治中表现出十分突出的效果。
以天然来源的化合物为先导，通过结构修饰和改造，实现更高的口服生物利用度、更好的药物靶向性、更加卓越的药效，同时具有更小的毒副作用，是天然产物研发领域的常规路径和终极目标。无论7,8-DHF的终端产品是以药物(或天然药物)、还是以功能(或保健)食品的形态出现，创造性地集成生物纳米材料 的制备技术(即采用物理的方法和手段，而非化学性改造)以提高7,8-DHF的口服生物利用度，进而防治老龄化社会日益高发的神经退行性疾病和其他代谢性疾病，都是人类慢病干预首选的最佳路径。
采用合适的纳米递质有效包封7,8-DHF，相较于化学法的结构修饰而言，具有工艺简单、过程环保、材料天然、产品安全性高等特点。迄今，除了发明人的研究工作外，国内外尚未见有关7,8-DHF复合生物纳米材料的研究报道。其技术关键在于：(1)在复合纳米粒子制备时，首选载体材料的安全性和可食性，并采用绿色、环保的制备工艺和方法。(2)确保7,8-DHF复合纳米粒子能耐受广阔的pH、离子强度和温度范围，具有优良的贮藏稳定性和商品性。(3)保证7,8-DHF复合纳米粒子口服后在消化道中的结构稳定性，使其更多地到达小肠吸收部位，进而提高其口服生物利用度。(4)如何使得7,8-DHF复合纳米粒子达到靶器官(如大脑)后，能高效透过血脑屏障、并在脑组织中有效释放目标物。(5)从中药配伍优化的理论出发，如何充分考量7,8-DHF与其他天然产物(如其他来源的生物黄酮)的协同增效作用，并以口服后在试验动物脑匀浆中能够检测到的7,8-DHF实际浓度作为唯一的刚性评价指标。
这些都是本发明需要解决的技术难题，并已达到预期效果。
本发明提供的一种高生物利用度的7,8-DHF复合纳米生物材料是指：以玉米醇溶蛋白和(糖基化)乳铁蛋白为包材，以7,8-DHF或与其他生物黄酮组成的复合物(即前者为君药、后者为臣药)为芯材，采用反溶剂沉淀法制备而成的复合纳米粒子。
该纳米材料具有远高于游离态7,8-DHF的生物利用度和脑靶向性。它在广泛的pH范围(pH 3～9)、不同的离子强度(0～500mmol/L NaCl)和高温(95℃)下均具有优良的稳定性，同时有着良好的贮藏稳定性。该纳米粒子的平均粒径在50～150nm之间，目标物(即芯材)的包封率在90％以上,冻干后可长期储存，并具有良好的复水性。
体外模拟消化试验结果显示，经过胃液和肠液消化后，该纳米材料仍能保持完整的结构到达小肠吸收部位。体内生物利用度试验结果显示，与游离态的7,8-DHF相比口服生物利用度增加了3～8倍，在脑组织中的有效浓度也显著增加，表现出良好的血脑屏障靶向性，这一点与纳米粒子表面存在的乳铁蛋白密切相关。 此外，将7,8-DHF与竹叶碳苷黄酮(如荭草苷、异荭草苷、牡荆苷、异牡荆苷)、槲皮素、山奈酚等生物黄酮复配，有利于提高肠粘膜细胞的吸收，显著提高其口服生物利用度。
本发明采用的乳铁蛋白是来源于人乳、牛乳或羊乳中的分子量约为80Kda的铁结合蛋白，具有一定的胃蛋白酶和胰蛋白酶抵抗性，并且在人的肠上皮细胞和血脑屏障中均有相应的受体。将乳铁蛋白与不同分子量(10、40和70kDa)的葡聚糖按质量比1:1复配，通过美拉德反应制备得到的糖基化乳铁蛋白具有更加强大的功能和更加稳定的结构。
该纳米材料的制备方法大致为：将玉米醇溶蛋白溶于高浓度的乙醇溶液中(如80％的乙醇水溶液)，按一定的体积比加入乳铁蛋白或糖基化乳铁蛋白，玉米醇溶蛋白与乳铁蛋白/糖基化乳铁蛋白的质量比大致为1:1。以其为载体、采用反溶剂沉淀法包封目标物(7,8-DHF和/或其它生物黄酮)，制备得到本发明的7,8-DHF复合纳米材料(7,8-DHF在纳米冻干粉中的质量占比大约为5～7％)。
本发明的纳米材料大幅度提高了7,8-DHF的生物利用度和脑靶向性，其生物学功效更为强大，可广泛应用于功能(保健)食品和新药领域，起到防治阿尔茨海默症、帕金森综合症、抑郁症、肥胖症、骨质疏松症和更年期综合征等慢性疾病。制剂可以以固体饮料、配方奶粉、压片糖果、片剂、颗粒剂、胶囊剂、冻干粉针剂等多种形态出现。
更具体地，本发明提供了一种高生物利用度的7,8-DHF复合纳米生物材料，它是以玉米醇溶蛋白(Zein)和(糖基化)乳铁蛋白(LF)为载体，以7,8-DHF或与其他植物黄酮的复合物(前者为君药、后者为臣药)为芯材，采用反溶剂沉淀法制备而成的复合纳米粒子，具有远高于原物质(7,8-DHF)的口服生物利用度和脑靶向性，同时具有高度的pH、离子强度、高温、贮藏稳定性及其抗消化性，平均粒径在60～150nm范围内，载体对目标物(即芯材)的包封率在90％以上，纳米混悬液冷冻干燥后可长期储存，冻干粉具有良好的复水性。
所述的复合纳米粒子是指以Zein和LF为包材、以7,8-DHF为芯材构建而成的复合纳米生物材料。优选地，是指以Zein和糖基化LF组成的二元递质包裹7,8-DHF制备而成的复合纳米生物材料。其中，糖基化LF是LF与不同分子量的葡聚糖(如10、40和70kDa)通过美拉德反应(如在温度60℃、相对湿度79％、 反应48h的条件下)得到的接枝产物，记作LF 10K、LF 40K和LF 70K。。
更为优选地，所述的复合纳米粒子是指以Zein和LF 40K组成的复合纳米递质(即包材)，负载以7,8-DHF或与竹叶黄酮的组合物(即芯材)后制备而成的复合纳米生物材料。
更为优选地，所述的芯材是由7,8-DHF与70％精度的竹叶黄酮制剂(BLF 70)组合而成，BLF 70中四个碳苷黄酮(荭草苷、异荭草苷、牡荆苷和异牡荆苷)合计占制剂总质量的65～75％(w/w)。
本发明还同时提供了上述复合纳米生物材料的制备方法：将芯材和Zein以不同的质量比分别溶解于80％的乙醇-水溶液中作为溶剂相，将LF或糖基化LF溶解于蒸馏水中作为反溶剂相，将溶剂相以1:3的体积比(v/v)快速加入反溶剂相中，搅拌30min，混合溶剂体系中的Zein与LF或糖基化LF的质量比控制在20:1～1:3之间；随后，用旋转蒸发仪在减压条件(40℃、-0.1Mpa)下除去多余的乙醇溶液，最后得到平均直径在60～150nm、芯材包封率为66～99.5％的复合纳米粒子的混悬液；将此混悬液在-80℃下预冻24h后真空冷冻干燥(-50℃、-0.1Mpa)36h，得到7,8-DHF复合纳米生物材料的冻干粉。
所述关键芯材与壁材的质量比中，7,8-DHF：Zein在1:5～1:15之间。
优选的，7,8-DHF与Zein的质量比为1:10(w/w)。
更为优选地，7,8-DHF:BLF 70:Zein的质量比为1:1:10(w/w)。
所述的反溶剂体系即为LF或糖基化LF的水溶液。
优选的，所述反溶剂体系为糖基化LF的水溶液。
所述Zein与LF或糖基化LF的质量比分别可为20:1、10:1、5:1、3:1,、2:1、1:1、1:2、1:3(w/w)。
优选的，所述Zein与LF的质量比为1:1(w/w)。
更优选地，所述Zein与糖基化LF的质量比为1:1(w/w)。
本发明中复合递质对芯材的包封率：当7,8-DHF与Zein的质量比在1:5～1:15之间，且Zein与LF或糖基化LF的质量比为1:1时，可实现66～99.5％的包封率。
优化的，所述复合递质对芯材的包封率为：7,8-DHF和Zein的质量比为1:10，且Zein与LF或糖基化LF的质量比为1:1时，可实现98～99.5％的包封率。
当使用复合芯材时，即当7,8-DHF:BLF 70:Zein的质量比为1:1:10时，且Zein 与糖基化LF的质量比为1:1时，纳米递质对二种芯材(即7,8-DHF与BLF 70的组合物)的包封率分别为96.21％(以7,8-DHF计)和92.13％(以BLF 70计)。
本发明的复合纳米生物材料的优化制备方法如下：将芯材(7,8-DHF或与其他植物黄酮的组合物)和Zein以1:10的质量比溶于80％的乙醇-水溶液中作为溶剂相，将LF或糖基化LF溶解于蒸馏水中作为反溶剂相，将溶剂相以1:3的体积比快速加入反溶剂相中搅拌30min(搅拌强度800rmp/min)，混合溶剂体系中Zein与LF(或糖基化LF)的质量比为1:1。随后，用旋转蒸发仪在减压(40℃、-0.1Mpa)条件下除去多余的乙醇溶液，得到平均粒径约为70～100nm、芯材包封率达70～99.5％的7,8-DHF复合生物纳米粒子的混悬液。将此混悬液在-80℃条件下预冻24h后进行真空冷冻干燥(-50℃、-0.1Mpa、36h)，即可得到7,8-DHF纳米粒子的冻干粉。
更为优选的制备方法为：将7,8-DHF:BLF 70:Zein以1:1:10的质量比共同溶于80％的乙醇-水溶液中作为溶剂相，将糖基化的LF溶于蒸馏水中作为反溶剂相，将溶剂相以1:3的体积比快速加入反溶剂相中，搅拌30min(搅拌强度800rmp/min)，随后用旋转蒸发仪在减压(40℃、-0.1Mpa)条件下除去多余乙醇，得到平均粒径在70～100nm之间、7,8-DHF和BLF包封率分别为96.21％和92.13％的复合生物纳米粒子的混悬液，将其在-80℃下预冻24h后进行真空冷冻干燥(-50℃，-0.1Mpa、36h)，即可得到7,8-DHF与竹叶黄酮复合的纳米粒子冻干粉。
本发明还同时提供了负载7,8-DHF(及其竹叶黄酮)后复合纳米生物材料的结构表征，符合以下所有或任一条件：
①复合纳米材料的微观和表面形态：复合纳米材料外观成球形，平均粒径在60～150nm范围内；并且,由于LF或糖基化LF的存在，LF的吸附显著改变了普通Zein纳米粒子的表面结构。如附图3和图4所示。
②复合纳米材料的热特性：糖基化LF与Zein组成的复合递质显著提高了纳米材料的热稳定性；同时，随着结枝时所用的葡聚糖碳链的增长(即分子量加大)，复合纳米递质的热稳定性也随之增加(熔融温度从68.57℃分别增加到75.74、80.89和85.60℃)。如附图5所示。
③复合纳米材料的晶体衍射特性：被上述纳米递质包封后的7,8-DHF分子从原先的结晶态转变为非结晶态(7,8-DHF在2θ值5°～90°范围内高度结晶形态的 特征衍射峰消失)。如附图6所示。
④复合纳米材料的红外特性:7,8-DHF通过氢键、疏水力和静电相互作用被包封于复合递质中，同时LF或糖基化LF与Zein以氢键和疏水相互作用形成了递质表面的特殊结构(类似于Zein为核、LF或糖基化LF为壳的核-壳结构)。如附图7所示。
本发明还同时提供了复合纳米递质及其负载目标物后的纳米粒子的理化稳定性及体外模拟的抗消化性：
理化稳定性：Zein与LF的复合纳米递质可在pH 3～9的范围内保持稳定，在中性和碱性条件下能耐受广阔范围的离子强度(0～500mmol/L NaCl)，具有良好的贮藏稳定性(纳米混悬液可稳定保持30d)和热稳定性(95℃下加热60min)。
优选的，所述的纳米递质的理化稳定性为Zein与糖基化LF(LF 10K、LF 40K和LF 70K)共同构建的复合递质可在pH 3～9的范围内保持稳定，在pH 3～9条件下能耐受广阔范围的离子强度(0～500mmol/L NaCl)，具有良好的贮藏稳定性(纳米混悬液可稳定保持30d)和热稳定性(95℃下加热60min)。
更优选的，所述的纳米递质的理化稳定性为Zein与LF 40K共同构建的复合纳米递质可在pH 3～9的范围内保持稳定，在pH 3～9条件下能耐受广阔范围的离子强度(0～500mmol/L NaCl)，具有良好的贮藏稳定性(纳米混悬液可稳定保持30d)和热稳定性(95℃下加热60min)。
负载7,8-DHF后的复合纳米粒子的体外模拟的抗消化性为负载7,8-DHF的Zein/LF复合纳米粒子经历胃肠道消化后，其平均粒径增大出现沉淀，载体结构变化明显。体外生物可及度相比较于游离7,8-DHF增加了3.52倍。
优选的，所述负载7,8-DHF后的复合纳米粒子的体外模拟的抗消化性为负载7,8-DHF的Zein/糖基化LF(LF 10K、LF 40K和LF 70K)复合纳米粒子经历胃肠道消化后，其平均粒径稳定无沉淀，载体结构变化不明显。体外生物可及度相比较于游离7,8-DHF增加了4.46～4.65倍。
更优选的，所述负载7,8-DHF后的复合纳米粒子的体外模拟的抗消化性为负载7,8-DHF的Zein/糖基化LF(LF 40K)复合纳米粒子经历胃肠道消化后，其平均粒径稳定无沉淀，载体结构仍为球形。体外生物可及度相比较于游离7,8-DHF增加了4.65倍。
本发明还同时提供了小鼠口服7,8-DHF复合纳米生物材料的生物利用度改善数据以及在主要靶器官(脑匀浆)中7,8-DHF水平的大幅度提升。
相比于7,8-DHF原物质，包封于Zein/LF复合纳米粒子中的同等质量的7,8-DHF(即DHF-Zein/LF)的相对口服生物利用度最高增加了5.28倍。
优选的，相比于7,8-DHF原物质，包封于Zein/LF 40K复合纳米粒子中的同等质量的7,8-DHF(即DHF-Zein/LF 40K)的相对口服生物利用度最高增加了8.46倍。
更优选的，相比于7,8-DHF原物质，包封于Zein/LF 40K复合纳米粒子中的同等质量7,8-DHF与BLF 70的复合物(即DHF-BLF 70-Zein/LF 40K)的相对口服生物利用度最高，增加了10.12倍。
实验动物脑匀浆(主要靶器官)中7,8-DHF含量水平的测定采用液质联用法：小鼠口服剂量为25mg/kg.bw，不同试样均折算成相同的7,8-DHF绝对质量。色谱条件：Shimadzu Nexerra UPLC系统，分析柱AcquityHSS T3 1.8μm 2.1×50mm；流动相流速：0.7mL/min，流动相A为0.1％甲酸铵溶液，流动相B为乙腈(含0.1％甲酸，v/v)。质谱条件：AB Sciex Qtrap 6500，离子源ESI(+)和(-)，扫描模式为MRM。
所述，当试验小鼠口服复合纳米粒子DHF-Zein/LF(折算成7,8-DHF的绝对质量为25mg/kg.bw)后，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量为45.1ng/g，2h时为25.5ng/g；与口服7,8-DHF原物质(溶解5％羧甲基纤维素钠水溶液中)的对照组相比，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量提高了1.75倍，2h时增加了1.86倍。
优选的，所述当试验小鼠口服复合纳米粒子DHF-Zein/LF 40K(折算成7,8-DHF的绝对质量为25mg/kg.bw)后，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量为70.1ng/g，2h时为42.5ng/g；与口服7,8-DHF原物质(溶解5％羧甲基纤维素钠水溶液中)的对照组相比，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量提高了2.73倍，2h时增加了3.10倍。
更优选的，所述当试验小鼠口服复合纳米粒子DHF-BLF 70-Zein/LF 40K(折算成7,8-DHF的绝对质量为25mg/kg.bw)后，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量为93.2ng/g，2h时为36.8ng/g；与口服7,8-DHF原物质(溶解5％羧甲基纤维素钠水溶液中)的对照组相比，0.5h时测得小鼠脑匀浆中7,8-DHF的实 际含量提高了3.63倍，2h时增加了2.67倍。
更具体地，本申请提供如下：
一种以玉米醇溶蛋白(Zein)和(糖基化)乳铁蛋白(LF)为载体，以7,8-DHF或与其他植物黄酮的复合物(前者为君药、后者为臣药)为芯材，采用反溶剂沉淀法制备而成的复合纳米生物粒子及其制备方法和用途。
该复合纳米生物粒子平均粒径在60～150nm，载体对目标物(即芯材)的包封率在90％以上，纳米混悬液冷冻干燥后可长期储存，冻干粉具有良好的复水性具有远高于原物质(7,8-DHF)的口服生物利用度和脑靶向性，同时具有良好的贮藏稳定性和抗消化性能。
该7,8-DHF复合纳米生物材料其特征是：所述的复合纳米生物材料是以Zein和LF组成的复合纳米载体，以7,8-DHF为芯材，制备而成的复合纳米粒子。优选以Zein和糖基化LF组成的二元递质包裹7,8-DHF制备而成的复合纳米生物材料。其中，糖基化LF是LF与不同分子量的葡聚糖(如10、40和70kDa)通过美拉德反应(如在温度60℃、相对湿度79％、反应48h的条件下)得到的接枝产物，记作LF 10K、LF 40K和LF 70K。
该7,8-DHF复合纳米生物材料其特征是：所述的复合纳米生物材料是以Zein和LF 40K组成的复合纳米递质(即包材)，负载以7,8-DHF或与竹叶黄酮的组合物(即芯材)后制备而成的复合纳米生物材料。其芯材为7,8-DHF与竹叶黄酮的组合物,优选70％精度的竹叶碳苷黄酮制剂(BLF 70)组合而成，即BLF 70中四个碳苷黄酮(荭草苷、异荭草苷、牡荆苷和异牡荆苷)合计占制剂总质量的65～75％(w/w)(其中各成分所占比例为荭草苷：异荭草苷：牡荆苷：异牡荆苷＝1:2.4:1.4:1)。
制备7,8-DHF复合纳米生物材料的反溶剂沉淀法：是将芯材和Zein以不同的质量比分别溶解于80％的乙醇-水溶液中作为溶剂相，将LF或糖基化LF溶解于蒸馏水中作为反溶剂相，将溶剂相以1:3的体积比(v/v)快速加入反溶剂相中，搅拌30min，混合溶剂体系中的Zein与LF或糖基化LF的质量比控制在20:1～1:3之间；随后，用旋转蒸发仪在减压条件(40℃、-0.1Mpa)下除去多余的乙醇溶液，最后得到平均直径在60～150nm、芯材包封率为66～99.5％的复合纳米粒子的混悬液；将此混悬液在-80℃下预冻24h后真空冷冻干燥(-50℃、-0.1Mpa)36h，得到7,8-DHF复合纳米生物材料的冻干粉。
7,8-DHF复合纳米生物材料的制备方法，其特征是：所述关键芯材与壁材的质量比中，7,8-DHF：Zein在1:5～1:15之间。特别地，优选7,8-DHF与Zein的质量比为1:10(w/w)，zein与LF或糖基化LF的质量比为1:1(w/w)，7,8-DHF:BLF 70:Zein的质量比为1:1:10(w/w)。
7,8-DHF复合纳米材料的结构表征，符合以下所有或任一条件：
微观和表面形态；
热特性；
晶体衍射特性；
红外特性。
该复合纳米生物材料具有良好的稳定性和储存性。Zein与糖基化LF共同构建的复合递质可在pH 3～9的范围内保持稳定，在pH 3～9条件下能耐受广阔范围的离子强度(0～500mmol/L NaCl)，具有良好的贮藏稳定性(纳米混悬液可稳定保持30d)和热稳定性(95℃下加热60min)。
该复合纳米生物材料具有良好的抗消化性能。负载7,8-DHF的Zein/LF复合纳米粒子经历胃肠道消化后，其平均粒径增大出现沉淀，载体结构变化明显，体外生物可及度相比较于游离7,8-DHF增加了3.52倍；负载7,8-DHF后的复合纳米粒子的体外模拟的抗消化性为负载7,8-DHF的Zein/糖基化LF(LF 40K)复合纳米粒子经历胃肠道消化后，其平均粒径稳定无沉淀，载体结构仍为球形。体外生物可及度相比较于游离7,8-DHF增加了4.65倍。
该复合纳米生物材料显著提高了7,8-DHF复合纳米生物材料的生物利用度改善数据以及主要靶器官(脑匀浆)中7,8-DHF水平的大幅度提升：
(1)相比于7,8-DHF原物质，包封于Zein/LF复合纳米粒子中的同等质量的7,8-DHF(即DHF-Zein/LF)的相对口服生物利用度最高增加了5.28倍；
(2)相比于7,8-DHF原物质，包封于Zein/LF 40K复合纳米粒子中的同等质量的7,8-DHF(即DHF-Zein/LF 40K)的相对口服生物利用度最高增加了8.46倍；
(3)相比于7,8-DHF原物质，包封于Zein/LF 40K复合纳米粒子中的同等质量的7,8-DHF与BLF 70复合物(即DHF-BLF 70-Zein/LF 40K)的相对口服生物利用度最高增加了10.12倍。
该复合纳米生物材料显著提高了7,8-DHF在主要靶器官(脑匀浆)中的有效浓度水平：
(1)当实验小鼠口服复合纳米粒子DHF-Zein/LF(折算成7,8-DHF的绝对质量为25mg/kg.bw)后，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量为45.1ng/g，2h时为25.5ng/g；与口服7,8-DHF原物质(溶解5％羧甲基纤维素钠水溶液中)的对照组相比，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量提高了1.75倍，2h时增加了1.86倍
(2)当实验小鼠口服复合纳米粒子DHF-Zein/LF 40K(折算成7,8-DHF的绝对质量为25mg/kg.bw)后，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量为70.1ng/g，2h时为42.5ng/g；与口服7,8-DHF原物质(溶解5％羧甲基纤维素钠水溶液中)的对照组相比，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量提高了2.73倍，2h时增加了3.10倍
(3)当试验小鼠口服复合纳米粒子DHF-BLF 70-Zein/LF 40K(折算成7,8-DHF的绝对质量为25mg/kg.bw)后，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量为93.2ng/g，2h时为36.8ng/g；与口服7,8-DHF原物质(溶解5％羧甲基纤维素钠水溶液中)的对照组相比，0.5h时测得小鼠脑匀浆中7,8-DHF的实际含量提高了3.63倍，2h时增加了2.67倍。
本发明提供的高生物利用度的7,8-DHF复合纳米生物材料与原物质(7,8-DHF)相比，生物学功效更为强大，适用于7,8-DHF原物质可能应用的绝大多数用途，如口服的药物、保健食品、功能食品、特殊医学用途食品、以及日用化学品或个人护理用品等，起到防治阿尔茨海默症、帕金森综合症、抑郁症、肥胖症、骨质疏松症和更年期综合征、改善睡眠和皮肤等多种作用。终端产品可以是胶囊、片剂、颗粒剂、粉剂、食品、饮品、糖果、凝胶等多种形态。
本发明公开了一种高生物利用度的7,8-DHF复合纳米生物材料，它是以玉米醇溶蛋白(Zein)和/或糖基化乳铁蛋白(LF)为载体，以7,8-DHF或与竹叶黄酮组成的复合物为芯材，采用反溶剂沉淀法制备而成的复合纳米粒子，包封率在90％以上，平均粒径在100nm左右，具有高度的理化稳定性和抗消化性能，冻干粉可长期稳定储存，并具有良好的复水性。与7,8-DHF原物质相比，该复合纳米材料的体外生物可及度提供了3～5倍。动物试验表明，与7,8-DHF原物质相比，口服本发明的复合纳米生物材料，7,8-DHF的相对生物利用度提高了5～10倍，在主要靶器官(脑组织)中的有效浓度提高了1.75～3.63倍。本发明的复合合纳米生物材料，除7,8-DHF既可以天然产物也可以是化学合成外，其他原辅材料均来自食物或新食品原料；制备过程除乙醇(食用酒精)外，无任何其他有机溶剂或化学催化剂，工艺绿色、环保。本发明的7,8-DHF复合纳米生物材料适用于7,8-DHF原物质可能应用的绝大多数用途，如口服的药物、保健食品、功能食品、特殊医学用途食品、以及日用化学品或个人护理用品等，起到防治阿尔茨海默症、帕金森综合症、抑郁症、肥胖症、骨质疏松症和更年期综合征、改善睡眠和皮肤等多种作用。终端产品可以是胶囊、片剂、颗粒剂、粉剂、食品、饮品、糖果、凝胶等多种形态。
与现有技术相比，本发明具有以下主要优点：
1)原辅材料均可食用、体内可降解、安全性高，同时制备方法便捷、绿色；对7,8-DHF而言，包封于Zein-LF的复合纳米递质中是一种物理过程(吸附与包埋)， 并未发生实质性的化学结构变化。
2)该复合纳米生物材料(DHF-Zein/LF、DHF-Zein/LF 40K和DHF-BLF 70-Zein/LF 40K)的平均粒径范围在60～150nm之间，目标物(即芯材)的包封率在90％以上。冻干后可长期储存，并具有良好的复水性。在宽泛的pH值(3～9)和离子强度(0～500mmol/L NaCl)范围内以及高温(95℃)下和30d贮藏过程中具有良好的体系稳定性，不易沉淀。
3)经过胃液和肠液消化后，该复合纳米粒子(DHF-Zein/LF 40K)仍能保持完整的结构到达试验动物的小肠吸收部位。
4)本发明的复合纳米生物材料大幅度提高了7,8-DHF的生物利用度和脑靶向性，是由于本发明的特定的复合纳米材料构建方式所带来的。主要优势表现在，①吸收度增加：由于纳米粒子分散系数高具有较大的表面积，使其与吸收部位的生物膜接触面积大增，提高了生物利用度；②透膜性增加：纳米粒子进入细胞主要通过内吞方式，有利于药物吸收度的提高和胞内作用的发挥；③靶向性增加：纳米载体中的LF和糖基化LF具有高度的靶向性，可识别肠道和血脑屏障细胞膜上的LF受体，通过LF受体介导、靶向性地与脑部位结合；④缓释性增加：纳米载药系统的释放效率可可调节7,8-DHF的释放，增强其体内保留时间。
5)本发明的复合纳米生物材料生物学功效较7,8-DHF更为强大，可广泛应用于功能(保健)食品和新药领域，起到防治阿尔茨海默症、帕金森综合症、抑郁症、肥胖症、骨质疏松症和更年期综合征等慢性疾病。可以以固体饮料、配方奶粉、压片糖果、片剂、颗粒剂、胶囊剂、冻干粉针剂等多种形态出现。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
试验材料与仪器
试验材料
Figure PCTCN2020127104-appb-000002
试验仪器
RE-52AA旋转蒸发仪 上海亚荣生化仪器厂
ALPHA 1-4 LD plus冷冻干燥剂 德国Christ公司
BS124S万分之一天平 德国SARTORIUS公司
Milli-Q超纯水仪 法国MERCK公司
PowerPac电泳仪 美国Bio-Rad公司
EL20pH计 瑞士METTLER TOLEDO公司
3K15高速离心机 德国Sigma公司
ACQUITY H-CLASS超高效液相色谱仪 美国Waters公司
Zetasizer Nano ZS90纳米粒度分析仪 英国Malvern公司
JEM-1200 EX透视电镜 日本JEOL公司
GeminiSEM 300场式扫描电镜 德国ZEISS公司
DSC1差式扫描量热仪 瑞士METTLER TOLEDO公司
TU-1810紫外可见分光光度计 北京普析通用仪器公司
SLHW-4磁力搅拌器 杭州仪表电机有限公司
D8X-射线衍射仪 德国Bruker公司
TU-1810紫外可见分光光度计 北京普析通用仪器公司
SLHW-4磁力搅拌器 杭州仪表电机有限公司
Nicolet iS10红外光谱 美国Thermo公司
J-1500 CD圆二色谱仪 日本JASCO公司
实施例1负载7,8-DHF的zein/LF和zein/糖基化LF二元纳米粒子的构建
1.1 Zein/LF二元纳米递质的构建
采用反溶剂沉淀法制备Zein一元和二元(Zein/LF)纳米递质。准确称量zein粉末置于80％的乙醇-水中，水浴超声5min，制备储存溶液(1％，w/v)。随后将储存溶液以1:3体积比快速加入蒸馏水(即反溶剂)中，于室温下连续搅拌(800rpm)30min。利用旋转蒸发仪在40℃、减压(-0.1Mpa)条件下除去多余的乙醇溶液。最后，得到zein一元递质，zein在体系中的终浓度为2.5mg/mL。同时配置不同zein/LF质量比(20:1、10:1、5:1、3:1,、2:1、1:1、1:2、1:3)的复合醇溶液，以LF的水溶液作为反溶剂，制备zein/LF二元递质。并将均匀分散的各纳米体系的pH调至6.0，测定其平均粒径和分散系数(PDI)。
利用纳米粒度分析仪在25℃下测定zein一元和zein/LF二元递质的平均粒径和PDI，测量角度90°，水的折射率为1.45。
不同zein和LF质量比条件下Zein/LF二元纳米递质的平均粒径和分散系数见图1。如图1A所示，Zein一元递质的平均粒径为169.56nm，随着LF与Zein质量比的逐渐增加，二元递质的平均粒径先减后增。当Zein与LF质量比为1:1时，平均粒径最小(74.63nm)。同时随着LF质量占比的增加，PDI值也表现出先升后降的态势，当Zein：LF的质量比为5:1时，PDI值最高。当质量比在2:1～1:3范围内，PDI值均处于一个较低的数值(＜0.200)，表明形成了稳定、尺寸均一的纳米胶体体系。
如图1B所示，将一元体系的pH值调节到Zein的等电点附近(pH约为6)，Zein一元胶体体系极不稳定，纳米粒子的平均粒径超过1000nm，并出现沉淀现象，推测是由于Zein之间疏水相互作用和静电斥力减弱所致。当加入LF后，体系PDI均处于较低水平，尤其是在1:1～1:3质量比范围内，Zein/LF二元纳米粒子的PDI值低于0.200，表明有足够多的LF吸附在Zein粒子表面，增加了粒子之间的空间位阻和静电斥力。同时当Zein与LF质量比为1:1时，平均粒径最小。综上所述，确定Zein与LF质量比为1:1为最佳配比。
1.2美拉德反应制备糖基化LF及其化学性质表征
将分子量为10、40和70kDa的葡聚糖(1.00％w/v)和LF(1.00％w/v)分别溶解在0.01mol/L的PBS(pH＝7.4)中，搅拌过夜。完全溶解后的样品按1:1的葡聚糖：LF质量比(w/w)混合。将混合物冻干后在60℃、相对湿度79％密封玻璃干燥器中孵育48h(干燥器中放入饱和KBr溶液)，48h后得到的美拉德产物再行冻干以去除多余水分，即得到LF-葡聚糖的接枝产物(即糖基化LF)，分别记做LF 10K、LF 40K和LF 70K。
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)：采用5％浓缩胶和8％分离胶。取5μL样品溶液(2mg/mL)与20μL蛋白上样缓冲液混合，置于沸水中加热5min，并快速冷却。取10μL混合样品加到凝胶电泳每个凹槽中，电压为80～120mV。电泳完毕后，用0.25％((w/v)的考马斯亮蓝R-250溶液染色，并用脱色液(10％乙酸、50％甲醇和40％蒸馏水，v/v)脱色过夜。
接枝效率：用邻苯二醛(OPA)法测定样品的接枝效率。OPA工作液配置如下：40mg的OPA(溶于1mL甲醇)、2.5mL 20％SDS溶液、25mL 0.1mol/L四硼酸钠缓冲液和100μLβ-巯基乙醇溶液混合均匀，用蒸馏水定容到50mL。将4.0mL OPA工作液与200μL乳铁蛋白-葡聚糖共轭物(2mg/mL)混合，室温下反应3min后测定340nm处的吸光度。氨基含量标准曲线以不同浓度的L-亮氨酸(0.25～2.5mmol/L)建立。接枝效率通过以下公式(4-5)计算：
Figure PCTCN2020127104-appb-000003
褐变程度：为了评估美拉德反应引起的褐变程度，将接枝物溶于蒸馏水(1.0mg/mL)中，在420nm处用紫外-可见分光光度计测量LF-葡聚糖接枝物的吸光度。
Zeta电动电位势：用动态光散射仪(DLS)在25℃下测试Zeta电动电位势。为了避免分析前的多重散射现象，测定前用蒸馏水将非接枝物和接枝物分别稀释至2.0mg/mL浓度。
圆二色谱：在室温、持续通氮气的条件下扫描，扫描速度为50nm/min，带宽1.0nm，路径长度为0.1cm。将LF-葡聚糖接枝物溶解在蒸馏水中，测定二级结构时的试样浓度为0.2mg/mL，在190～260nm波长范围内进行扫描。并对获得的数据进行分析。
红外光谱：将1％LF-葡聚糖接枝产物与KBr混合后置于玛瑙研钵中充分研磨 均匀，利用FTIR对样品进行扫描，扫描范围为500～4000cm -1，分辨率为4cm -1。使用OMNIC软件8.0版进行分析。
如图2A所示，使用SDS-PAGE测定LF和糖基化LF的分子量。LF对应的主要区带约在80kDa处(条带0)。相比较于LF，三个LF-葡聚糖接枝物在80kDa的条带减少，颜色变淡，而在高分子量处的区带颜色变深，导致高的迁移率产生，说明两者通过美拉德反应生成了高分子量的糖化蛋白(条带1～3)。同时发现分子量为10和40kDa的葡聚糖与LF共价结合后在高分子量区域的条带颜色要深于70kDa糖化产物，表明随着葡聚糖分子量的增加，还原性羰基末端的数量减少，从而导致美拉德反应的减弱。
当表征形成蛋白质-多糖接枝物时，评估氨基含量是分析多糖结合程度很重要的指标。美拉德反应发生时，蛋白质的游离氨基和与还原糖的羰基通过共价连接形成席夫碱。采用OPA方法测定游离氨基的数量。如图2B所示，LF与分子量为10、40和70kDa葡聚糖的接枝效率分别为24.96、16.54和11.39％，表明葡聚糖的分子量大小对LF接枝效率有明显影响。
美拉德反应产物的颜色深浅能够直观地反映美拉德反应的程度，通常用420nm波长处的吸光度值大小作为褐变产物多少的指标。如图3B所示，10、40和70kDa的葡聚糖与LF共价结合后，A 420分别为0.081、0.041和0.030。此外，LF-葡聚糖接枝产物的zeta电位电动势要明显低于LF与葡聚糖的混合物(p<0.05)(图3C)，这是由于LF上带正电的-NH 2基参与了席夫碱的形成，从而导致zeta电位电动势降低。
CD能够反映蛋白质的二级结构信息。研究了糖基化对LF二级结构的影响，结果如图4.12D所示。LF的远紫外区域的CD扫描在208nm和215nm处显示了负的最小值，在190-195nm处显示最大值，是典型的含α-螺旋和β-折叠结构较多的蛋白质光谱。图3D中的插表显示糖基化修饰后，LF的α-螺旋和β-折叠减少，无序结构增加，表明糖基化LF的二级结构发生了一定变化。
FTIR光谱数据显示，四种LF试样在3300～3600cm -1和950～1150cm -1附近差异明显(图3E)，3300～3600cm -1的吸收峰被认为是N-H拉伸振动和O-H拉伸振动。天然LF的吸收峰主要在3291cm -1处(酰胺A带，代表N-H拉伸结合氢键)。与LF相比，糖基化的LF在3351cm -1处的吸收峰出现明显蓝移，LF 10K、LF 40K 和LF 70K分别增加了54、66和72cm -1，说明LF的-NH 2基团参与了美拉德反应。950～1150cm -1处的吸收峰反映了C-O拉伸和O-H变形振动，原LF在1060cm -1处的吸收峰，在LF 10K、LF 40K和LF 70K中分别红移了42、44和43cm -1，说明葡聚糖的还原羰基参与了美拉德反应。此外，LF在1500～1700cm -1波段出现了两个典型峰，分别为1651cm -1(酰胺I带)、1538cm -1(酰胺II带)。酰胺I带主要与C＝O伸缩振动有关，而酰胺II带主要与C-N的伸缩振动有关。美拉德反应后，C＝O和C-N伸缩振动强度发生了变化。综上所述，葡聚糖的接枝反应改变了LF的二级结构和一级结构。
1.3负载7,8-DHF的Zein/LF和Zein/糖基化LF二元纳米粒子构建
具体操作参考1.1，不同之处在于将7,8-DHF与Zein按10:1和5:1的质量比(w/w)充分溶解到80％的乙醇-水溶液中，分别以蒸馏水、LF和糖基化LF水溶液作为反溶剂，分别制备负载7,8-DHF的一元(DHF-Zein)和二元(DHF-Zein/LF、DHF-Zein/LF 10K、DHF-Zein/LF 40K和DHF-Zein/LF 70K)纳米粒子。
包封率(EE)测定：通过高速离心(4℃、10000×g、10min)去除纳米悬浮液中游离的7,8-DHF。取上清液(含负载7,8-DHF的纳米粒子)，用甲醇破乳稀释5倍，并用甲醇将等量的初始纳米悬浮液稀释5倍。7,8-DHF由UPLC法测定方法。EE和LC计算公式(4-1和4-2)如下:
Figure PCTCN2020127104-appb-000004
表1、DHF-Zein/糖基化LF二元纳米体系的理化特性
Figure PCTCN2020127104-appb-000005
Figure PCTCN2020127104-appb-000006
注：各行不同的字母，表示存在显著性关系(p<0.05)。
表1总结了不同纳米递送体系负载的7,8-DHF的EE、平均粒径和PDI值。当Zein与7,8-DHF质量比为5:1时，DHF-Zein、DHF-Zein/LF DHF-Zein/LF 10K、DHF-Zein/LF 40K和DHF-Zein/LF 70K纳米粒子的EE分别为37.27、66.10、72.41、84.75和83.61％。LF、LF 10K、LF 40K和LF 70K与Zein复合使用包封率提高了约1.7、1.9、2.3和2.2倍。当Zein与7,8-DHF质量比为10:1时，DHF-Zein、DHF-Zein/LF DHF-Zein/LF 10K、DHF-Zein/LF 40K和DHF-Zein/LF 70K纳米粒子的EE分别为46.38、98.31、98.66、99.41、99.21％。表明LF和糖基化LF稳定了Zein纳米粒子，通过非共价相互作用(氢键和疏水力)形成稳定的二元递质体系从而增强了包封7,8-DHF的效果，尤其是LF 40K与Zein复合为载体的包封效果最佳。此外，Zein一元体系的平均粒径为169.56nm，而Zein/糖基化LF二元体系的平均粒径下降至78.63～87.24nm。与Zein/LF粒子相比，Zein/LF 10K、Zein/LF 40K和Zein/LF 70K的平均粒径略微增大，与LF上结合的糖链有关。PDI也表现出与平均粒径相似的变化趋势。
此外，负载7,8-DHF的Zein/LF和Zein/糖基化LF二元纳米粒子显示良好的水复溶性，复溶的EE有所下降，但仍高于90％，同时平均粒径为103.6-110.3nm范围内，PDI在0.265-0.295范围内，展现粒径低，分散均匀的稳定体系。其中以DHF-Zein/LF 40K纳米粒子的效果最好。
实施例2负载7,8-DHF的Zein/LF和Zein/糖基化LF二元纳米粒子的结构表征
2.1材料与方法
2.1.1微观和表面形态
利用透射电镜(TEM)观察样品的微观结构和形貌。将新鲜制备的纳米悬浮液滴在Formvar-carbon载样铜网上，风干5min。然后，用2％乙酸双氧铀染色样品，用滤纸除去多余的染色剂。电镜加速电压为120kV。采用场发射扫描电镜(FE-SEM)捕获冻干纳米粒子的表面形貌。分析之前，利用溅射涂布机于真空条件下在样品表面涂上3-6nm厚的金层。电镜加速电压为10.0kV。
2.1.2热特性分析
利用示差扫描量热仪对冻干试样进行热特性分析。准确称取6-10mg样品放入铝坩埚中，密封。用相同条件的空坩埚作参比。采用N 2氛围以10℃/min的升温速度从20℃升温至300℃进行扫描量热分析。
2.1.3晶体衍射特性分析
X-射线衍射(XRD)分别进行粉末状7,8-DHF、干燥空载或负载纳米粒子(冻干粉)的XRD分析。仪器的铜阳极产生铜Kα辐射，加速电压为40kV，管电流为40mA。Soller狭缝设置为2.5°，散度狭缝设置为0.5°。2θ角范围为5°～70°，步长0.02°，步长时间0.2s。
2.14红外特性分析
傅里叶红外光谱(FTIR)分析：将1％冻干样品与KBr混合，置于玛瑙研钵中充分研磨均匀，利用傅里叶红外光谱分析仪(FTIR)对样品进行扫描。光谱扫描范围为500-4000cm -1，分辨率为4cm -1，使用OMNIC软件8.0版进行分析。
2.2试验结果
2.2.1纳米粒子的微观和表面形态
TEM展示了胶体递质的形状、大小、均匀性和完整性。从图3可以看出，在DHF-Zein(负载7,8-DHF的Zein纳米粒子)纳米粒子的平均粒径在100nm以上3。此外，DHF-Zein纳米粒子之间呈相互连接状态，这可能是由于Zein胶体体系被稀释10倍后，Zein粒子之间的疏水相互作用导致了聚集。然而，在添加糖基化LF后，DHF-Zein/糖基化LF纳米粒子均呈现70～100nm范围内的球形形态(图3的C、D和E)，并且分散均匀，表明糖基化乳铁蛋白的吸附可增加静电排斥和空间排斥效应。从而可以防止DHF-Zein纳米粒子的聚集。此外，与DHF-Zein/LF纳米粒子相比，DHF-Zein/糖基化LF纳米粒子的平均粒径相对较大(图3B)。
为进一步观察不同复合纳米粒子的表面形态，利用FE-SEM进行表征。如图4所示，Zein和DHF-Zein纳米粒子均呈典型的球形，且大小均匀。然而，糖基化LF后，DHF-Zein/糖基化LF纳米粒子相比Zein和DHF-Zein纳米粒子发生了巨大的变化，表面粗糙且不规则(图3的D、E和F)。表明糖基化LF通过非共价力吸附到了DHF-Zein纳米粒子表面。这一结果与天然LF吸附到DHF-Zein纳米粒子表面的结果一致(图4C)。并且相比较于Zein一元体系，负载后二元体系的平均 粒径均有所下降。
2.2.2热特性分析
如图5所示，包封7,8-DHF后，DHF-Zein、DHF-Zein/LF、DHF-Zein/LF 10K、DHF-Zein/LF 40K、DHF-Zein/LF 70K纳米粒子中均未出现7,8-DHF的吸热峰，说明7,8-DHF从结晶态转变为非晶态。此外，添加LF和糖基化LF后，DHF-Zein/LF、DHF-Zein/LF 10K、DHF-Zein/LF 40K和DHF-Zein/LF 70K的吸热峰值分别为72.61、75.74、80.89℃和85.60℃，说明它们提高了纳米粒子的热稳定性，尤其是LF 40K和LF 70K。
2.2.3晶体衍射特性分析
利用X-射线衍射在2θ值5°-90°范围内测定各试样的晶体衍射模式。如图6所示，LF 10K、LF 40K和LF 70K也出现了与LF类似的峰型，表明这些接枝蛋白质均以非结晶形态存在。然而，在负载7,8-DHF的复合纳米粒子(DHF-Zein/LF、DHF-Zein/LF 10K、DHF-Zein/LF 40K和DHF-Zein/LF 70K)中，并没有观察到7,8-DHF结晶形态的特征衍射峰，表明其被有效包封后呈现为非晶态，这一现象与热特性分析结果吻合。与DHF-Zein相比，在19.6°衍射角度，DHF-Zein/LF 70K纳米粒子的峰值显著增加，而DHF-Zein/LF 10K和DHF-Zein/LF 40K纳米粒子的特征峰几乎消失。这一结果证实了Zein、LF和糖基化LF之间存在着氢键、疏水相互作用等非共价力。
2.2.4红外特性分析
如图7展示了4000-500cm -1波数范围内不同样品的吸收峰。Zein的O-H基团拉伸特征峰为3306cm -1(图7A)，当Zein与糖基化LF结合形成Zein/LF 10K、Zein/LF 40K和Zein/LF 70K纳米粒子后，其氢键特征峰分别从3306cm -1转移至3406、3404和3417cm -1，表明Zein和糖基化LF的结合有氢键的参与，同时对比Zein/LF纳米粒子，其氢键结合能力更为强烈。此外，2953cm -1处被认为是Zein疏水性的C-H基团的拉伸振动峰。形成Zein/LF 10K、Zein/LF 40K和Zein/LF 70K纳米粒子后，其特征峰从2953cm -1年红移至2932、2930和2931cm -1，表明更强的疏水相互作用参与了Zein/糖基化LF二元纳米粒子的形成。此外，Zein波段在1652cm -1(酰胺I带)和1538cm -1(酰胺II带)出现另外两个特征峰。随着糖基化LF的加入，与Zein相比，酰胺I和酰胺II的特征峰没有变化，说明静电相互作用不参与Zein/糖基化LF二元纳米粒子的形成。这一结果与上一部分Zein与LF的结合方式相同。如图7B所示，DHF-Zein、DHF-Zein/LF、DHF-Zein/LF 10K、DHF-Zein/LF 40K、 DHF-Zein/LF 70K纳米粒子中7,8-DHF的典型特征峰全部消失，说明7,8-DHF被成功包封于纳米粒子中。相比较于未负载的纳米粒子，负载后的纳米粒子几个主要特征峰的波数均发生了变化，说明7,8-DHF的存在也会在一定程度上改变复合载体间的非共价结合能力。
实施例3纳米递质的理化稳定性和负载7,8-DHF的Zein/LF和Zein/糖基化LF复合纳米粒子的胃肠道稳定性
3.1材料与方法
3.1.1纳米载体的理化稳定性
pH值的影响：用2mmol/L NaOH或2mmol/L HCl分别调节Zein一元、Zein/LF和Zein/糖基化LF二元纳米体系的pH值分别至3-9，静止2h后进行平均粒径、浊度和分散系数测定。
离子强度的影响：室温下，分别添加不同浓度的NaCl溶液(0、10、25、50、100、200和500mmol/L)处理24h，评价不同离子强度和不同pH条件下纳米粒子的稳定性。
贮存时间的影响：将新鲜制备的纳米悬浮液调节pH至3.0-9.0，在25℃下见光保存30d。
贮藏温度的影响：新鲜制备的纳米悬浮液置于95℃加热60min，室温冷却。研究不同贮藏温度对试样稳定性的影响，利用DLS测定贮藏过程中平均粒径的变化，并以未加热的样品作为对照。
3.1.2负载7,8-DHF的Zein/LF和Zein/糖基化LF二元纳米粒子的胃肠道稳定性和体外生物可及度
分别将10mL的7,8-DHF溶液、DHF-Zein/LF、DHF-Zein/LF 10K、DHF-Zein/LF 40K和DHF-Zein/LF 70K纳米悬浮液混合于10mL的模拟胃液(SGF，配方组成为：2mg/mL NaCl和3.2mg/mL胃蛋白酶，pH＝2.5)中，置于水浴摇床中37℃孵化60min，转速为100rpm；调整pH至7.4后,取上述模拟胃消化液10mL与等体积的模拟肠液(SIF，配方组成为：4mg/mL胰酶、5mg/mL胆汁盐、8.8mg/mL NaCl和6.8mg/mL K 2HPO 4，pH＝7.4)混合，置于水浴摇床中37℃孵化120min,转速为100rpm。最后，将最终消化液在20000×g的离心力下离心1h，收集上清液(即混合胶束 相，内含溶解的7,8-DHF)，利用UPLC法测定其7,8-DHF含量，生物可及度按下式计算:
Figure PCTCN2020127104-appb-000007
此外，每隔30和60(胃消化液)、120和180min(肠消化液)收集2mL消化液)，测定其平均粒径。
另将最终消化液制成冻干粉，进行FE-SEM表征，并观察其微观表面形态。
3.2试验结果
3.2.1 Zein/LF和Zein/糖基化LF纳米递质的理化稳定性
pH稳定性：Zein一元纳米体系在pH 5～7范围内极其不稳定，添加LF后的二元体系可有效改善其稳定性。进一步将LF糖基化后，在更宽的pH范围内(3～9)，Zein/LF 10K、Zein/LF 40K和Zein/LF 70K的二元体系均能保持稳定，纳米粒子处在一个较低的平均粒径范围(80-120nm)(图8A)。
离子强度稳定性：图8的B、C、D、E和F显示了不同pH条件下离子强度在对纳米载体稳定性的影响。Zein一元体系对离子强度高度敏感(图8B)，添加LF后的二元体系虽然有所改善，但在低pH(3～5)条件下依然不稳定(图8C)，而LF糖基化后则发挥了不同程度的稳定效果。如在Zein/LF 10K的二元体系中，纳米粒子的平均粒径随着离子强度的增强而不断增大，在高浓度NaCl(500mmol/L)和低pH(3和4)条件下,其平均粒径超过350nm(图8D)。然而随着葡聚糖分子量的增大，其对Zein纳米粒子的稳定性越来越好，Zein/LF 40K(图8E)和Zein/LF 70K(图8F)的平均粒径在广阔范围的pH(3-9)和离子强度(0-500mmol/L NaCl)下分别小于250nm和200nm，Zein/LF 70K表现了最佳的稳定效果。表明随着葡聚糖分子量的增大，其链长相应增加，从而产生了更大的空间位阻，阻止了纳米粒子的团聚效应，同时糖基化的LF在粒子表面形成的界面层也起到了屏蔽外界电荷的作用。
贮藏稳定性：Zein一元体系在30天的储存过程(25℃常温光照)中不稳定。与Zein/LF相比，Zein/糖基化LF的二元体系在30天的储存过程中更加稳定，说明LF的糖接枝化能有效提高纳米粒子的稳定性。
热稳定性：95℃加热60min后，Zein一元体系的平均粒径＞400nm。之前的研究也表明，未包覆的Zein纳米粒子在加热条件下不稳定。然而，在相同的加 热条件下，二元体系(Zein/LF和Zein/糖基化LF)的平均粒径与加热前先比均无显著变化(p>0.05)，表明糖基化LF的存在显著改善了Zein纳米粒子的热稳定性。
3.2.2负载7,8-DHF的Zein/LF和Zein/糖基化LF复合纳米粒子的外抗消化性和生物可及度
目标物在模拟胃肠道中的有效保护和持续释放是评价纳米载体有效性的关键。本研究采用模拟胃液(SGF)和模拟肠液(SIF)的体外消化体系，测定了负载7,8-DHF的一元、二元纳米粒子在模拟消化过程(30、60、120、180min)中的平均粒径变化。如图9A所示，在SGF中消化60min后，一元纳米粒子(DHF-Zein)均粒径显著增大(p<0.05)继续在SIF相中消化后，其粒径减小，可能是由于肠液中胰酶的作用，水解了聚集物，同时由于含有胆汁盐，具有较强的乳化能力，可以乳化水解的聚合物。二元纳米粒子(DHF-Zein/LF)在SIF消化过程中发生了聚集，当LF经过糖基化处理后的体系(DHF-Zein/LF  10K、DHF-Zein/LF 40K、DHF-Zein/LF 70K)，经过SGF消化后的粒径略有增大，继而经SIF消化后也无显著变化。这可能是由于纳米粒子表明的LF经过接枝化处理后，既提供了强大的空间斥力以克服粒子间的相互吸引(疏水相互作用和范德华力)，又在粒子表明形成了新的界面，屏蔽了酸、碱及酶的降解作用。
通过体外胃肠道消化模拟试样，还对游离7,8-DHF及其不同建构的复合纳米材料进行了体外生物可及度的评价。如图9B所示，7,8-DHF对照样的溶解度相对较低，经体外消化模拟后，其生物可及度为18.06％。纳米化技术表现出显著的改善作用，一元纳米粒子(DHF-Zein)的生物可及度即提升至31.85％(p<0.05)，二元纳米粒子中由于LF存在，7,8-DHF的生物可及度增加到63.51％，比对照样(游离7,8-DHF)提高了3倍(p<0.05)。进一步将LF糖基化后，生物可及度超过80％，如DHF-Zein/LF 40K达到最大值(84.05％)。
FE-SEM图像显示，体外消化模拟对负载纳米粒子的表面形态有重要影响。如图10所示，体外消化后，DHF-Zein和DHF-Zein/LF颗粒的形貌发生了明显的变化，尤其是DHF-Zein(原颗粒呈球形，消化后呈块状结构)。对于DHF-Zein/LF二元粒子经过胃肠道消化后，由球形变为方形，是由于DHF-Zein/LF二元粒子在SIF消化过程中，大量聚集所致。然而，消化后的DHF-Zein/LF 40K和DHF-Zein/LF 70K 虽然粒径有所增大，但仍保持相对的球状。表明糖基化LF的引入有助于提高纳米粒子的抗消化能力。
实施例4 7,8-DHF复合纳米生物材料的口服生物利用度及其在靶器官中的含量
4.1基于纳米化技术的7,8-DHF口服生物利用度的显著提升
42只SD雄性大鼠，体重250-300g，随机分成7组，每组6只。经过一周适应后(12h光暗循环，22±2℃恒温，RH 55±5％，SPF屏障系统)，口服灌胃进行药代动力学实验。动物试样批准的伦理号为：ZJU 20190101。
将7,8-DHF(制成0.5％CMC的悬浮液)、DHF-Zein、DHF-Zein/LF和DHF-Zein/LF 40K试样分别灌胃，灌胃剂量为50mg/kg(均以7,8-DHF绝对含量计算)。分别于0.08、0.25、0.5、0.75、1、1.5、2、4、6、10和24h时眼眶取血(200μL)至K2-EDTA预处理抗凝管中。收集的血液样品于4000×g离心10min后收集上层血浆样品。将血样存储于-80℃下，待测。
将100μL血样与20μL山柰酚(内标，300ng/mL)混合，加入280μL纯甲醇震荡2min以去除内源蛋白。13800×g离心10min后，将上清液转移至新的1.5mL试管中，室温下用氮气吹干。加入100μL纯甲醇复溶并震荡2min后，于13800×g离心10min。最后，取5μL上清液注入UPLC系统分析。
UPLC测试条件：色谱柱：C 18,1.7μm,2.1mm×50mm。流动相为甲醇(溶剂A)和0.05％三氟乙酸(溶剂B)。梯度洗脱程序如下：20％溶剂A(0～1min),20～80％溶剂A(1～5min),80～100％溶剂A(5～7min)，100～20％溶剂A(7～8min)和20％溶剂A(8min)。流速为0.2mL/min，温度为37℃，PDA检测波长为330nm。校准曲线经三次试验获得。回归方程为y＝37.93x+299.32，相关系数r为0.9998，其中y为峰面积，x为血样中7,8-DHF浓度(ng/mL)。UPLC法的7,8-DHF定量范围为10～10000ng/mL。
药代动力学参数分析采用非房室模型，达峰浓度(浓度最大值，C max)、达峰时间(达到最大浓度的时间，T max)、半衰期(浓度下降一半所需时间，t 1/2)、消除速率常数(K e)、平均驻留时间(MRT)、总清除率(Cl)、表观分布容积(V d)、浓度时间曲线下面积(AUC)和相对生物利用度(F rel)利用Kinetica 4.4.1软件模拟获得。相对生物利用度通过下式计算得到:
Figure PCTCN2020127104-appb-000008
药代试验的血浆浓度-时间曲线如图11所示。对照样(游离态的7,8-DHF)口服后吸收迅速，在0.25h(T max)时达到127.36ng/mL的峰值浓度(C max)。排除半衰期(t 1/2)、排除率常数(K e)、平均保留时间(MRT)、曲线下面积(AUC (0-t))分别为2.23h、0.20h -1、2.67h、278.98ng·h/mL(表2)。
而被包封于Zein、Zein/LF和Zein/LF 40K纳米递质中以后，7,8-DHF的C max分别为241.74、306.69和450.84ng/mL，相对生物利用度(F rel)分别增加到252.95、528.33和846.20％，表明在复合纳米材料中7,8-DHF的口服生物利用度得到了大幅度提升。同时，T max、t 1/2、MRT值也分别增加，说明纳米材料延长了7,8-DHF的在血液中的保留时间。其中，二元体系中的DHF-Zein/LF 40K对7,8-DHF口服生物利用度的改善作用最为突出。
表2、口服7,8-DHF负载纳米粒子后的药代动力学参数
Figure PCTCN2020127104-appb-000009
4.2 7,8-DHF复合纳米生物材料的构建和口服生物利用度
7,8-DHF:竹叶黄酮(BLF):Zein以1:1:10的质量比溶解于80％的乙醇-水溶液中作为溶剂体系，将糖基化LF溶解于蒸馏水中作为反溶剂体系，将溶剂体系以1:3体积比快速加入反溶剂体系中搅拌30min，混合溶剂体系中Zein和糖基化LF的质量比为1:1，随后利用旋转蒸发仪在40℃、减压(-0.1Mpa)条件下除去多余的乙醇溶液，得到7,8-DHF复合纳米生物材料(DHF-BLF-Zein/LF 40K)。同时测定其包封率、平均粒径和口服生物利用度(表3)。
表3、7,8-DHF复合纳米生物材料包封率、平均粒径和相对口服生物利用度
Figure PCTCN2020127104-appb-000010
Figure PCTCN2020127104-appb-000011
如表3所示，同时包封7,8-DHF和竹叶黄酮(BLF)构建7,8-DHF复合纳米生物材料后，其7,8-DHF的包封率为96.21％，相比较于DHF-Zein/LF和DHF-Zein/LF 40K有所下降，但仍保持较高的包封率。同时BLF的包封率也达到了92.13％。同时，7,8-DHF复合纳米生物材料相比较于DHF-Zein/LF和DHF-Zein/LF 40K，平均粒径增大，达到97.8nm。此外，7,8-DHF复合纳米生物材料的相对口服生物利用度达到1012.41％，相对于DHF-Zein/LF和DHF-Zein/LF 40K也发生了增大。
4.3 7,8-DHF复合纳米生物材料在试验小鼠脑组织中的含量
40只1.5-2月龄的C57BL/6小鼠，雌雄各半，体重20-30g。分成7,8-DHF(制成0.5％CMC的悬浮液)、DHF-Zein/LF、DHF-Zein/LF 40K和DHF-BLF 70-Zein/LF 40K共4组，又分为0.5h和2h两个时间节点，每组在每个时间节点取5只小鼠。将试样分别灌胃，灌胃剂量为25mg/kg(均以7,8-DHF的绝对含量计)。每个时间点将小鼠处死取脑组织，测定脑匀浆中的7,8-DHF含量。
脑组织中7,8-DHF含量定量分析及样品前处理方法：称取0.1g脑组织，加入3倍体积(v/w)的PBS(1X，pH 7.4)，匀浆，取50μL脑匀浆液，加入200μL乙腈溶液，涡旋振荡1min，4℃、3000rmp离心10min，取100μL上清液转移至新试管中，加入100μL超纯水，振荡混匀后备用。
7,8-DHF的LC-MS/MS检测条件参数，液相条件：Shimadzu Nexerra UPLC(分析柱：AcquityHSS T3 1.8μm 2.1×50mm)，流速：0.7mL/min；流动相A：0.1％甲酸铵溶液，流动相B：乙腈(含0.1％甲酸，v/v))。质谱条件：AB Sciex Qtrap6500，离子源ESI(+)和(-)，扫描模式MRM。
表4、不同组别实验小鼠在不同时间节点时脑匀浆中的7,8-DHF含量水平
Figure PCTCN2020127104-appb-000012
如表4所示，DHF-Zein/LF、DHF-Zein/LF 40K和DHF-BLF-Zein/LF 40K的脑组织相比较于游离7,8-DHF组均有所增加，0.5小时分别提高了1.75、2.76和3.63倍；2小时时，虽然7,8-DHF水平有所降解，但含量仍高于7,8-DHF对照组分别，含 量分别为25.5，42.5和368ng/g。尤其是DHF-BLF 70-Zein/LF 40K组，脑部7,8-DHF的含量最高，表现出高度的脑靶向性和极强的透过血脑屏障的能力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种复合纳米生物材料，其特征在于，包含如下组分：
    1)药物载体，所述药物载体包含玉米醇溶蛋白和乳铁蛋白；和
    2)药物，所述药物为7,8-二羟基黄酮；
    所述药物载体包封所述药物。
  2. 如权利要求1所述的复合纳米生物材料，其特征在于，所述乳铁蛋白是糖基化的乳铁蛋白。
  3. 如权利要求2所述的复合纳米生物材料，其特征在于，所述糖基化的乳铁蛋白为葡聚糖糖基化的乳铁蛋白。
  4. 如权利要求3所述的复合纳米生物材料，其特征在于，所述葡聚糖的分子量为5-100Kda。
  5. 如权利要求1所述的复合纳米生物材料，其特征在于，所述药物还包含选自下组的生物黄酮：竹叶碳苷黄酮、橙皮素、柚皮素、EGCG、黄芩素、漆黄素、山奈酚、鹰嘴豆芽素A、槲皮素、杨梅素、染料木素或其组合。
  6. 如权利要求1所述的复合纳米生物材料，其特征在于，所述药物载体包封所述药物，且包封率＞60％。
  7. 如权利要求1所述的复合纳米生物材料，其特征在于，所述药物载体中，所述玉米醇溶蛋白和所述乳铁蛋白的质量比为0.8-1.5。
  8. 如权利要求1所述的复合纳米生物材料，其特征在于，具有选自下组的一个或多个特征：
    1)所述复合纳米生物材料的平均粒径为50-150nm；
    2)所述复合纳米生物材料的分散系数为0.2-0.5；
    3)所述复合纳米生物材料为非晶态。
  9. 一种权利要求1所述的复合纳米生物材料的制备方法，其特征在于，包括步骤：
    1)提供第一混合液和第二混合液；
    所述第一混合液包含第一溶剂、药物和玉米醇溶蛋白；
    所述第二混合液包含第二溶剂和乳铁蛋白；
    2)将所述第一混合液加入所述第二混合液中，搅拌得到第三混合液；
    3)旋转蒸发所述第三混合液，得到所述的复合纳米生物材料。
  10. 一种权利要求1所述的复合纳米生物材料的用途，其特征在于，用于选自下组的用途：
    1)用于制备预防和/或治疗选自下组的疾病的药物制剂或功能食品：阿尔茨海默症、帕金森综合症、亨廷顿舞蹈症、Rett综合症、抑郁症、肥胖症、糖尿病、骨质疏松症、更年期综合征；
    2)用于制备预防和/或治疗BDNF/TrkB信号相关疾病的药物。
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CN114235980B (zh) * 2021-11-16 2024-06-11 武汉人福创新药物研发中心有限公司 检测广金钱草提取物中黄酮类化合物含量的方法
CN114948869A (zh) * 2022-05-26 2022-08-30 中国药科大学 一种染料木素纳米混悬液的制备方法及该方法制备的染料木素纳米混悬液

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