CN108420793B

CN108420793B - Blank mixed micelle and preparation method and application thereof

Info

Publication number: CN108420793B
Application number: CN201711432287.5A
Authority: CN
Inventors: 王丹; 李翀; 王亚华; 詹华杏
Original assignee: Xiamen Ginposome Pharmaceutical Co ltd
Current assignee: Xiamen Ginposome Pharmaceutical Co ltd
Priority date: 2017-12-26
Filing date: 2017-12-26
Publication date: 2021-09-24
Anticipated expiration: 2037-12-26
Also published as: WO2019128608A1; CN108420793A

Abstract

The invention discloses a blank mixed micelle and a preparation method and application thereof. The blank mixed micelle comprises an amphiphilic copolymer and ginsenoside shown in a formula I. The blank mixed micelle has the advantages of high efficiency, safety, stability, strong targeting property, good uniformity, stable quality, simple and convenient preparation process and the like, and can be used for encapsulating one or more of active substances and substances with health care effect in medicaments and cosmetics to form the mixed micelle loaded with the active substances. Compared with the traditional nano micelle, the mixed micelle loaded with the active substance has more outstanding drug forming property and multi-drug resistance, good stability, good uniformity, high safety and small particle size, and has better curative effect on drug-resistant cells after being loaded with the active drug.

Description

Blank mixed micelle and preparation method and application thereof

Technical Field

The invention relates to a blank mixed micelle and a preparation method and application thereof.

Background

Polymeric micelles are generally characterized by having an amphiphilic character, i.e., having both hydrophilic and hydrophobic groups, the hydrophobic group generally forming a core in the middle and the hydrophilic group generally being disposed on the outside to form a shell. The polymer micelle can wrap fat-soluble medicines in a hydrophobic center of the polymer micelle to form the polymer micelle carrying the medicines, and can dissolve the hydrophilic end of the polymer micelle in water or alcohol. The polymer micelle can wrap the medicine inside the micelle, prolong the circulation time and the biological half-life of the medicine in blood, increase the accumulation of the medicine in a pathological change part, reduce adverse reaction, or be connected with a special carrier, an antibody or a ligand, so that the polymer micelle can be combined with a receptor of a target cell, and the treatment effect is improved.

Genex-PM developed by Samyang corporation of Korea takes methoxy polyethylene glycol-polylactic acid copolymer (mPEG-PDLLA) as a membrane material to prepare the taxol freeze-dried nano micelle, which can increase the water solubility of taxol and reduce the toxic and side effects of medicines. CN201110105540.2 discloses that methoxy polyethylene glycol 2000-polyester block copolymer (mPEG-PDLLA) forms micelle through molecular self-assembly, and then insoluble tumor drug is encapsulated in hydrophobic core formed by polyester to prepare polymer micelle, thereby improving maximum tolerance dose of drug and reducing toxic and side effect. CN201611083457.9 takes PEO-PCL copolymer as micelle carrier to prepare florfenicol micelle preparation, thus improving the bioavailability of florfenicol. CN201611054732.4 provides an amphiphilic triblock copolymer inclusion adriamycin with pH and reduction double sensitivity, and the drug has good biocompatibility, reduces the toxicity of anticancer drugs and reduces the damage to normal tissues. CN201610847822.2 discloses a PEG-PLA or a mixed polymer of PEG-PLA and folate-PEG-PLA to include nanoparticles of paclitaxel. CN201110367315.6 discloses a preparation method of a paclitaxel micelle taking a segmented copolymer of polyether and polyester as an auxiliary material.

As described in the research progress of the preparation method of the drug-loaded polymer micelle of the zhanghai, university of chinese pharmacy, the polymer micelle really enters into application to solve two problems, one is the stability problem of the polymer micelle, and the other is the low concentration problem of the polymer micelle. As described in the Miao nationality of Chinese pharmacy university, bee front, research on the micellization mechanism and physical stability of the block polymer micelle, the stability of the polymer micelle is an important index of the preparation. Meanwhile, the problems of biodegradability, potential toxicity, high cost, great difficulty in organic synthesis, non-unity of polymer molecular weight and the like of the amphiphilic polymer material still exist. Therefore, the search for a micelle membrane material with good drug-loading capacity, stable structure and in vivo, no toxicity, single molecule, low cost and good drug effect is always an important research direction of the drug-loading technology.

According to patents CN201310155639.2, CN201380026612.1, US20150297727A1, PCT/CN2013/088558, JP2015-514348 and the like, part of ginsenoside derivatives (such as Rg5, Rk1 and the like) can form micelles by themselves under certain conditions and can entrap paclitaxel and other pharmaceutical active ingredients. However, further research shows that the ginsenoside derivative nano-micelle has strong hemolytic property, and the use of the ginsenoside derivative nano-micelle as an intravenous injection is limited.

Therefore, the research and development of novel micelles with stronger drug-loading capacity, higher biocompatibility and better safety, or the reduction of the dosage of the existing amphiphilic polymer to reduce the biocompatibility and other defects are problems which need to be solved urgently in the field.

Disclosure of Invention

The invention aims to overcome the defects of high hemolysis, instability in animal bodies and the like of the existing ginsenoside nano-micelle drug-loading system, and simultaneously provides a blank mixed micelle, a preparation method and application thereof in order to solve the problems of insufficient biodegradability of a high-molecular polymer micelle membrane material, non-single molecular structural component of the membrane material, large particle size, easy potential toxicity caused by the membrane material, high price of the membrane material and the like. The blank mixed micelle has the advantages of high efficiency, safety, stability, strong targeting property, good uniformity, stable and reliable quality, simple and convenient preparation process and the like, and can be used for encapsulating one or more of active substances and substances with health care effect in medicaments and cosmetics to form the mixed micelle loaded with the active substances. When the active substance encapsulated by the mixed micelle is an anti-tumor medicament, the obtained mixed micelle loaded with the active substance has the targeting effect on tumor cells, the multi-drug resistance effect, the synergistic attenuation and the medicament synergistic effect. Specifically, compared with the traditional nano micelle, the active substance loaded mixed micelle is more prominent in the aspects of drug formation and multi-drug resistance, and has the advantages of good stability, good uniformity, high safety and small particle size, and has better curative effect on drug-resistant cells after the active drug is loaded.

The invention provides a blank mixed micelle, which comprises an amphiphilic copolymer and ginsenoside shown in a formula I:

wherein R is¹And R²Each independently is H, -OH, R¹⁰、R¹¹、R¹²Or R¹³However, R¹And R²Not H or-OH simultaneously;

R³is composed of

R⁴Is H, -OH, ketone (═ O), methoxy (-OCH)₃) Ethoxy (-OEt), acetoxy (-OAc), n-propoxy (n-propoxy), isopropoxy (iso-propoxy), n-propionyloxy (n-propionyloxy), isopropionyloxy (iso-propionyloxy), n-butoxy (n-butyloxy), isobutoxy (iso-butyloxy), n-butyryl (n-butyloxy), isobutyryl (iso-butynyl), benzoyl (-OBz), fluorine (-F), chlorine (-Cl), bromine (-Br), iodine (-I), amino (-NH)₂) Or a thio group (-SH);

R⁵is H, -OH, ketone (═ O), methoxy (-OCH3), or acetoxy (-OAc);

R⁶is-OH, methoxy (-OCH3), hydroperoxy (-OOH), acetoxy (-OAc) or benzoyl (-OBz);

R⁷and R⁸Independently is H, -OH, -OCH₃-OCHO, -OAc or-OBz;

R¹⁰is any one of the following groups: O-Glc, -O-Rha, -O-Lyx, -O-Xyl, -O-Ara (p), -O-Ara (f), -O-Glc (2 → 1) Glc (the number indicates the carbon position and the "→ indicates the linkage, the same applies hereinafter), -O-Glc (6 → 1) Glc, -O-Glc (2 → 1) Rha, -O-Glc (2 → 1) Xyl, -O-Glc (6 → 1) Rha, -O-Glc (2 → 1) Ara (p), -O-Glc (6 → 1) Ara (p), -O-Glc (2 → 1) Ara (f), -O-Glc (6 → 1) Ara (f), -O-Glc (2 → 1), -O-Glc (2 → 1) Xyl, -O-Glc (6 → 1) Xyl, -O-Glc (2 → 1) Glc (4 → 1) Xyl, -O-Glc (2 → 1) Lyx, -O-Glc (6 → 1) Lyx, -O-Glc (2 → 1) Rha, -O-Glc (2 → 1) Lyx, -O-Glc (2 → 1) ara (f), -O-Glc (2 → 1) ara (p), -O-Glc (2 → 1) Glc (6 → 1) Glc, -O-Glc (2 → 1) Glc (6 → 1) Rha, -O-Glc (2 → 1) Glc (6 → 1) Glc (35) Glc (2 → 1) Glc → 1), -O-Glc (2 → 1) Glc (6 → 1) Ara (f), -O-Glc (2 → 1) Glc (6 → 1) Ara (p), -O-Glc (6 → 1) Glc (2 → 1) Glc, -O-Glc (6 → 1) Glc (2 → 1) Rha, -O-Glc (6 → 1) Glc (2 → 1) Xyl, -O-Glc (6 → 1) Glc (2 → 1) Lyx, -O-Glc (6 → 1) Glc (2 → 1) Ara (f), -O-Glc (6 → 1) Glc (2 → 1) Ara (p), -O-Glc (6 → 1) Glc, -O-Glc (6 → 1) Rha, -O-Glc (6 → 1) or-Glc (6 → 1) arh → 1) Glc (6 → 1) or-1) Ara (6 → 1) Glc (6 → 1) Glc (6 → 1) and (1) Glc (1) and (6 → 1) Glc (1) and (1) a ) (ii) a

R¹¹Is R¹⁰With more than one hydroxy group of R¹⁰Substituted, each R¹⁰(when there are two or more) are each independently the same or different;

R¹²is any one of the following groups;

I) -mPEG, -Z-mPEG, -mPEO, -Z-PEO, -mVP, -Z-PVP, -mEPEG or-Z-EPEG; wherein m is H, alkyl or acyl, Z is-CO (CH)₂)_aCO-、-NH(CH₂)_aCO-、-NH(CH₂)_bX-or-CO-Ar-CH₂-; wherein X is O, S or NH, Ar is aryl, a is 1, 2, 3, 4, 5,6.7 or 8, b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

II)C₄-C₂₂fatty acyl, phosphate, succinate, n-butyrate, sulfonate, malate, or sodium sulfate salts of (a);

III) a group formed by dehydrogenation of the carboxyl group in Boc-glycine, Boc-alanine, Boc-arginine, Boc-lysine, Boc-serine, acetylphenylalanine, acetylproline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine or valine;

IV) -O-PEO, -O-PVP, -O-PEG, -O-MPEG, -O-EPEG, -O-Glc (2 → 1) Glc (6 → 1) Mal or-O-Glc (2 → 1) Glc (6 → 1) Ac;

R¹³is any one of the following groups; (N, N-dimethylaminoethyl) -carbamoyl (DC for short, the molecular structural formula is

(N, N-dimethylaminopropyl) -carbamoyl (DMAPA for short, and the molecular structural formula is

N- (N ', N' -dimethyl) ethylsuccinic acid monoamide (molecular structural formula is shown in the specification)

Or N- (N ', N' -dimethyl) propyl succinic acid monoamide (molecular structural formula is shown in the specification)

In a preferred embodiment of the invention, R¹preferably-OH,

In a preferred embodiment of the invention, R²Preferably H, -OH,

In a preferred embodiment of the invention, R³Preferably, it is

Further preferred is

More preferably

In a preferred embodiment of the invention, R⁴preferably-OH, -OAc or ═ O.

In a preferred embodiment of the invention, R⁵Preferably H or-OH.

In a preferred embodiment of the present invention, one or more hydroxyl groups of the ginsenoside represented by formula I are optionally substituted by R¹¹Substituted; each R¹¹(when two or more are present) are each independently the same or different.

In a preferred embodiment of the present invention, one or more hydroxyl groups of the ginsenoside represented by formula I are optionally substituted by R¹²Substituted; each R¹²(when there are two or more) are each independently the same or different.

Wherein Glc is glucopyranosyl, Xyl is xylopyranosyl, Rha is rhamnopyranosyl, ara (p) is arabinopyranosyl, ara (f) is arabinofuranosyl, and Lyx is lyxosyl.

Wherein Mal is malonyl, Ac is acetyl, PEG is polyethylene glycol, PEO is polyoxyethylene, MPEG is monomethoxy-terminated polyethylene glycol, EPEG is epoxy-terminated polyethylene glycol, and PVP is povidone.

Wherein, in-O-Glc, Glc has the structural formula:

in-O-Ara (p), Ara (p) has the formula:

in-O-Lyx, Lyx has the formula:

in the formula of-O-Ara (f), Ara (f)

In the formula-O-Rha, Rha has the structural formula

In the-O-Xyl group, the formula of Xyl is

Mal has the structural formula

Wherein the PEG, the PEO, the PVP and the EPEG are preferably respectively and independently 200-20000 in number average molecular weight.

The fatty acyl group may be an acyl group of a naturally occurring saturated or unsaturated fatty acid or an acyl group of an artificially synthesized saturated or unsaturated fatty acid, and is preferably a stearoyl group or a palmitoyl group.

In a preferred embodiment of the present invention, the ginsenoside represented by formula I is preferably one or more compounds of table 1, more preferably 20(S) -ginsenoside Rg3, 20(S) -ginsenoside Rh2, protopanaxadiol, protopanaxatriol, ginsenoside Rg5, ginsenoside Rk1, ginsenoside Rh3, ginsenoside Rg2, ginsenoside Rg4, ginsenoside Rh4, ginsenoside Rh1, damelin a, ginsenoside Rg5H, ginsenoside Rg5H1(E), ginsenoside Rg5H1(Z), ginsenoside Rk1H, ginsenoside Rh3 Rg H, ginsenoside Rh3H1(E), ginsenoside Rh3H1(Z), ginsenoside Rp1, 25-methyl-isoginsenoside Rg3, isoginsenoside Rg3(E), isoginsenoside 3(Z), isoginsenoside Rh3H1, Rh2, ginsenoside Rh 8656, ginsenoside Rp 84695 (Z828653), ginsenoside rjp 5927 (Z) and ginsenoside rj 3, One or more of ginsenoside Rp3, pseudoginsenoside GQ, pseudoginsenoside HQ, ginsenoside SC-Rp1 and ginsenoside DC-Rp 1.

TABLE 1

In the blank mixed micelle, the HPLC purity of the ginsenoside shown in the formula I is preferably greater than or equal to 90%, more preferably more than 95%, and the percentage refers to the percentage of the mass of the ginsenoside shown in the formula I in the total mass of the blank mixed micelle.

The amphiphilic copolymer can be a diblock copolymer and/or a triblock copolymer which are commonly used in common nano-micelles. The amphiphilic copolymer preferably refers to a polymer in which a hydrophilic group and a hydrophobic group are connected, wherein the hydrophilic group is preferably one or more of polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), polyvinyl alcohol (PVA), chitosan and derivatives thereof (such as chitosan-cholic acid), cell membrane-like Phosphorylcholine (PC) and water-soluble cyclodextrin derivatives; the hydrophobic group is preferably one or more of polylactic acid (PLA, e.g., poly-D-lactic acid (PDLA), poly-L-lactic acid (PLLA), poly-D, L-lactic acid (PDLLA), polylactide-glycolide (PLGA), poly-epsilon-caprolactone (PCL), poly-benzyl aspartic acid (PBLA), poly benzyl glutamate (PBLG), polystyrene (Pst), polyisopropylacrylamide (pipa), polylysine (Plys), polyaspartic acid (Pasp), polyhistidine (phil), phospholipids, Phosphatidylethanolamine (PE), Distearoylphosphatidylethanolamine (DSPE), Cholesterol (CHO), and hydrophobic cyclodextrin derivatives (ethyl- β -CD).

In a preferred embodiment of the present invention, the amphiphilic copolymer in the blank micelle mixture is preferably mPEG-DSPE, mPEG-PDLLA, mPEG-PLA, PVP-PNIPAM (PNIPA ═ poly-N-isopropylacrylamide), mPEG-PAsp, PEG-DSPE-NH2, PEG-P, or a mixture thereofAsp, PEG-Phis, PEG-PLGA, PEG-PBLG, PEG-PLA, PEG-PBLA, PEG-PCL, PEG-PCLA, PEO-PASp, PEO-PGlu, PNIPPA-PAA, PCLA-PEG-PCLA, PEO-PPO-PEO, PEO-PLA-PEO, PEG-PLGA-PEG, phosphatidylethanolamine-polyethylene glycol (DMPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG), C8 Ceramide-polyethylene glycol (C8Ceramide-PEG), C16 Ceramide-polyethylene glycol (C16Ceramide-PEG), distearoylphosphatidylethanolamine-polyethylene glycol-Succinyl (DSPE-SuPEG), Distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG) Carboxylic Acid), distearoylphosphatidylethanolamine-polyethylene glycol-Maleimide (DSPE-PEG Maleimide), distearoylphosphatidylethanolamine-polyethylene glycol-propionamide dimercaptopyridine (DSPE-PEG PDP), distearoylphosphatidylethanolamine-polyethylene glycol-cyanuric chloride (DSPE-PEG Cyanur), distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG Amine), distearoylphosphatidylethanolamine-polyethylene glycol-Biotin (DSPE-PEG Biotin), distearoylphosphatidylethanolamine-polyethylene glycol-folic Acid (DSPE-PEG Folate), Dilauroyl phosphatidyl ethanolamine-polyethylene glycol (DLPE-PEG), distearoyl phosphatidyl ethanolamine-polyethylene glycol-active ester (DSPE-PEG-NHS), phosphatidyl ethanolamine-polyethylene glycol-active ester (DMPE-PEG-NHS), dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-active ester (DPPE-PEG-NHS), dilauroyl phosphatidyl ethanolamine-polyethylene glycol-active ester (DLPE-PEG-NHS), distearoyl phosphatidyl ethanolamine-polyethylene glycol-Maleimide (DSPE-PEG-Maleimide), phosphatidyl ethanolamine-polyethylene glycol-Maleimide (DMPE-PEG-Maleimide), dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-Maleimide (DPPE-PEG-Maleimide), Dilauroyl phosphatidyl ethanolamine-polyethylene glycol-Maleimide (DLPE-PEG-Maleimide), distearoyl phosphatidyl ethanolamine-polyethylene glycol-Biotin (DSPE-PEG-Biotin), distearoyl phosphatidyl ethanolamine-polyethylene glycol-fluorescein (DSPE-PEG-FITC), distearoyl phosphatidyl ethanolAmine-polyethylene glycol-hydroxy (DSPE-PEG-OH), distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH)₂) Phosphatidylethanolamine-polyethylene glycol-amino (DMPE-PEG-NH)₂) Dipalmitoyl phosphatidylethanolamine-polyethylene glycol-amino (DPPE-PEG-NH)₂) Dilauroyl phosphatidyl ethanolamine-polyethylene glycol-amino (DLPE-PEG-NH)₂) Distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH), phosphatidylethanolamine-polyethylene glycol-carboxyl (DMPE-PEG-COOH), dipalmitoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DPPE-PEG-COOH), dilauroylphosphatidylethanolamine-polyethylene glycol-carboxyl (DLPE-PEG-COOH), distearoylphosphatidylethanolamine-polyethylene glycol-thio (DSPE-PEG-SH), distearoylphosphatidylethanolamine-polyethylene glycol-Silane (DSPE-PEG-Silane), distearoylphosphatidylethanolamine-polyethylene glycol-azide (DSPE-PEG-N)₃) Cholesterol-polyethylene glycol (Cholesterol PEG), methoxy-polyethylene glycol-Cholesterol (mPEG-CLS), Cholesterol-polyethylene glycol-active ester (Cholesterol PEG NHS ester), Cholesterol-polyethylene glycol-maleimide (CLS-PEG-Mal), Cholesterol-polyethylene glycol-Biotin (Cholesterol PEG Biotin), Cholesterol-polyethylene glycol-fluorescein (Cholesterol PEG fluorescein), Cholesterol-polyethylene glycol-carboxyl (Cholesterol PEG COOH), Cholesterol-polyethylene glycol-amino (Cholesterol PEG NH₂) And Cholesterol-polyethylene glycol-thio (Cholesterol PEG SH), more preferably one or more of mPEG-DSPE, mPEG-PDLLA, mPEG-PLA, PEG-DSPE-NH2, PEG-PASp, PEG-PBLA, PEG-PBLG, PEG-PCL, PEG-Phis, PEG-PLGA, PEO-PASp and PEO-PPO-PEO. Wherein, the number average molecular weight of the polyethylene glycol is preferably 300-50000, more preferably 500-10000, such as 300, 350, 500, 550, 1000, 2000, 3400, 5000, 10000, 20000, 30000, 40000 or 50000.

In the present invention, the number average molecular weight of the mPEG-DSPE is preferably 2000. The number average molecular weight of the mPEG-PDLLA is preferably 2000 or 4000, and more preferably 2000. The number average molecular weight of the mPEG-PLA is preferably 2400. The number average molecular weight of the PEG-DSPE is preferably 2000 or 4000. The number average molecular weight of the PEG-DSPE-NH2 is preferably 4000. The number average molecular weight of the PEG-PASp is preferably 4800. The number average molecular weight of the PEG-PBLA is preferably 2000. The number average molecular weight of the PEG-PBLG is preferably 4000. The number average molecular weight of the PEG-PCL is preferably 2000. The number average molecular weight of the PEG-Phis is preferably 4000. The number average molecular weight of the PEG-PLGA is preferably 2000. The number average molecular weight of the PEO-PASp is preferably 4800. The number average molecular weight of the PEO-PPO-PEO is preferably 4800. The DMPE-PEG preferably has a number average molecular weight of 350, 550, 750, 1000, 2000, 3000 or 5000. The number average molecular weight of the DPPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000 or 5000. The number average molecular weight of the DSPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000, 5000, 10000, 20000, 30000 or 40000. The number average molecular weight of DOPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000 or 5000. The number average molecular weight of the C8Ceramide-PEG is preferably 750, 2000 or 5000. The number average molecular weight of the C16Ceramide-PEG is preferably 750, 2000 or 5000. The number average molecular weight of the DLPE-PEG is preferably 2000 or 5000. The number average molecular weight of the DSPE-PEG-NHS is preferably 1000, 2000, 5000, 10000, 20000, 30000 or 40000. The number average molecular weight of the DMPE-PEG-NHS is 3400 or 5000. The number average molecular weight of the DPPE-PEG-NHS is preferably 3400 or 5000. The number average molecular weight of the DLPE-PEG-NHS is preferably 3400 or 5000. The number average molecular weight of the DSPE-PEG-Maleimide is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the DMPE-PEG-Maleimide is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the DPPE-PEG-Maleimide is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the DLPE-PEG-Maleimid is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the DSPE-PEG-Biotin is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the DSPE-PEG-FITC is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the DSPE-PEG-OH is preferably 2000, 3400 or 5000. The number average molecular weight of the DSPE-PEG-NH2 is preferably 2000, 3400 or 5000. The number average molecular weight of the DMPE-PEG-NH2 is preferably 2000, 3400 or 5000. The number average molecular weight of the DPPE-PEG-NH2 is preferably 2000, 3400 or 5000. The number average molecular weight of the DLPE-PEG-NH2 is preferably 2000, 3400 or 5000. The number average molecular weight of the DSPE-PEG-COOH is preferably 2000, 3400 or 5000. The number average molecular weight of the DMPE-PEG-COOH is preferably 2000, 3400 or 5000. The number average molecular weight of the DPPE-PEG-COOH is preferably 2000, 3400 or 5000. The number average molecular weight of DLPE-PEG-COOH is preferably 2000, 3400 or 5000. The number average molecular weight of the DSPE-PEG-SH is preferably 5000. The number average molecular weight of the DSPE-PEG-Silane is 3400 preferably. The number average molecular weight of the DSPE-PEG-N3 is preferably 2000, 3400 or 5000. The number average molecular weight of the mPEG-CLS is preferably 1000, 2000, 5000, 10000 or 20000. The number average molecular weight of Cholesterol PEG NHS ester is preferably 1000, 2000, 3400, 5000 or 10000. The CLS-PEG-Mal preferably has a number average molecular weight of 2000, 3400, 5000 or 10000. The CLS-PEG-Biotin preferably has a number average molecular weight of 2000, 3400 or 5000. The CLS-PEG-FITC preferably has a number average molecular weight of 2000, 3400 or 5000. The number average molecular weight of Cholesterol PEG COOH is preferably 3400. The number average molecular weight of the Cholesterol PEG amine is 3400 preferably. The number average molecular weight of Cholesterol PEG Thiol/Sulfhydril is 3400 preferably.

In a preferred embodiment of the present invention, the diblock copolymer is preferably one or more of mPEG-DSPE, mPEG-PDLLA, mPEG-PLA, PEG-DSPE-NH2, PEG-PASp, PEG-PBLA, PEG-PBLG, PEG-PCL, PEG-Phis, PEG-PLGA, PEO-PASp and PEO-PPO-PEO.

In a preferred embodiment of the present invention, the triblock copolymer is preferably one or more of poloxamer (PEO-PPO-PEO), PEG-PLGA-PEG, PCLA-PEG-PCLA, PEO-PLA-PEO and PCL-PEG-PCL. Wherein, the number average molecular weight of PEG is required to be between 200 and 20000, preferably between 1000 and 15000, and the ratio of the number average molecular weight of the hydrophobic group to the number average molecular weight of the hydrophilic group is preferably 1:1 to 0.8: 1.

In a preferred embodiment of the present invention, the mass ratio of the amphiphilic copolymer to the ginsenoside represented by formula I is preferably 100:1-0.01:1, preferably 10:1-0.1:1, more preferably 10:1-0.25:1, such as 4:1, 3:1, 2:1, 1.67:1, 0.67:1, 0.5:1, 0.25: 1.

In a preferred embodiment of the present invention, the blank mixed micelle may further comprise one or more of an antioxidant, a lyoprotectant, an emulsifier, and a co-emulsifier.

In the present invention, the antioxidant may be an antioxidant which is conventional in the art, and is preferably one or more of sodium metabisulfite, sodium thiosulfate, propyl gallate, α -tocopherol, α -hydroxy acid, flavonoid, phenylpropanoid phenolic compound, vitamin e (ve), vitamin c (vc), fumaric acid, cysteine, methionine, Butylated Hydroxyanisole (BHA), dibutylhydroxytoluene (BHT), thiodipropionic acid, sulfite (e.g., sodium sulfite), bisulfite (e.g., sodium bisulfite), dithioaminobenzoic acid compound, citric acid, malic acid, sorbitol, glycerol, propylene glycol, hydroquinone, hydroxycoumarin, ethanolamine, phosphoric acid, and phosphorous acid. The content of the antioxidant in the blank mixed micelle is generally less than or equal to 25%, preferably 0.001-15%, such as 3%, 6%, 14%, 0.01-10%, 0.01-5% or 0.1-1%; the percentage (%) refers to the percentage of the mass of antioxidant to the total mass of the blank mixed micelles.

In a preferred embodiment of the invention, the antioxidant is vitamin E and/or vitamin C.

In the present invention, the lyoprotectant may be a lyoprotectant conventional in the art, and typically is one or more of a sugar, a polyol, an amino acid, and a buffer. Wherein, the sugar is preferably one or more of monosaccharide, disaccharide and polysaccharide. The monosaccharide is preferably one or more of glucose, mannitol, xylitol and sorbitol. The disaccharide is preferably one or more of sucrose, lactose, galactose and maltose. The polysaccharide is preferably trehalose. The polyol is preferably propylene glycol and/or glycerol. The amino acid is preferably an alpha-amino acid, such as one or more of threonine, glycine, glutamic acid, arginine and histidine. The buffer is generally referred to as a buffer solution. The buffer solution may be a buffer solution conventional in the art, preferably having a pH of between 3 and 10, more preferably between 5 and 7. The buffer solution is preferably normal saline, an ethanol-acetic acid buffer solution, a tris buffer solution, a barbiturate buffer solution, a sodium formate buffer solution, a phthalate buffer solution, a citrate buffer solution, a citric acid-disodium hydrogen phosphate buffer solution, an ammonia-ammonium chloride buffer solution, a borax-calcium chloride buffer solution, an acetate buffer solution, an acetic acid-lithium salt buffer solution, an acetic acid-sodium acetate buffer solution, an acetic acid-ammonium acetate buffer solution, a phosphoric acid-triethylamine buffer solution or a phosphate buffer solution. The content of the lyoprotectant in the blank mixed micelle is generally less than or equal to 80%, such as 61.73-75.76%, and also such as 61.73%, 65.36%, 65.57%, 74.07%, 75.19%, 75.76%, 0.5-60%, 5-60% or 30-60%; the percentage (%) refers to the mass of lyoprotectant in the total mass of the blank mixed micelle.

In a preferred embodiment of the present invention, the lyoprotectant is one or more of a 5% dextrose aqueous solution, physiological saline, and a phosphate buffer solution.

In the present invention, the emulsifier is preferably one or more selected from gum arabic, tragacanth, gelatin, albumin, casein, soybean lecithin, cholesterol, sorbitan fatty acid (lipophilic), polysorbate (20, 40, 60, 80), polyoxyethylene fatty acid ester (hydrophilic), polyoxyethylene fatty alcohol ether, polyoxyethylene polyoxypropylene copolymer, sucrose fatty acid ester, and glyceryl monostearate, for example, cholesterol. The content of the emulsifier in the blank mixed micelle is generally less than or equal to 10 percent, such as 0.01 to 10 percent, 0.1 to 5 percent or 1 to 5 percent; the percentage (%) refers to the percentage of the mass of the emulsifier to the total mass of the blank mixed micelle.

In the invention, the coemulsifier is preferably one or more of n-butyl alcohol, ethylene glycol, ethanol, propylene glycol, glycerol and polyglycerol ester. The content of the co-emulsifier in the blank mixed micelle is generally less than or equal to 10 percent, such as 0.01 to 10 percent, 0.1 to 5 percent or 1 to 5 percent; the percentage (%) is the mass of co-emulsifier in the total mass of the blank mixed micelle.

In a preferred embodiment of the present invention, the blank mixed micelle comprises an amphiphilic copolymer and ginsenoside represented by formula I.

In a preferred embodiment of the present invention, the blank mixed micelle comprises PEG-DSPE (number average molecular weight of 2000) and ginsenoside Rk 1.

In a preferred embodiment of the present invention, the blank mixed micelle comprises mPEG-PDLLA (number average molecular weight of 2000) and protopanaxatriol PPT.

In a preferred embodiment of the present invention, the blank mixed micelle comprises an amphiphilic copolymer, ginsenoside represented by formula I, an antioxidant and a lyoprotectant.

In a preferred embodiment of the present invention, the blank mixed micelle comprises mPEG-DSPE (number average molecular weight of 2000), damulin A, VE and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the blank mixed micelle comprises PEG-DSPE (number average molecular weight of 4000), protopanaxadiol PPD, VC and 5% aqueous glucose solution.

In the present invention, the blank mixed micelle can be prepared by a conventional preparation method in the art, and generally, a direct dissolution method or a dialysis method can be used. The present invention preferably employs the following method one or method two:

the first method comprises the following steps:

(1) mixing water, amphiphilic copolymer and ginsenoside represented by formula I, optionally adding one or more of antioxidant, emulsifier and co-emulsifier to obtain a clear solution;

(2) forming a film or forming micelles by itself, then mixing the film or micelles with water or an aqueous solution containing a freeze-drying protective agent, optionally adding one or more of an antioxidant, an emulsifier and an auxiliary emulsifier to obtain a solution, filtering, and freeze-drying to obtain the blank mixed micelles;

the second method comprises the following steps:

(1) mixing organic solvent or mixed solvent of water and organic solvent with amphiphilic copolymer, ginsenoside represented by formula I, optionally adding one or more of antioxidant, emulsifier and co-emulsifier to obtain a clear solution;

(2) removing the organic solvent of the clear solution obtained in the step (1), forming a film, then mixing the film with water or an aqueous solution containing a freeze-drying protective agent, optionally adding one or more of an antioxidant, an emulsifier and a co-emulsifier to obtain a solution, filtering, and freeze-drying to obtain the blank mixed micelle.

In the first method or the second method, the amphiphilic copolymer, the ginsenoside represented by the formula I, the antioxidant, the lyoprotectant, the emulsifier and the coemulsifier are defined as the same as the above.

In step (1) of the second method, the organic solvent may be an organic solvent that is conventional in the art for preparing mixed micelles, and is preferably one or more of a nitrile solvent, a C1-C4 alcohol solvent, a ketone solvent, an ether solvent, and a halogenated hydrocarbon solvent, and more preferably one or more of a C1-C4 alcohol solvent, a nitrile solvent, an ether solvent, and a halogenated hydrocarbon solvent. The nitrile solvent is preferably acetonitrile. The C1-C4 alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and n-butanol. The ether solvent is preferably diethyl ether or tetrahydrofuran. The halogenated hydrocarbon solvent is preferably chloroform and/or dichloromethane. The ketone solvent is preferably acetone and/or butanone. The amount of the organic solvent may be the amount conventionally used in the preparation method of mixed micelles in the art, and is not particularly limited, and generally, the organic solvent and all components are mixed to obtain a clear solution, and preferably, the volume-to-mass ratio of the organic solvent to all components in step (1) of the second method is 4-10 mL/g.

In the first or second process, step (1), the temperature of the mixing may be a temperature conventional in the art, and is generally 0 to 80 ℃, preferably 10 to 80 ℃, more preferably 30 to 60 ℃ (e.g., 30, 37, 40, 45, 50, 55, 60 ℃). According to the common knowledge in the art, in some cases, in order to reach a mixing temperature of 80 ℃, it is necessary to carry out under heating; alternatively or in the case where there are temperature sensitive materials such as proteinaceous materials in all the components of the raw materials except the lyoprotectant, mixing at 0 ℃ is generally chosen.

In step (2) of the second method, the operation of removing the organic solvent from the clarified solution in step (1) may be a conventional operation in the art, and the organic solvent is removed by using a rotary evaporator, a film evaporator or membrane dialysis. Wherein the temperature for removing the organic solvent is conventionally selected according to the organic solvent to be removed, and is generally 25-80 ℃.

In the step (2) in the first or second method, the film formation may be performed by vacuum rotary concentration.

In step (2) of the first or second method, the filtration may be performed in a manner conventional in the art of mixed micelle preparation, and is intended to remove bacteria, solid particles, etc. In the present invention, the filtration is preferably a microfiltration membrane filtration. The pore size of the microporous filter membrane is preferably 0.22 microns.

In the first method or the second method, when the membrane is mixed with the aqueous solution of the lyoprotectant in the step (2), the aqueous solution of the lyoprotectant is the aqueous solution formed by mixing the lyoprotectant with water. The aqueous solution of the lyoprotectant is preferably 5% -10% of the aqueous solution of the lyoprotectant, and the percentage refers to the percentage of the mass of the lyoprotectant in the total mass of the aqueous solution of the lyoprotectant. The lyoprotectant is preferably a 5% aqueous glucose solution, physiological saline, or phosphate buffer. The amount of the aqueous solution of the lyoprotectant is not particularly limited as long as it does not affect the formation of mixed micelles, and is preferably the same as the amount of the organic solvent used in step (1).

In a preferred embodiment of the present invention, in the second method, when the aqueous solution of the lyoprotectant is a buffer, the lyoprotectant is directly mixed with the lyoprotectant after the film-forming operation in step (2) is finished.

In the first or second method, in the step (2), the drying operation may be a conventional operation in the art, and preferably freeze-drying, and generally a freeze-dryer is used for freeze-drying. The temperature and time of the freeze-drying are those conventional in the art and may not be particularly limited.

The invention also provides an application of the blank mixed micelle in the preparation of the mixed micelle loaded with the active substance, wherein the active substance in the mixed micelle loaded with the active substance is one or more of active substances in medicines and cosmetics and substances with health care functions. Therefore, the invention also provides an active substance loaded mixed micelle. The active substance-loaded mixed micelle generally means that one or more active substances (active substances) in a medicament are wrapped in the blank mixed micelle.

In the present invention, when there are a plurality of active substances to be supported, except for the combination medication mode used clinically, it is sufficient if the supported active substances do not react with each other, thereby preventing a decrease in pharmacological activity or an adverse reaction from occurring.

In the mixed micelle loaded with the active substance, the mass ratio of the ginsenoside shown in the formula I to the amphiphilic copolymer and the medicament is preferably 100:1-1:1 (such as 20:1, 16.7:1, 16:1, 12:1, 10:1, 8.3:1, 6:1 and 4:1), more preferably 25:1-5:1 (such as 20:1, 16.7:1, 16:1, 12:1, 10:1, 8.3:1 and 6:1), and most preferably 15:1-5:1 (such as 12:1, 10:1, 8.3:1 and 6: 1).

In the active substance, the drug can be a conventional drug in the field, and is preferably one or more of antitumor drugs, anti-inflammatory drugs, antibacterial drugs, sedative hypnotic drugs, antipsychotic drugs, hormone drugs, antibiotic drugs, calcium ion antagonists, antiviral drugs, immunosuppressants, anesthetics, cardiovascular and cerebrovascular and vasodilatation drugs, polynucleotides and oligonucleotides (including ribonucleotides and deoxyribonucleotides).

In the active substance, the anti-tumor drug may be a conventional anti-malignant tumor drug in the field, and is preferably paclitaxel, docetaxel, cabazitaxel, irinotecan hydrochloride, camptothecin, hydroxycamptothecin, aminocamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan hydrochloride, Lurtotecan (Lurtotecan), topotecan, belotecan, cisplatin, carboplatin, oxaliplatin, Nedaplatin (Nedaplatin), Lobaplatin (Lobaplatin), Satraplatin (Satraplatin), miriplatin, pentylplatin, arosplatin (L-NDDP), carmustine, chlorambucil, melphalan, cephalotaxine, homoharringtonine, triptolide, tacrolimus, daunorubicin, pingycin, doxorubicin hydrochloride, idarubicin, fluorouracil, cytarabine, methotrexate, etoposide phosphate, podophyllotoxin, vinorelbine tartrate, Vincristine sulfate, vinblastine, vindesine sulfate, temozolomide, tegafur, cyclophosphamide, ifosfamide, dacarbazine, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, bortezomib, gemcitabine hydrochloride, fludarabine phosphate, capecitabine, one or more of decitabine, pemetrexed disodium, recombinant human interferon a2b, arabinoside cytosine, all-trans retinoic acid, interleukin-2, etoposide, thymidylate synthase inhibitor, mitoxantrone, minoxidil, azithromycin, epirubicin hydrochloride, doxorubicin hydrochloride (adriamycin), amrubicin hydrochloride, KRN-5500, tamoxifen, 5-aminolevulinic acid (5-ALA), 3 ', 5' -cyclocytidine dipalmitate and curcumenol.

In a preferred embodiment of the present invention, the anti-tumor drug is one or more of paclitaxel, docetaxel, camptothecin, homoharringtonine, doxorubicin, cisplatin, oxaliplatin, epothilone C, irinotecan hydrochloride, and all-trans retinoic acid.

In the active substance, the anti-inflammatory drug is preferably one or more of indomethacin, naproxen, ketochromic acid, aspirin, acetaminophen, diclofenac, ibuprofen, bifendate, nimesulide, rofecoxib and celecoxib.

In a preferred embodiment of the invention, the anti-inflammatory drug is one or more of indomethacin, naproxen and bifendate.

In the active substance, the antibacterial drug is preferably amphotericin B, gentamicin, penicillin G, econazole nitrate, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, raviconazole, caspofungin, micafungin, anidulafungin, cefpiramide sodium, cefotaxime sodium, ceftriaxone, cefoperazone, cefditoren pivoxil, cefoxitin sodium, cephalexin, cefuroxime sodium, cefixime, cefpodoxime, cefepime, cefodizime, cefsulodin, ceftizoxime, cefetamet pivoxil, cefteram pivoxil, cefdinir, cefamandole, cefotiam, ceforanide, ceforanian, ceforanide, cefonicid, cefotaxime, cefprozil, cefazolin sodium, cefadroxil, cephalothin, cefotiamidine, cefotaxime, cefotiazine, cefotiam, One or more of cefapirin, cefpirome, cefaclor, cefepime, sodium fusidate, florfenicol and tigecycline.

In a preferred embodiment of the invention, the antibacterial agent is amphotericin B.

In the active substances, the anti-sedation hypnotic drug is preferably one or more of clonazepam, diazepam, nitrazepam, estazolam, alprazolam, barbital, phenobarbital, amobarbital, somnolence and thiopentoxyfylline.

In a preferred embodiment of the invention, the anti-sedative hypnotic agent is clonazepam.

In the active substance, the antipsychotic drug is preferably one or more of haloperidol, chlorpromazine, risperidone, agomelatine, fluoxetine, paroxetine, duloxetine, sertraline, fluvoxamine, citalopram, escitalopram, venlafaxine, mirtazapine, imipramine, amitriptyline, chlorimipramine, doxepin, remmeturon, venlafaxine, phenelzine, isocarboxazid and tranylcypromine.

In a preferred embodiment of the invention, the antipsychotic agent is haloperidol.

In the active substance, the hormone medicine is preferably dihydrotestosterone and/or progesterone.

In a preferred embodiment of the invention, the hormonal agent is dihydrotestosterone.

In the active substance, the antibiotic is preferably cyclosporin A, nystatin, penicillin V, amoxicillin, ampicillin, oxacillin, cloxacillin, procaine penicillin, benzathine penicillin, piperacillin, mezlocillin, ticarcillin, azlocillin, mezlocillin, carbenicillin, sulbenicillin, furacilin, nafcillin, dicloxacillin, pivampicillin, apacillin, aspoxicillin, pimecrillin, methicillin, lenamicillin, zicillin, flucloxacillin, kanamycin, natamycin, mitomycin, butylamine, tylosin, Verteporfin, cefpiramide sodium, netilmicin sulfate, azithromycin, ofloxacin, ciprofloxacin, enoxacin, lomefloxacin, pefloxacin, rufloxacin, sarafloxacin, fleroxacin, moxifloxacin, ampicillin, mefenacillin, oxacillin, mezlocillin, mecillin, and a, mecillin, and a, mecillin, and a, One or more of gefloxacin, trovafloxacin, levamifloxacin, gemifloxacin, gatifloxacin, tosufloxacin, pazufloxacin, sparfloxacin, clarithromycin, clindamycin, polymyxin, tobramycin, vancomycin, azithromycin, doxycycline, tetracycline, oxytetracycline, minocycline, chlortetracycline, guanmecycline, demeclocycline, metacycline, etimicin, netilmicin, sisomicin, amikacin, arbekacin, dibekacin, aztreonam, meropenem, imipenem, thienamycin, panipenem, ertapenem, neomycin, paromomycin and spectinomycin.

In a preferred embodiment of the invention, the antibiotic is cyclosporin a.

In the active substance, the calcium ion antagonist is preferably one or more of fenofibrate, nimodipine, nifedipine, nicardipine, nitrendipine, verapamil, amlodipine, diltiazem, flunarizine, prenylamine, galopam and tiapamil.

In a preferred embodiment of the present invention, the calcium antagonist is fenofibrate.

In the active substance, the anesthetic is preferably one or more of desflurane, sevoflurane, isoflurane, enflurane, propofol, fentanyl, uratan, lidocaine, procaine, tetracaine, bupivacaine, sodium pentobarbital, chloral hydrate, ketamine, aldochloroketone and morphine.

In a preferred embodiment of the invention, the anesthetic is propofol.

In the active substances, the cardiovascular and cerebrovascular and vasodilatation drugs are preferably one or more of dabigatran etexilate, alogliptin, alginic acid diester sodium, bilobalide, ginkgetin, ginkgo biloba extract, asarone, olmesartan medoxomil, repaglinide, lipoic acid, breviscapine, urapidil, nicotinic acid, captopril, losartan, puerarin, tanshinone IIA, sarpogrelate hydrochloride, tropicamide, fluvastatin, pravastatin, simvastatin, lovastatin, simvastatin, mevastatin, cerivastatin, rosuvastatin calcium and rosuvastatin calcium.

In a preferred embodiment of the invention, the cardiovascular and cerebrovascular and vasodilatation drug is puerarin.

In the active substance, the polynucleotide or oligonucleotide preferably refers to a fragment having a genetic function and the like, which is composed of several bases A, T, C, G and U, such as SiRNA, antisense nucleic acid or an RNAi sequence of microglia NLRP3 gene.

In a preferred embodiment of the invention, the polynucleotide or oligonucleotide is SiRNA.

Among the active substances, the active substances in the cosmetics generally refer to active substances having effects of nourishing, improving skin conditions and preventing skin diseases in the cosmetics, and are preferably one or more of ursolic acid, superoxide dismutase (SOD), bioprotein T4N5, vitamin D2, vitamin K3, methyl nicotinate, refined snake oil, hyaluronic acid, essential oils and ceramides.

In a preferred embodiment of the present invention, the active substance in the cosmetic is vitamin K3.

In a preferred embodiment of the present invention, the mixed micelle comprises an amphiphilic copolymer, ginsenoside represented by formula I, and an active substance.

In a preferred embodiment of the present invention, the mixed micelle comprises mPEG-PDLLA (number average molecular weight of 4000), ginsenoside Rg5H and docetaxel.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-DSPE (number average molecular weight of 2000), ginsenoside Rg3Me and propofol.

In a preferred embodiment of the present invention, the mixed micelle comprises mPEG-PDLLA (number average molecular weight of 4000), chitosan-cholic acid, ginsenoside Rg4 and homoharringtonine.

In a preferred embodiment of the invention, the mixed micelle comprises mPEG-PLA (with the number average molecular weight of 2400), ginsenoside Rh4 and adriamycin.

In a preferred embodiment of the invention, the mixed micelle comprises PEG-PBLG (with the number average molecular weight of 4000), ginsenoside Rg2(Z) and all-trans retinoic acid.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PAsp (number average molecular weight of 4800), ginsenoside Rg3H and cisplatin.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PBLA (number average molecular weight of 2000), ginsenoside Rg3E and irinotecan hydrochloride.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-Phis (number average molecular weight of 4000), ginsenoside Rg2 and epothilone C.

In a preferred embodiment of the present invention, the mixed micelle comprises an amphiphilic copolymer, ginsenoside represented by formula I, an active substance and an antioxidant.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-DSPE (number average molecular weight of 2000), ginsenoside Rg5, paclitaxel and vitamin e (ve).

In a preferred embodiment of the invention, the mixed micelle comprises an amphiphilic copolymer, ginsenoside represented by formula I, an active substance and a freeze-drying protective agent.

In a preferred embodiment of the present invention, the mixed micelle comprises PEO-PAsp (number average molecular weight of 4800), ginsenoside Rh1, camptothecin, and a 5% aqueous glucose solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PLGA (number average molecular weight 2000), ginsenoside Rh2, oxaliplatin and 5% aqueous glucose solution.

In a preferred embodiment of the invention, the mixed micelle comprises mPEG-PDLLA (with the number average molecular weight of 4000), ginsenoside Rg3, indomethacin and 5% glucose aqueous solution.

In a preferred embodiment of the invention, the mixed micelle comprises PEG-Phis (number average molecular weight 4000), Rh3, naproxen and 5% aqueous glucose solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PCL (number average molecular weight 2000), isopanasaponin Rh2(E), haloperidol and 5% aqueous glucose solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PBLA (number average molecular weight 2000), ginsenoside Rg3(Z), dihydrotestosterone and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PAsp (number average molecular weight of 4800), pseudoginsenoside GQ, vitamin K3 and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PBLG (number average molecular weight 4000), ginsenoside Rp2, biphenyldicarboxylate and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-DSPE-NH2 (number average molecular weight of 4000), pseudoginsenoside HQ, puerarin and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PCL (number average molecular weight 2000), ginsenoside Rp3, cyclosporin a and 5% aqueous glucose solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PCL (number average molecular weight of 2000), ginsenoside Rh2(Z), fenofibrate and 5% aqueous glucose solution.

In a preferred embodiment of the invention, the mixed micelle comprises PEG-PCL (with a number average molecular weight of 2000), isoginsenoside SC-Rp1, amphotericin B and a 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-PCL (number average molecular weight of 2000), ginsenoside DC-Rp1, SiRNA and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises poloxamer 188(PEO-PPO-PEO) (number average molecular weight of 4800), ginsenoside Rg2(E), adriamycin and 5% glucose aqueous solution.

In a preferred embodiment of the present invention, the mixed micelle comprises an amphiphilic copolymer, ginsenoside represented by formula I, an active substance, an antioxidant, a lyoprotectant and an emulsifier.

In a preferred embodiment of the present invention, the mixed micelle comprises PEG-DSPE (number average molecular weight of 4000), ginsenoside Rp1, clonazepam, vitamin E, saturated phosphate buffer solution and cholesterol.

In a preferred embodiment of the present invention, the mixed micelle may further comprise other auxiliary materials and is encapsulated in a film. The other auxiliary materials can be other auxiliary materials which are conventionally added in the field for preparing the micelle except for the antioxidant and the freeze-drying protective agent, such as one or more of an emulsifier or a co-emulsifier.

The invention also provides a preparation method of the ginsenoside mixed micelle modified by the amphiphilic copolymer loaded with the active substance and shown in the formula I.

In the present invention, the preparation method of the active material-loaded mixed micelle may employ chemical conjugation and physical encapsulation methods (e.g., solvent evaporation, dialysis, emulsification) which are conventional in the art. The present invention is preferably any of the following methods:

method a (solvent evaporation method) comprises the following steps:

mixing an amphiphilic copolymer, ginsenoside shown as a formula I, an active substance and an organic solvent, optionally adding one or more of an antioxidant, an emulsifier and a co-emulsifier, removing the organic solvent, forming a film, mixing with water or an aqueous solution containing a freeze-drying protective agent, optionally adding one or more of the antioxidant, the emulsifier and the co-emulsifier to form a mixed micelle solution loaded with the active substance, filtering, and freeze-drying to obtain the active substance-loaded mixed micelle solution;

method B (dialysis) comprises the following steps:

mixing an amphiphilic copolymer, ginsenoside shown in formula I and an organic solvent, mixing with an active substance, dialyzing with water or a water solution containing a freeze-drying protective agent to obtain a mixed micelle solution loaded with the active substance, optionally adding one or more of an antioxidant, an emulsifier and a co-emulsifier, filtering, and freeze-drying a dialysate;

method C (emulsification) comprises the following steps:

mixing an active substance with an organic solvent to obtain a mixture A, mixing an amphiphilic copolymer, ginsenoside shown as a formula I and water or a buffer solution to obtain a mixture B, dropwise adding the mixture A into the mixture B to form an oil/water (O/W) mixed emulsion, optionally adding one or more of an antioxidant, a freeze-drying protective agent, an emulsifier and a co-emulsifier, removing the organic solvent, filtering, and freeze-drying;

method D (chemical bonding method) comprises the following steps:

mixing an active substance, an amphiphilic copolymer, ginsenoside shown in formula I and a solvent, wherein the active substance is covalently combined with the amphiphilic copolymer or active groups on the ginsenoside shown in formula I, optionally adding one or more of an antioxidant, a freeze-drying protective agent, an emulsifier and a co-emulsifier, and when the solvent contains an organic solvent, removing the organic solvent, filtering, and freeze-drying.

Method E (direct dissolution method) comprises the following steps:

when the active substance is easily soluble in water, mixing the active substance, amphiphilic copolymer, ginsenoside represented by formula I and water, optionally adding one or more of antioxidant, emulsifier and co-emulsifier, filtering, and freeze drying.

In method A, B, C, D, E, the conditions and parameters are referred to in the first or second method of the blank mixed micelle preparation method.

In the method B, the dialysis operation may be a conventional operation in the art of the preparation method of mixed micelles, and the present invention preferably dialyzes the mixed micelle solution in an aqueous glucose solution (e.g., 0.15mol/L) or in pure water. The dialysis time may be a time conventional in the art of mixed micelle preparation, and is preferably 5 to 20 hours, more preferably 12 hours.

In each of the above methods, the organic solvent or solvent is preferably one or more of dichloromethane, chloroform, methanol, ethanol, diethyl ether, acetonitrile, acetone, ethyl acetate, Tetrahydrofuran (THF), Dimethylformamide (DMF), N-dimethylacetamide (DMAc), Dimethylsulfoxide (DMSO), and pyridine.

In each of the above processes, the active substance may also be used preferably in the form of an aqueous solution of the active substance or an organic solution of the active substance, depending on the lipid solubility or water solubility of the active substance. The mass fraction of the aqueous solution of the active substance or the organic solution of the active substance is not particularly limited, and is preferably 1-20% by mass volume of the aqueous solution or the organic solution, and the percentage refers to the mass (g) of the active substance in the total volume (mL) of the aqueous solution of the active substance or the organic solution of the active substance. The organic solvent in the organic solution of the active substance may be an organic solvent that is conventional in the art, as long as it can dissolve the active substance well. In the present invention, the organic solvent is preferably a sulfoxide-based solvent, such as dimethyl sulfoxide (DMSO).

In each of the above methods, the definitions of the amphiphilic copolymer, the ginsenoside represented by formula I, the antioxidant, the lyoprotectant, the emulsifier and the co-emulsifier are the same as those described above, and the amounts and proportions of the components are the same as those described above.

A preferred embodiment of the method D may be that PEO-PPO-PEO and doxorubicin are dissolved in DMSO, DMAP (lutidine) is added at 30-50 ℃ and stirred for 3-4hrs to prepare a conjugate of micelle and doxorubicin, PBS buffer solution in which ginsenoside represented by formula I is dissolved at pH 7.4 is added, the mixture is depressurized and concentrated to remove the organic solvent, and freeze-dried to obtain the mixed micelle carrying the active substance.

The particle size of the blank mixed micelle and the active material-supporting mixed micelle is preferably 10 to 200nm (e.g., 24.5, 40.9, 24.5, 28.6, 16.7, 24.5, 33.4, 35.5, 66, 27.3, 18.8, 26.9, 23.9, 22.5, 20.7, 46.5, 32.9, 18.3, 23.6, 27, 26.2, 29.6, 28.2, 21.1, 30.7, 42.2, 28.3, 24.7), and more preferably 16.7 to 30.7 nm.

The encapsulation efficiency of the active material-supporting mixed micelle is preferably 80% or more, more preferably 90% or more, and most preferably 95% or more.

When the active substance in the active substance-loaded mixed micelle is a drug and/or a substance with a health-care function, the administration route of the active substance-loaded mixed micelle can be the conventional administration route in the field, and is preferably injection administration, oral administration or transdermal administration, and is used for treating diseases and/or medical health care. Therefore, the active substance-loaded mixed micelles are usually prepared in the form of injections, lyophilized injections, oral solid preparations, oral liquids, liniments, ointments, tinctures or aerosols. The injection administration mode is preferably intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. Generally, the active substance-loaded mixed micelle is added to physiological saline, a phosphate buffer solution or a 5% aqueous glucose solution to prepare an injection solution for injection administration.

In the mixed micelle loaded with the active substance, when the active substance is an anti-tumor medicament, the mixed micelle loaded with the active substance generally has a targeting effect on tumor cells, an anti-multidrug resistance effect, a synergistic attenuation effect and a medicament synergistic effect.

In the present invention, the expression "comprising … …" can also be said to "consist of … …".

For example: in a preferred embodiment of the present invention, the mixed micelle comprises an amphiphilic copolymer, ginsenoside represented by formula I, an active substance, an antioxidant, a lyoprotectant and an emulsifier.

It can also be expressed as: in a preferred embodiment of the invention, the mixed micelle consists of an amphiphilic copolymer, ginsenoside represented by formula I, an active substance, an antioxidant, a freeze-drying protective agent and an emulsifier.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

In the present invention, room temperature means 10 to 30 ℃.

In the present invention, the density of the aqueous solution of the lyoprotectant or the aqueous solution of the active substance is calculated as 1g/mL (i.e., the density of water), and therefore, the total mass m of the aqueous solution of the lyoprotectant or the aqueous solution of the active substance is ρ ═ V.

In the present invention, the density of the organic solution of the active material is calculated according to the kind of the organic solvent, and when the organic solvent is DMSO, for example, the density of the organic solution of the active material is 1.1 g/mL.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

(1) the blank mixed micelle has the advantages of high efficiency, safety, stability, good uniformity, reliability, simple and convenient preparation process and the like, and can be used for encapsulating one or more of active substances and substances with health care effect in medicaments and cosmetics to form the blank mixed micelle loaded with the active substances. When the active substance encapsulated by the blank mixed micelle is an anti-tumor medicament, the obtained mixed micelle loaded with the active substance has the targeting effect on tumor cells, the multi-drug resistance effect, the synergistic attenuation and the medicament synergistic effect. Specifically, compared with the traditional mixed micelle, the micelle has more excellent indexes, especially in the aspects of patent drug property, multi-drug resistance, synergy and attenuation, drug synergistic action and the like.

(2) The mixed micelle can effectively improve the hemolysis of the ginsenoside after gelling, and the hemolysis of the mixed micelle is not seen at 1 mg/ml.

(3) The mixed micelle can effectively improve the stability of the micelle, is turbid after being placed for 8-12 hours or even more than 24 hours, has the particle size of 15-66 nm, and has performance remarkably superior to that of a ginsenoside nano micelle or an amphiphilic copolymer nano micelle.

(4) The mixed micelle disclosed by the invention has better activity on drug-resistant strains such as human lung cancer paclitaxel drug-resistant strains (A549/T), and is low in effect concentration and good in drug effect.

Drawings

FIG. 1 is a graph showing the cell survival rate of mixed empty, Genex-PM, paclitaxel mixed micelle against human lung cancer cell (A549). Wherein, the upper X axis represents the concentration of ginsenoside (ng. mL)^-1) Logarithmic value of (D), lower X-axis represents paclitaxel concentration (ng. mL)^-1) The logarithmic value of (c).

FIG. 2 is a graph showing the cell survival rate of mixed empty, Genex-PM, paclitaxel mixed micelle against human lung cancer paclitaxel resistant strain (A549/T). Wherein, the upper X axis represents the concentration of ginsenoside (ng. mL)^-1) Logarithmic value of (D), lower X-axis represents paclitaxel concentration (ng. mL)^-1) The logarithmic value of (c).

FIG. 3 is a graph showing the tumor inhibition curves of human lung cancer cells A549 in the Control group, the Genex-PM group and the paclitaxel mixed micelle group.

FIG. 4 is a tumor-suppressing curve chart of Control group, Genex-PM group, and paclitaxel mixed micelle group against human lung cancer paclitaxel resistant strain (A549/T).

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

1. Experimental drugs: 20(S) -ginsenoside Rg3, 20(S) -ginsenoside Rh2, protopanaxadiol, protopanaxatriol, ginsenoside Rg5, ginsenoside Rk1, ginsenoside Rh3, ginsenoside Rg2, ginsenoside Rg4, ginsenoside Rh4, ginsenoside Rh1, damelin A, ginsenoside Rp1, 25-methyl-isoginsenoside Rg3, isoginsenoside Rg3(E), isoginsenoside Rg3(Z), isoginsenoside Rh2(E), isoginsenoside Rh2(Z), isoginsenoside Rg2(E), isoginsenoside Rg2(Z), ginsenoside Rp2, ginsenoside Rp3, pseudoginsenoside GQ, pseudoginsenoside HQ, ginsenoside SC-Rp1, ginsenoside DC-Rp1, taxol, docetaxel, trans-camptothecin, tretinoin, irinotecan, oxaliplatin, and oxaliplatin, Indomethacin, naproxen, homoharringtonine, doxorubicin, clonazepam, haloperidol, dihydrotestosterone, cyclosporin a, fenofibrate, biphenyldicarboxylate, propofol, puerarin, vitamin K3 are conventional commercially available in the art, for example, from shanghai ben medicine science and technology limited.

The ginsenoside Rg5H and the isoginsenoside Rg3H can be prepared according to preparation examples in the application.

PEG-DSPE, mPEG-PDLLA, mPEG-PLA, PEO-PASp, PEG-DSPE-NH2, Poloxamer 188(PEO-PPO-PEO), PEG-PCL, PEG-PLGA, PEG-PBLA, PEG-PBLG, PEG-PASp, PEG-PHIs, vitamin E are routinely commercially available in the art, for example, from Saian Rexi Biotech Ltd.

Conventional SiRNA was purchased from leber.

Genex-PM, polyethylene glycol-polylactic acid copolymer (mPEG-PDLLA) as a membrane material paclitaxel lyophilized nanomicelles were prepared from Samyang Biopharmaceutics Corporation of Korea.

2. The following examples and application examples used instruments that were owned by the institute of medicine, southwest university, with equipment models and source information as follows:

high performance liquid chromatography (Agilent 1100);

electronic balance (TB-215, Denver Instrument, USA);

ultrasonic cleaning machine (SB3200DT, Ningbo Xinzhi Biotech Co., Ltd.);

nitrogen blower (HGC-12A, constant Olympic technology development Co., Ltd., Tianjin);

a rotary evaporator (RE-2000A, Shanghai Yangrong Biochemical Instrument factory);

an ultrapure water production system (ULUP-IV-10T, Ulpu technologies, Sichuan);

a constant temperature oscillator (SHA-C, Australian instruments, Inc., Changzhou);

ultrasonic cell crusher (JY92-II, Ningbo Xinzhi Biotech GmbH);

high pressure homogenisers (B15, AVESTIN, canada);

laser particle size analyzer (Nano ZS, malvern instruments, uk);

micro-extruders (Mini-extruder, Avanti Polar Lipids Inc);

photoelectric microscopes (XDS-1B, Chongqing photoelectric instruments, Inc.);

clean bench (SW-CJ-1FD, air technologies, Inc., Antai, Suzhou);

cell culture incubator (CCL-170B-8, Singapore ESCO);

fluorescence inverted microscope (IX-73, Orlybar, Japan);

small animal live imaging system (FX PRO, Bruker, usa);

3. experimental cell lines:

a549 human lung cancer cell (south kyo kaki organism);

the method for establishing the A549/T human lung cancer paclitaxel resistant strain comprises the following steps:

and inducing the parent A549 cell to establish a human lung cancer drug-resistant cell line A549/Taxol by adopting a low-concentration dosage continuous induction method. The newly recovered A549 cells are cultured for 2 generations or 3 generations under the conventional condition, so that the cells grow stably. When the culture medium is renewed the next day after the cells are digested and passaged, the parent A549IC is replaced by Taxol₅₀1/10 paclitaxel was added at the starting concentration. The next day after adding the medicineAnd (4) new culture medium and maintaining the concentration of the paclitaxel for conventional subculture. After the cells with each paclitaxel concentration stably grow, the drug concentration is increased to continue culturing until the cells can stably grow in a culture medium containing 2.5mg/L paclitaxel for twelve months.

4. And (3) hemolysis detection:

preparation of 2% erythrocyte suspension: taking blood of healthy rabbit, putting into conical flask containing glass beads, shaking for 10min, or stirring blood with glass rod to remove fibrinogen to obtain defibrinated blood. Adding about 10 times of 0.9% sodium chloride solution, shaking, centrifuging for 15 minutes at 1000-1500 rpm, removing supernatant, and washing the precipitated red blood cells with 0.9% sodium chloride solution for 2-3 times until the supernatant is not red. The red blood cells were made up to 2% suspension in 0.9% sodium chloride solution for testing.

The inspection method comprises the following steps: 5 clean glass test tubes are taken, wherein the number 1 and the number 2 are used as test article tubes, the number 3 is used as a negative control tube, the number 4 is used as a positive control tube, and the number 5 is used as a test article control tube. The suspension of red blood cells (2%), sodium chloride solution (0.9%) and purified water were added in this order as shown in the following table, mixed well and immediately incubated in a 37 + -0.5 deg.C incubator. After 3 hours hemolysis and coagulation reactions were observed.

Test tube numbering	1、2	3	4	5
					2% erythrocyte suspension/ml	2.5	2.5	2.5	/
0.9% sodium chloride solution/ml	2.2	2.5	/	4.7
					Purified water/ml	/	/	2.5	/
Test solution/ml	0.3	/	/	0.3

If the solution in the test tube is clear red, no cells are left at the bottom of the tube, indicating hemolysis; if the erythrocytes sink completely, the supernatant is colorless and transparent, or although the supernatant is colored and clear, no obvious difference is observed by naked eyes in the tubes 1, 2 and 5, which indicates that no hemolysis occurs.

If there is a reddish-brown or reddish-brown flocculent precipitate in the solution, it is not dispersed after gently inverting 3 times, indicating that there is probably erythrocyte agglutination, and observing under a microscope, if the erythrocyte agglutination is visible.

And (5) judging a result: when the negative control tube has no hemolysis or condensation and the positive control tube has hemolysis, if the solution in 2 test sample tubes has no hemolysis or condensation within 3 hours, the test sample is judged to be in accordance with the specification; if the solution of 1 test tube is hemolyzed and/or condensed within 3 hours, 4 test tubes are set for retesting, and the solution of the test tube is not hemolyzed and/or condensed within 3 hours, otherwise, the test tube is judged to be not in accordance with the regulations.

In a specific experiment, the concentration of the test sample (ginsenoside) can be adjusted according to actual conditions.

5. Experimental animals: kunming mice (or normal mice), purchased from the animal center of third department of military medical university;

BALB/C-nu/nu mice (or nude mice) were purchased from Shanghai Spiker laboratory animals, Inc.

6. The cell culture method comprises the following steps: the cell lines involved were placed in a 5% CO solution₂In a 37 ℃ incubator, the cells were cultured in DMEM or RPMI1640 complete medium (containing 10% fetal bovine serum, 100U/ml penicillin, 100. mu.g/ml streptomycin), digested with 0.25% trypsin-EDTA and passaged 2 to 3 times per week.

7. Administration: for each experiment, a negative control group (mixed blank micelle, mixed blank for short), a positive control group (Genex-PM), and a mixed micelle group were set. More than 6 concentration gradients, one-half or five-fold dilutions, triplicate wells per concentration were set.

8. Tumor cell inhibitory concentration IC₅₀The experimental method comprises the following steps: tumor cells in logarithmic growth phase are digested with pancreatin to prepare cell sap with a certain concentration, and the cell sap is inoculated into a 96-well plate according to 5000 cells per well, and 100 mul is added into each well. The next day, fresh medium containing samples at different concentrations and corresponding solvent controls was added, 100. mu.l per well (DMSO final concentration)<0.5 percent), setting 10 dose groups for each sample, setting three parallel holes for each group, continuously culturing for 72 hours in an incubator at 37 ℃, removing supernatant, adding 100 mu l of PBS and 10 mu l of CCK-8 into each hole, uniformly shaking by using a micro-oscillator, continuously culturing for 3 hours, measuring Optical Density (OD) value by using an enzyme-labeling instrument at a reference wavelength of 630nm and a detection wavelength of 450nm, taking tumor cells treated by solvent control as a control group, and calculating IC according to an intermediate effect equation₅₀。

9. In vitro cell assay methods: tumor cells were collected in logarithmic growth phase and resuspended in DMEM complete medium (containing 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL of chain)Mycin) at a final concentration of 4X 10⁴one/mL. In a 96-well cell culture plate, 200. mu.l of the above cell suspension (8X 10) was added per well³Individual cells/well) at 37 ℃ in 5% CO₂The cell culture box is used for culturing for 48 hours, the DMEM complete culture medium is respectively changed into 200 mul of antitumor drugs containing different concentrations, the final concentration of the drugs is set to be more than 6 groups, the DMEM complete culture medium is used as a negative control group, each concentration is provided with 4 multiple wells, and the experiment is repeated for 3 times. Cells were incubated at 37 ℃ with 5% CO₂After culturing for 72 hours in the cell culture box, 20. mu.l of MTT solution of 5mg/mL was added to each well, the cell culture box was placed for further culturing for 4 hours, the supernatant was discarded, 150. mu.l of DMSO was added to each well, and after shaking for 10 minutes, the OD at 490nm was measured using a continuous spectrum multifunctional microplate reader (Tecan, Switzerland), and the cell survival rate was calculated according to the following formula: (cell survival (%) ═ OD_Medicine/OD_Control×100％)。

Cell survival (%) ═ OD_{490 (sample)}/OD_{490 (control)}×100％；

Wherein, OD_{490 (sample)}OD value for the experimental group, OD_{490 (control)}The OD value of the blank control group.

10. The in vivo efficacy test method comprises the following steps: take 1X 10⁷-10×10⁷Injecting the tumor cells of logarithmic growth phase into the right axilla of 18-20g nude mice slowly by using a 1mL injector, injecting 100 mul of each nude mouse, observing the growth of tumor mass until the volume of the tumor mass is about 100mm³. Animals were randomized into groups to begin dosing. Tumor volumes were weighed and determined every two days, and the longest and shortest tumor diameters were measured with a vernier caliper, nude mice were sacrificed, tumor mass volumes were determined, Relative Tumor Volume (RTV), relative tumor proliferation rate (T/C) and percent tumor inhibition were calculated and statistically analyzed.

Tumor volume calculation formula: and V is abh/2. Wherein, a is the tumor diameter, b is the tumor transverse diameter, and h is the tumor height.

Relative tumor volume RTV calculation formula: RTV is Vt/V0. Where Vt is the tumor volume at a certain time and V0 is the tumor volume at the beginning of the administration.

Formula for calculating relative tumor proliferation rate: T/C (%) ═ TRTV/CRTV × 100%. Wherein TRTV is treatment RTV and CRTV is solvent control RTV.

Formula for percentage of tumor inhibition: the percentage of tumor inhibition was (tumor weight of solvent control group-tumor weight of administration group)/tumor weight of solvent control group × 100%.

The evaluation standard of the curative effect is as follows: T/C (%) >60 is ineffective; T/C (%) ≦ 60, and tumor volume statistically treated P <0.05 was valid compared to the solvent control.

In the following application examples, C (μ M) refers to concentration, wherein the concentration of Taxol + Rg5 refers to the concentration of paclitaxel and the concentration of ginsenoside Rg5 in the mixed micelles, for example, 5+30 refers to the concentration of paclitaxel in the ginsenoside Rg5 and the concentration of ginsenoside Rg5 is 5 μ M and 30 μ M; time (d) refers to the time (days).

In the following application examples, when blank mixed micelles of ginsenoside Rg5 appear, if not specifically mentioned, the blank mixed micelles (mixed blank for short) formed by PEG-DSPE and ginsenoside Rg5 prepared by the method of example 1 are all referred to; when the ginsenoside Rg5 and paclitaxel mixed micelle appear, unless otherwise specified, the paclitaxel mixed micelle formed by PEG-DSPE prepared by the method in example 5 and ginsenoside Rg5 (Taxol + Rg5 for short) is referred to.

In the following examples, the operating temperature and pressure are generally room temperature and normal pressure unless otherwise specified. Wherein the room temperature is 10-30 ℃; atmospheric pressure refers to one standard atmosphere.

Preparation of ginsenoside compound shown in formula I

Preparation example 1 preparation of ginsenoside Rg5H

Dissolving 10g of 20(R) -ginsenoside Rg3 in 20mL of pyridine, dropwise adding 10mL of acetic anhydride in an ice-water bath, then adding an appropriate amount (generally catalytic amount, for example, 1g) of catalyst DMAP, slowly heating to room temperature, reacting for 10 hours, detecting by TLC until a raw material point disappears, concentrating under reduced pressure to remove an organic solvent, extracting by 200 mL/ethyl acetate for 3 times, combining organic phases, washing by 2M hydrochloric acid for 3 times, washing by saturated sodium bicarbonate for 3 times, drying by anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain an Rg3 acetylated product.

Dissolving 10g of Rg3 acetylated product in 50mL of dichloromethane, sequentially adding 1g of boron trifluoride diethyl etherate and 1g of triethylsilane in an ice water bath, slowly heating to room temperature, continuing to react for 5-10min at room temperature, cooling to 0 ℃, adding ice water, quenching the reaction, washing for 3 times by using 100 mL/time saturated sodium bicarbonate solution, drying by anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain an Rg5H acetylated product.

10g of Rg5H acetylated product was taken and dissolved in 50mL of methanol and dioxane 1:1, adding 2g of potassium hydroxide into the mixed solution, stirring for dissolving, heating for reflux reaction for 10 hours, detecting by TLC until a raw material point disappears, neutralizing by using saturated citric acid aqueous solution until the pH value is 7, extracting by using 100 mL/ethyl acetoacetate for 3 times, combining organic phases, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure until the raw material point is dry, separating by high pressure chromatography, performing chromatographic separation by using C18, performing gradient elution by using methanol water, detecting by using an evaporative light diffuser (ELSD), and concentrating a product section until the product section is dry to obtain the ginsenoside Rg 5H.

Ginsenoside Rg 5H:

¹H NMR(δ,500M):5.37(1H,d,J＝7.5Hz),5.30(1H,t,J＝6.0Hz),4.91(1H,d,J＝7.5Hz),4.55(1H,m),4.45-4.49(2H,m),4.23-4.33(5H,m),4.12-4.14(2H,m),3.90-3.92(3H,m),3.27(1H,m)，2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.64(3H,s),1.61(3H,s),1.41(3H,d,J＝2.8),1.35-1.50(8H,m),1.28(3H,s),1.20(1H,m),1.09(3H,s),1.03(1H,m),0.94(3H,s),0.95(3H,s),0.78(3H,s),0.73(1H,m).

¹³C NMR(δ,125M):130.8,126.4,106.1,105.1,89.0,83.4,78.4,78.2,78.3,78.0,71.7,71.0,62.9,62.8,77.2,56.4,54.8,51.7,50.4,49.2,48.6,40.0,39.7,39.2,36.9,9,35.2,32.1,31.4,28.2,27.1,26.9,26.8,25.8,23.0,18.5,17.7,17.0,16.6,16.4,15.9.

ESI:770.13(M+H)⁺,HR-ESI-MS:792.0215(C₄₂H₇₂NaO₁₂),Cal 792.0230.

preparation example 2 preparation of ginsenoside Rg5H1(E), Rg5H1(Z), Rk1H

Weighing 10g of 20(R) -Rg3 acetylated product, dissolving in 50mL of methanol, adding 1g of palladium carbon, introducing hydrogen at normal temperature and normal pressure, stirring for 4-6 hours until the reaction solution does not absorb hydrogen, detecting by TLC until the raw material point disappears, filtering out the palladium carbon, reducing pressure, concentrating to remove the methanol, and drying to obtain the 20(R) -Rg3 acetylated hydrogenation product.

Weighing 10g of 20(R) -Rg3 acetylated hydrogenation product, dissolving in 50mL of toluene, adding 10g of p-toluenesulfonic acid, slowly heating to 90 ℃ for refluxing, reacting for 4 hours, detecting by TLC until the raw material point disappears, cooling, washing for 3 times by 100 mL/saturated sodium bicarbonate solution, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain a crude acetylated product mixture (acetylated product mixture of Rg5H1(E type), Rg5H1(Z type) and Rk 1H).

Weighing 5g of crude acetylation product mixture, dissolving in 20mL of methanol, adding 5g of sodium methoxide, reacting at room temperature for 10 hours, detecting by TLC until the raw material point disappears, concentrating under reduced pressure to dryness, re-dissolving with ethyl acetate, washing with 100 mL/time water for 3 times, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain crude product mixture (mixture of Rg5H1(E type), Rg5H1(Z type) and Rk 1H).

Weighing 5g of crude product mixture, separating by high pressure chromatography, eluting with C18 as filler, methanol water gradient, evaporating the light diffuser (ELSD), and concentrating the product segment to dryness to obtain 1.8g of Rg5H1(E type) with HPLC purity of above 98%, 0.2g of Rg5H1(Z type) with HPLC purity of above 98% and 0.8g of Rk1H with HPLC purity of above 98%, respectively.

Ginsenoside Rg5H1(Z)

¹H NMR(δ,500M):5.38(1H,d,J＝7.5Hz),5.10(1H,t,J＝6.6Hz),4.91(1H,d,J＝7.5Hz),4.55(1H,m),4.45-4.49(2H,m),4.23-4.33(5H,m),4.12-4.14(2H,m),3.90-3.92(3H,m),3.27(1H,m)，2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.64(3H,s),1.61(3H,s),1.41(3H,s),1.35-1.50(8H,m),1.28(3H,s),1.20(1H,m),1.09(3H,d,J＝2.8Hz),1.02(1H,m),0.97(3H,s),0.96(3H,d,J＝2.8Hz),0.79(3H,s),0.74(1H,m).

¹³C NMR(δ,125M):138.79,125.86,106.3,105.3,89.2,83.6,78.6,78.4,78.2,78.0,71.9,71.2,63.1,63.0,77.4,56.6,55.0,51.9,50.6,49.4,48.8,40.2,39.9,39.4,37.1,35.4,32.3,31.6,28.4,27.3,26.9,26.9,25.9,23.2,18.7,17.9,17.2,16.8,16.6,16.3.

ESI:770.13(M+H)⁺,HR-ESI-MS:770.0321(C₄₂H₇₃O₁₂),Cal 770.0315.

Ginsenoside Rg5H1(E)

¹H NMR(δ,500M):5.37(1H,d,J＝7.5Hz),5.11(1H,t,J＝7.0Hz),4.91(1H,d,J＝7.5Hz),4.55(1H,m),4.45-4.49(2H,m),4.23-4.33(5H,m),4.12-4.14(2H,m),3.90-3.92(3H,m),3.27(1H,m)，2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.64(3H,s),1.61(3H,s),1.41(3H,s),1.35-1.50(8H,m),1.28(3H,s),1.20(1H,m),1.09(3H,d,J＝2.8Hz),1.02(1H,m),0.97(3H,s),0.96(3H,d,J＝2.8Hz),0.79(3H,s),0.74(1H,m).

¹³C NMR(δ,125M):138.81,126.55,106.3,105.3,89.2,83.6,78.6,78.4,78.2,78.0,71.9,71.2,63.1,63.0,77.4,56.6,55.0,51.9,50.6,49.4,48.8,40.2,39.9,39.4,37.1,35.4,32.3,31.6,28.4,27.3,26.9,26.9,25.9,23.2,18.7,17.9,17.2,16.8,16.6,16.3.

ESI:770.13(M+H)⁺,HR-ESI-MS:770.0321(C₄₂H₇₃O₁₂),Cal 770.0315.

Ginsenoside Rk1H

¹H NMR(δ,500M):5.37(1H,d,J＝7.5Hz),5.04(1H,br.s),4.91(1H,d,J＝7.5Hz),4.80(1H,m),4.55(1H,m),4.45-4.49(2H,m),4.23-4.33(5H,m),4.12-4.14(2H,m),3.90-3.92(3H,m),3.27(1H,m),2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.64(3H,s),1.61(3H,s),1.35-1.50(8H,m),1.28(3H,s),1.20(1H,m),1.09(3H,d,J＝2.8Hz),1.02(1H,m),0.97(3H,s),0.96(3H,d,J＝2.8Hz),0.79(3H,s),0.74(1H,m).

¹³C NMR(δ,125M):155.6,108.2,106.3,105.3,89.2,83.6,78.6,78.4,78.2,78.0,71.9,71.2,63.1,63.0,77.4,56.6,55.0,51.9,50.6,49.4,48.8,40.2,39.9,39.4,37.1,35.4,32.3,31.6,28.4,27.3,26.9,26.9,25.9,23.2,18.7,17.9,17.2,16.8,16.6,16.3.

ESI:770.13(M+H)⁺,HR-ESI-MS:770.0321(C₄₂H₇₃O₁₂),Cal 770.0315.

Preparation example 3 preparation of ginsenoside Rh3H

Ginsenoside Rh3H was obtained by the same method as in preparation example 1, using 20(R) -Rh2 and 20(S) -Rh2 as the starting materials, respectively.

The ginsenoside Rh3H is added into the raw materials of the ginseng,

¹H NMR(δ,500M):5.11(1H,t,J＝7.0Hz),4.95(1H,d,J＝8.0Hz),4.40(1H,d,J＝11.5Hz),4.40(1H,m),4.24(1H,m),4.21(1H,m),4.05(1H,m),4.02(1H,m),3.93(1H,m),3.38(1H,dd,J＝11.5,4.5Hz),2.46-2.53(2H,m),2.40(1H,dd,J＝21.5,10.5Hz),2.21(1H,m),1.95(1H,m),2.03(2H,m),1.82(1H,m),1.72(2H,m),1.71(3H,s),1.69(3H,s),1.65(3H,s),1.59(1H,m),1.52(2H,m),1.49(2H,m),1.43(2H,m),1.36(1H,m),1.32(3H,s),1.24(1H,m),1.06(1H,m),1.01(3H,s),1.00(H,s),0.99(3H,s),0.81(3H,s),0.76(1,m),0.74(1H,d,J＝10.5Hz).

¹³C NMR(δ,125M):131.7,127.0,107.9,89.7,79.7,79.3,76.8,72.8,71.8,64.0,57.3,52.7,51.6,51.3,50.1,44.2,41.0,40.6,40.1,37.9,36.1,33.1,32.4,29.1,27.7,27.6,26.8,23.7,23.6,19.4,18.7,18.3,17.7,17.4,16.8,

ESI:607.83(M+H)⁺,HR-ESI-MS:607.8921(C₃₆H₆₇O₇),[M+H]⁺,Cal 607.8915.

preparation example 4 preparation of ginsenoside Rh3H1(E), ginsenoside Rh3H1(Z) and ginsenoside Rk2H

Ginsenoside Rh3H1(E), ginsenoside Rh3H1(Z) and ginsenoside Rk2H with HPLC purity of more than 98% are respectively obtained by the same method as in preparation example 2 by using 20(R) -Rh2 acetylated product or 20(S) -Rh2 acetylated product as raw material.

Ginsenoside Rh3H1(E),

¹H NMR(δ,500M):5.04(1H,br.s),4.95(1H,d,J＝8.0Hz),4.80(1H,m),4.40(1H,d,J＝11.5Hz),4.40(1H,m),4.24(1H,m),4.21(1H,m),4.05(1H,m),4.02(1H,m),3.93(1H,m),3.38(1H,dd,J＝11.5,4.5Hz),2.46-2.53(2H,m),2.40(1H,dd,J＝21.5,10.5Hz),2.21(1H,m),1.95(1H,m),2.03(2H,m),1.82(1H,m),1.72(2H,m),1.69(3H,s),1.65(3H,s),1.59(1H,m),1.52(2H,m),1.49(2H,m),1.43(2H,m),1.39(3H,m),1.36(1H,m),1.32(3H,s),1.24(1H,m),1.06(1H,m),1.01(3H,d,J＝2.8Hz),1.00(H,s),0.99(3H,d,J＝2.8Hz),0.81(3H,s),0.76(1,m),0.74(1H,d,J＝10.5Hz).

¹³C NMR(δ,125M):138.7,125.9,107.9,89.7,79.7,79.3,76.8,72.8,71.8,64.0,57.3,52.7,51.6,51.3,50.1,44.2,41.0,40.6,40.1,37.9,36.1,33.1,32.4,29.1,27.7,27.6,26.8,23.7,23.6,19.4,18.7,18.3,17.7,17.4,16.8,

ginsenoside Rh3H1(Z),

¹H NMR(δ,500M):5.32(1H,t,J＝7.0Hz),4.95(1H,d,J＝8.0Hz),4.40(1H,d,J＝11.5Hz),4.40(1H,m),4.24(1H,m),4.21(1H,m),4.05(1H,m),4.02(1H,m),3.93(1H,m),3.38(1H,dd,J＝11.5,4.5Hz),2.46-2.53(2H,m),2.40(1H,dd,J＝21.5,10.5Hz),2.21(1H,m),1.95(1H,m),2.03(2H,m),1.82(1H,m),1.72(2H,m),1.70(3H,s),1.69(3H,s),1.59(1H,m),1.52(2H,m),1.49(2H,m),1.43(2H,m),1.39(3H,m),1.36(1H,m),1.32(3H,s),1.24(1H,m),1.06(1H,m),1.01(3H,d,J＝2.8Hz),1.00(H,s),0.99(3H,d,J＝2.8Hz),0.81(3H,s),0.76(1,m),0.74(1H,d,J＝10.5Hz).

ginsenoside Rk2H

¹H NMR(δ,500M):4.95(1H,d,J＝8.0Hz),4.40(1H,d,J＝11.5Hz),4.40(1H,m),4.24(1H,m),4.21(1H,m),4.05(1H,m),4.02(1H,m),3.93(1H,m),3.38(1H,dd,J＝11.5,4.5Hz),2.46-2.53(2H,m),2.40(1H,dd,J＝21.5,10.5Hz),2.21(1H,m),1.95(1H,m),2.03(2H,m),1.82(1H,m),1.72(2H,m),1.70(3H,s),1.69(3H,s),1.59(1H,m),1.52(2H,m),1.49(2H,m),1.43(2H,m),1.39(3H,m),1.36(1H,m),1.32(3H,s),1.24(1H,m),1.06(1H,m),1.01(3H,d,J＝2.8Hz),1.00(H,s),0.99(3H,d,J＝2.8Hz),0.81(3H,s),0.76(1,m),0.74(1H,d,J＝10.5Hz).

¹³C NMR(δ,125M):155.6,108.2,107.9,89.7,79.7,79.3,76.8,72.8,71.8,64.0,57.3,52.7,51.6,51.3,50.1,44.2,41.0,40.6,40.1,37.9,36.1,33.1,32.4,29.1,27.7,27.6,26.8,23.7,23.6,19.4,18.7,18.3,17.7,17.4,16.8,

Preparation example 5 preparation of ginsenoside Rk4H

By the same method as preparation example 1, 20(R) -Rg2 and 20(S) -Rg2 were used as raw materials, respectively, to obtain ginsenoside Rk 4H.

The ginsenoside Rk4H is added into the raw materials,

¹H NMR(δ,500M):6.46(1H,brs),5.33(1H,t,J＝8.3),5.25(1H,d,J＝7.2),4.92(1H,m),4.77(1H,m),4.66(1H,m),4.52(1H,d,J＝9.8),4.69(1H,m),4.34(1H,m),4.37(1H,m),4.28(1H,m),4.20(1H,m),3.96(1H,m),3.90(1H,m),3.47(1H,dd,J＝4.1,11.6),2.59(1H,m),2.30(1H,m),2.26(1H,m),2.26(3H,m),2.10(3H,s),2.01(1H,m),1.98(2H,m),1.86(1H,m),1.79(3H,m),1.78(3H,d,J＝5.7),1.67(3H,s),1.63(3H,s),1.64(2H,m),1.53(3H,m),1.41(3H,d,J＝2.8Hz),1.39(1H,m),1.35(3H,s),1.29(2H,m),1.20(3H,s),0.98(3H,s),0.95(3H,s),0.96(3H,m).

¹³C NMR(δ,125M):130.8,126.4,102.0,101.9,79.5,78.7,78.4,75.9,78.9,71.3,67.3,74.4,73.1,71.1,63.0,60.9,54.7,51.8,49.7,39.7,48.3,46.1,41.2,40.1,39.4,32.1,35.9,32.2,31.4,27.1,26.7,27.8,25.9,23.0,17.7,17.0,17.2.

ESI:770.23(M+H)⁺,HR-ESI-MS:770.0341(C₃₆H₆₇O₇),[M+H]⁺,Cal 770.0328.

preparation example 6 preparation of ginsenoside Rk4H1(E), ginsenoside Rk4H1(Z) and ginsenoside Rg6H

By adopting the same method as preparation example 2, the raw materials are 20(R) -Rg2 acetylated product and 20(S) -Rg2 acetylated product, and ginsenoside Rk4H1(E), ginsenoside Rk4H1(Z) and ginsenoside Rg6H with HPLC purity of more than 98% are respectively obtained.

The ginsenoside Rk4H1E is contained in the Chinese medicinal composition,

¹H NMR(δ,500M):6.46(1H,brs),5.40(1H,t,J＝8.3),5.25(1H,d,J＝7.2),4.92(1H,m),4.77(1H,m),4.66(1H,m),4.52(1H,d,J＝9.8),4.69(1H,m),4.34(1H,m),4.37(1H,m),4.28(1H,m),4.20(1H,m),3.96(1H,m),3.90(1H,m),3.47(1H,dd,J＝4.1,11.6),2.59(1H,m),2.30(1H,m),2.26(1H,m),2.26(3H,m),2.10(3H,s),2.01(1H,m),1.98(2H,m),1.86(1H,m),1.79(3H,m),1.78(3H,d,J＝5.7),1.71(3H,s),1.67(3H,s),1.63(3H,s),1.64(2H,m),1.53(3H,d,J＝10.5Hz),1.41(3H,d,J＝2.8Hz),1.39(1H,m),1.29(2H,m),1.20(3H,s),0.98(3H,s),0.95(3H,s),0.83(3H,d,J＝10.5Hz).

¹³C NMR(δ,125M):140.2,123.6,102.0,101.9,79.5,78.7,78.4,75.9,78.9,71.3,67.3,74.4,73.1,71.1,63.0,60.9,54.7,51.8,49.7,39.7,48.3,46.1,41.2,40.1,39.4,32.1,35.9,32.2,31.4,27.1,26.7,27.8,25.9,23.0,17.7,17.0,17.2.

the ginsenoside Rk4H1Z is contained in the Chinese medicinal composition,

¹H NMR(δ,500M):6.46(1H,brs),5.42(1H,t,J＝8.3),5.25(1H,d,J＝7.2),4.92(1H,m),4.77(1H,m),4.66(1H,m),4.52(1H,d,J＝9.8),4.69(1H,m),4.34(1H,m),4.37(1H,m),4.28(1H,m),4.20(1H,m),3.96(1H,m),3.90(1H,m),3.47(1H,dd,J＝4.1,11.6),2.59(1H,m),2.30(1H,m),2.26(1H,m),2.26(3H,m),2.10(3H,s),2.01(1H,m),1.98(2H,m),1.86(1H,m),1.79(3H,m),1.78(3H,d,J＝5.7),1.67(3H,s),1.65(3H,s),1.64(2H,m),1.53(3H,d,J＝10.5Hz),1.41(3H,d,J＝2.8Hz),1.39(1H,m),1.29(2H,m),1.20(3H,s),0.98(3H,s),0.95(3H,s),0.83(3H,d,J＝10.5Hz).

¹³C NMR(δ,125M):140.2,120.4,102.0,101.9,79.5,78.7,78.4,75.9,78.9,71.3,67.3,74.4,73.1,71.1,63.0,60.9,54.7,51.8,49.7,39.7,48.3,46.1,41.2,40.1,39.4,32.1,35.9,32.2,31.4,27.1,26.7,27.8,25.9,23.0,17.7,17.0,17.2.

ginsenoside Rg6H

¹H NMR(δ,500M):6.46(1H,brs),5.25(1H,d,J＝7.2),5.04(1H,br.s),4.92(1H,m),4.80(1H,m),4.77(1H,m),4.66(1H,m),4.52(1H,d,J＝9.8),4.69(1H,m),4.34(1H,m),4.37(1H,m),4.28(1H,m),4.20(1H,m),3.96(1H,m),3.90(1H,m),3.47(1H,dd,J＝4.1,11.6),2.5(1H,m),2.30(1H,m),2.26(1H,m),2.26(3H,m),2.10(3H,s),2.01(1H,m),1.98(2H,m),1.86(1H,m),1.79(3H,m),1.78(3H,d,J＝5.7),1.63(3H,s),1.64(2H,m),1.53(3H,d,J＝10.5Hz),1.41(3H,d,J＝2.8Hz),1.39(1H,m),1.35(3H,s),1.29(2H,m),1.20(3H,s),0.98(3H,s),0.95(3H,s),0.83(3H,d,J＝10.5Hz).

¹³C NMR(δ,125M):155.6,108.2,102.0,101.9,79.5,78.7,78.4,75.9,78.9,71.3,67.3,74.4,73.1,71.1,63.0,60.9,54.7,51.8,49.7,39.7,48.3,46.1,41.2,40.1,39.4,32.1,35.9,32.2,31.4,27.1,26.7,27.8,25.9,23.0,17.7,17.0,17.2.

Preparation example 7 preparation of Isoginsenoside Rg3H

Dissolving 1.5g of acetylated product of ginsenoside Rg3 in 150mL of ethanol, adding 300mg of 5% palladium carbon, stirring, introducing hydrogen at 40 ℃, and reacting for 6 hours. After the reaction is finished, filtering to remove palladium carbon, extracting the filtrate for 3 times by using ethyl acetate, combining organic phases, and concentrating under reduced pressure until the organic phases are dried to obtain a crude product of the isoginsenoside Rg 3H. Separating by high pressure chromatography, eluting with C18 as filler, detecting with methanol water gradient, detecting with Evaporative Light Scattering Device (ELSD), and concentrating product segment to dry to obtain 0.36g +0.55g isoginsenoside Rg3H with HPLC purity of above 98%.

The isoginsenoside Rg3H can be extracted from radix Ginseng,

¹H NMR(δ,500M):5.37(1H,d,J＝7.5Hz),4.91(1H,d,J＝7.5Hz),4.55(1H,m),4.45-4.49(2H,m),4.23-4.33(5H,m),4.12-4.14(2H,m),3.90-3.92(3H,m),3.27(1H,m),2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(3H,m),1.78(3H,d,J＝5.7),1.67-1.71(3H,m),1.64(3H,s),1.61(3H,s),1.41(6H,s),1.35-1.50(10H,m),1.28(3H,s),1.20(1H,m),1.09(3H,m),1.02(1H,m),0.97(3H,s),0.96(3H,m),0.79(3H,s),0.74(1H,m).

¹³C NMR(δ,125M):106.3,105.3,89.2,83.6,82.3,78.6,78.4,78.2,78.0,71.9,71.2,63.1,63.0,77.4,56.6,55.0,51.9,50.6,49.4,48.8,40.2,39.9,39.4,37.1,35.4,32.3,31.6,30.0,29.8,27.3,26.9,26.9,25.9,23.2,18.7,17.9,17.2,16.8.

ESI:788.05(M+H)⁺,HR-ESI-MS:788.0721(M+H)⁺,(C₄₂H₇₃O₁₃),Cal 788.0728.

preparation example 83 preparation of sodium sulfate-ginsenoside Rg5H (SC-Rg5H)

Dissolving 10g of ginsenoside Rg5H in 50mL of ethanol, adding 1g of NaOH, heating to 80 ℃, refluxing, introducing air, reacting for 5 days, and detecting by TLC until the raw material point disappears. After the reaction is finished, extracting for 3 times by 100 mL/n-butanol, combining organic phases, concentrating under reduced pressure to dryness, crystallizing for 3 times by using ethanol, and drying to obtain 2.6g of ginsenoside Rg5H mother nucleus with the HPLC purity of more than 98%.

Weighing 10g of Rg5H mother nucleus, dissolving in 100mL of pyridine, placing the reaction bottle in a saline-ice bath, cooling to 0 ℃, slowly adding 30mL of chlorosulfonic acid dropwise, reacting at room temperature for 2 hours, and detecting by TLC until the raw material point disappears. After the reaction was completed, 0.1M NaOH was added to adjust pH to 7.0, n-butanol was extracted 3 times, and organic phases were combined and concentrated to dryness under reduced pressure. Separating by high pressure chromatography, eluting with C18 as filler and methanol water gradient, evaporating light diffuser (ELSD), and concentrating product segment to dryness to obtain 11.4g Rg5H mother nucleus with HPLC purity of above 98%.

¹H NMR(δ,500M):5.30(1H,t,J＝6.0Hz),3.96(1H,m),3.80(1H,m),2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.64(3H,s),1.61(3H,s),1.41(3H,d,J＝2.8),1.35-1.50(8H,m),1.28(3H,s),1.20(1H,m),1.09(3H,s),1.03(1H,m),0.94(3H,s),0.95(3H,s),0.78(3H,s),0.73(1H,m).

¹³C NMR(δ,125M):139.7,125.7,78.2,78.3,72.8,69.7,56.6,51.7,51.1,51.0,44.4,40.4,39.7,37.6,35.6,32.8,32.4,30.2,29.0,28.9,28.4,23.8,18.9,17.2,16.7,16.5,16.0,13.2.

LRMS(ESI):523.7916[C₃₀H₅₁O₆S])；HRMS(ESI):found 523.7923,[C₃₀H₅₁O₆S]).

Preparation example 93 preparation of sodium sulfate-Isoprotopanaxadiol PPD (E) (SC-IsoPPD (E)))

Weighing 10g of iso-protopanaxadiol PPD (E type), dissolving in 100mL of pyridine, placing the reaction bottle in a saline-ice bath, cooling to 0 ℃, slowly adding 30mL of chlorosulfonic acid dropwise, removing the ice bath, reacting at room temperature for 2 hours, and detecting by TLC until the raw material point disappears. After the reaction was completed, 0.1M NaOH was added to adjust pH to 7.0, n-butanol was extracted 3 times, and organic phases were combined and concentrated to dryness under reduced pressure. Separating by high pressure chromatography, eluting with C18 as filler and methanol water gradient, Evaporating Light Scatterer (ELSD), and concentrating product segment to dryness to obtain 11.4g 3-sodium sulfate-isopinoglycol PPD (E type) with HPLC purity of above 98%.

¹H NMR(δ,500M):5.40(1H,t,J＝6.0Hz),3.96(1H,m),3.80(1H,m),2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.71(3H,s),1.57(3H,s),1.51(3H,s),1.35-1.50(8H,m),1.20(1H,m),1.09(3H,s),1.03(1H,m),0.94(3H,s),0.95(3H,s),0.78(3H,s),0.73(1H,m).

¹³C NMR(δ,125M):123.8,123.6,78.2,72.8,70.7,56.6,51.7,51.1,51.0,44.4,40.4,39.7,37.6,35.6,32.8,32.4,30.2,29.0,28.9,28.4,23.8,18.9,17.2,16.7,16.5,16.0,13.2.

ESI-MS:539.79[M-Na]^-.

Preparation example 103 preparation of (N, N-dimethylaminoethyl) -carbamoyl-ginsenoside Rg5H (DC-Rg5H)

By the same method as that in embodiment 23, the raw material is Rg5H mother nucleus, and the DC-ginsenoside Rg5H is obtained.

Weighing 10g of Rg5H mother nucleus, dissolving in 200mL of dry dichloromethane, adding 10g of DMAP, cooling to 0 ℃ in an ice bath, dropwise adding 50mL of triphosgene (10g) dissolved in dichloromethane, controlling the reaction temperature to be 0-5 ℃, reacting for 2 hours, and detecting by TLC until the raw material point disappears. Adding purified water to terminate the reaction, extracting with ethyl acetate for 3 times, mixing organic phases, concentrating under reduced pressure to dryness, crystallizing with ethanol for 2 times, and oven drying to obtain 6.5g of 3-chloroformyl-ginsenoside Rg 5H.

Weighing 5g of 3-chloroformyl-ginsenoside Rg5H, dissolving in 50mL of dichloromethane, cooling to 0 ℃ in an ice bath, slowly dropwise adding 15mL of chloroform solution containing 5mL of N, N-dimethylethylenediamine, controlling the reaction temperature at 0-5 ℃, reacting for 4 hours, and detecting by TLC until the raw material point disappears. Adding purified water to terminate the reaction, extracting with chloroform for 3 times, mixing organic phases, concentrating under reduced pressure to dryness, crystallizing with ethanol for 3 times, and oven drying to obtain 3.6g of 3- (N, N-dimethylaminoethyl) -carbamoyl-ginsenoside Rg 5H.

¹H NMR(δ,500M):5.20(1H,t,J＝6.9Hz),3.80(1H,m),3.75(4H,m),3.42(6H,s),2.70(1H,m),2.20-2.28(4H,m),2.08(1H,m),1.98(2H,m),1.95(1H,m),1.72(1H,m),1.60(1H,m),1.57(3H,s),1.51(3H,s),1.45(3H,m),1.38(1H,m),1.32(3H,m),1.19(1H,m),0.98(1H,m),0.92(3H,s),0.92(3H,d,J＝6.5Hz),0.72(3H,s),0.68(1H,m),0.63(1H,m).

¹³C NMR(δ,125M):156.1,131.2,125.3,89.0,72.5,60.3,56.5,52.5,51.2,50.8,48.2,46.1,40.2,39.8,39.3,37.1,35.4,33.9,32.6,30.7,28.1,27.1,26.7,25.7,18.5,17.7,17.0,16.6,16.4,15.8.

ESI:559.90(M+H)⁺,HR-ESI-MS:559.9011(C₃₅H₆₃N2O₃),Cal 559.9020.

Preparation example 11 preparation of pseudo-ginsenoside GP

Dissolving 10g of ginsenoside GQ acetylated product in 200mL of toluene, adding 1.5g of boron trifluoride diethyl etherate, heating to 90 ℃, refluxing for 4 hours, detecting by TLC until a raw material point disappears, cooling, washing for 3 times by using 100 mL/time saturated sodium bicarbonate solution, drying by anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain a GP acetylated product.

Dissolving 10g of GP acetylated product in 50mL of methanol, adding 2g of sodium methoxide, stirring and dissolving, reacting for 10 hours at room temperature, detecting by TLC until a raw material point disappears, extracting for 3 times by using 100 mL/ethyl acetate, combining organic phases, drying by using an appropriate amount of anhydrous sodium sulfate, concentrating to be dry under reduced pressure, crystallizing for 2 times by using an appropriate amount of methanol, and drying to obtain 2.2g of pseudo ginsenoside GP with the HPLC purity of more than 98%.

¹H NMR(δ,500M):5.25(1H,d,J＝7.8Hz),4.81(1H,d,J＝7.8Hz),4.45(1H,d,J＝10.2Hz),4.36(2H,m),4.21–4.24(3H,m),4.09–4.14(2H,dd,J＝19.2,9.6Hz),3.99–4.05(2H,m),3.79–3.83(3H,m),3.59(1H,td,J＝10.2,4.8Hz),3.16(1H,dd,J＝12.0,4.2Hz),1.33(3H,d,J＝6.5Hz),1.15(s,6H),1.13(3H,d,J＝6.5Hz)1.08(s,3H),0.96(s,3H),0.84(s,3H),0.78(s,3H),0.64(s,3H).

¹³C NMR(C₅D₅N,150MHz)d:106.2,105.2,88.9,86.8,85.7,83.6,78.4,78.3,78.2,78.1,77.3,71.8,71.7,71.2,70.4,62.9,62.8,56.5,52.2,50.8,49.8,48.5,40.0,39.8,39.3,37.0,35.2,32.9,32.5,31.7,28.9,28.1,27.7,27.3,27.0,26.8,25.5,18.5,18.4,16.6,16.6,15.6.

ESI-MS:m/z 786.03[M+1]⁺.

Preparation of mixed micelle

EXAMPLE 1 preparation of PEG-DSPE-containing ginsenoside Rk1 blank Mixed micelle

Dissolving 100mg of PEG-DSPE (number average molecular weight of 2000) and 200mg of ginsenoside Rk1 in 20ml of ethanol, concentrating under reduced pressure at 37 ℃ to form a rotary film, then continuing to evaporate to dryness, adding 20ml of purified water, stirring and hydrating at 37 ℃, and filtering through a 0.22 mu m filter membrane after dissolution to obtain a blank nano mixed micelle solution. After detection, the average particle size is 24.5 nm.

EXAMPLE 2 preparation of protopanaxatriol PPT blank Mixed micelles containing mPEG-PDLLA

200mg of mPEG-PDLLA (the number average molecular weight is 2000) and 100mg of protopanaxatriol PPT are dissolved in 20ml of methanol, and the mixture is concentrated into a membrane at 50 ℃ under reduced pressure, then is continuously evaporated to dryness, 20ml of purified water is added, the mixture is stirred and hydrated at 50 ℃, and is filtered through a 0.22 mu m filter membrane after dissolution, so that a blank mixed micelle solution is obtained. After detection, the average particle size is 40.9 nm.

Example 3 preparation of Damirin A blank Mixed micelles containing mPEG-DSPE

200mg of mPEG-DSPE (the number average molecular weight is 2000), 400mg of damulin A and 20mg of VE are taken to be dissolved in 20ml of ethanol, decompressed, concentrated and rotated to form a film at 60 ℃, then the film is continuously evaporated to dryness, 20ml of 5 percent glucose aqueous solution is added, the mixture is stirred and hydrated at 60 ℃, and after the dissolution, the mixture is filtered through a 0.22 mu m filter membrane to obtain a blank nano mixed micelle solution. After detection, the average particle size is 24.5 nm.

EXAMPLE 4 preparation of protopanaxadiol PPD blank mixed micelle containing PEG-DSPE

Dissolving 100mg of PEG-DSPE (number average molecular weight is 4000) and 200mg of protopanaxadiol PPD and 20mg of VC in 20ml of acetonitrile, concentrating under reduced pressure at 50 ℃ to form a film by rotation, then continuously evaporating to dryness, adding 20ml of 5% glucose aqueous solution, stirring and hydrating at 50 ℃, and filtering through a 0.22 mu m filter membrane after dissolution to obtain a blank nano mixed micelle solution. The average particle size was 28.6nm after detection.

Example 5 preparation of PEG-DSPE containing paclitaxel ginsenoside Rg5 Mixed micelle

Taking 200mg of PEG-DSPE (the number average molecular weight is 2000), 400mg of ginsenoside Rg5, 100mg of paclitaxel and 100mg of VE, dissolving in 200ml of methanol: chloroform-1: 1 (volume ratio) at 60 ℃, rotary concentrating to form a film, then continuing to evaporate to dryness, adding 20ml of purified water, and stirring and hydrating at 60 ℃ to dissolve to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 16.7nm, and the encapsulation rate is not less than 95%.

Example 6 preparation of docetaxel ginsenoside Rg5H Mixed micelle containing mPEG-PDLLA

Dissolving 200mg of mPEG-PDLLA (with the number average molecular weight of 4000), 400mg of ginsenoside Rg5H and 50mg of docetaxel in 20ml of chloroform, performing rotary concentration at 45 ℃ under reduced pressure to form a film, continuing to evaporate to dryness, adding 20ml of purified water, and performing stirring hydration at 45 ℃ to dissolve the purified water to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 24.5nm, and the encapsulation rate is not less than 95%.

Example 7: preparation of camptothecin ginsenoside Rh1 mixed micelle containing PEO-PASp

Dissolving 400mg of PEO-PASp (with number average molecular weight of 4800) and 100mg of ginsenoside Rh1 and 25mg of camptothecin in 20ml of dichloromethane, performing rotary concentration at 40 ℃ under reduced pressure to form a membrane, then continuing to evaporate to dryness, adding 20ml of 5% glucose aqueous solution, and stirring and hydrating at 40 ℃ to dissolve the solution to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 33.4nm, and the encapsulation rate is not less than 95%.

Example 8: preparation of PEG-DSPE-containing propofol 25-methyl-isoginsenoside Rg3 (isoRg 3Me) mixed micelle

100mg of PEG-DSPE (the number average molecular weight is 2000), 400mg of iso-Rg 3Me and 25mg of propofol are taken to be dissolved in 20ml of ethanol, and are subjected to rotary concentration at 60 ℃ under reduced pressure to form a membrane, and then the membrane is continuously evaporated to dryness, 20ml of purified water is added, and the mixture is stirred and hydrated at 40 ℃ to be dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 35.5nm, and the encapsulation rate is not less than 95%.

Example 9 preparation of homoharringtonine ginsenoside Rg4 Mixed micelles containing mPEG-PDLLA and Chitosan-cholic acid

Dissolving 500mg of mPEG-PDLLA (with the number average molecular weight of 4000), 100mg of chitosan-cholic acid, 300mg of ginsenoside Rg4 and 50mg of homoharringtonine in 20ml of diethyl ether, performing rotary concentration at 30 ℃ under reduced pressure to form a film, then continuously evaporating to dryness, adding 20ml of purified water, and stirring and hydrating at 30 ℃ to obtain a clear micelle solution after dissolution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 66nm, and the encapsulation rate is not less than 90%.

EXAMPLE 10 preparation of doxorubicin ginsenoside Rh4 mixed micelles containing mPEG-PLA

100mg of mPEG-PLA (the number average molecular weight is 2400), 400mg of ginsenoside Rh4 and 50mg of adriamycin are dissolved in 20ml of methanol, are subjected to rotary concentration at 50 ℃ under reduced pressure to form a membrane, are continuously evaporated to dryness, are added with 20ml of purified water, are stirred and hydrated at 40 ℃, and are dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 27.3nm, and the encapsulation rate is not less than 95%.

Example 11 preparation of oxaliplatin ginsenoside Rh2 mixed micelles containing PEG-PLGA

100mg of PEG-PLGA (the number average molecular weight is 2000), 200mg of ginsenoside Rh2 and 50mg of oxaliplatin are dissolved in 20ml of THF, and are subjected to rotary concentration at 55 ℃ under reduced pressure to form a film, then the film is continuously evaporated to dryness, 20ml of 5% glucose aqueous solution is added, and the mixture is stirred and hydrated at 55 ℃ to be dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 18.8nm, and the encapsulation rate is not less than 95%.

EXAMPLE 12 preparation of PEG-DSPE containing clonazepam ginsenoside Rp1 Mixed micelles

Dissolving 100mg of PEG-DSPE (number average molecular weight is 4000), 400mg of ginsenoside Rp1, 10mg of vitamin E, 10mg of cholesterol and 50mg of clonazepam in 20ml of tetrahydrofuran, concentrating under reduced pressure at 60 ℃ to form a rotary film, continuously evaporating to dryness, adding 20ml of saturated phosphate buffer solution, hydrating at 40 ℃, and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 26.9nm, and the encapsulation rate is not less than 90%.

Example 13 preparation of Indometacin ginsenoside Rg3 Mixed micelle containing mPEG-PDLLA

100mg of mPEG-PDLLA (the number average molecular weight is 4000), 400mg of ginsenoside Rg3 and 30mg of indometacin are dissolved in 20ml of chloroform, the mixture is subjected to vacuum concentration at 45 ℃ to form a rotary film, then the rotary film is continuously evaporated to dryness, 20ml of 5% glucose aqueous solution is added, and the rotary film is hydrated at 30 ℃ to be dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 23.9nm, and the encapsulation rate is not less than 95%.

EXAMPLE 14 preparation of PEG-PHIS-CONTAINING NAPPESSEN GINSOSIDE Rh3 MIXED PELLET

100mg of PEG-Phis (number average molecular weight is 4000) and 200mg of ginsenoside Rh3 and 30mg of naproxen are taken and dissolved in 20ml of methanol, and the mixture is subjected to reduced pressure concentration at 50 ℃ to form a rotary film, and then is continuously evaporated to dryness, and 20ml of 5% glucose aqueous solution is added, and the mixture is hydrated at 30 ℃ and dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 22.5nm, and the encapsulation rate is not less than 90%.

Example 15 preparation of PEG-PCL-containing haloperidol Isoginsenoside Rh2(E) Mixed micelle

400mg of PEG-PCL (the number average molecular weight is 2000), 100mg of isoginsenoside Rh2(E) and 30mg of haloperidol are dissolved in 20ml of chloroform, vacuum concentration and rotary film formation are carried out at 45 ℃, then evaporation to dryness is carried out continuously, 20ml of 5% glucose aqueous solution is added, hydration is carried out at 30 ℃, and the clear micelle solution is obtained by dissolution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 20.7nm, and the encapsulation rate is not less than 95%.

EXAMPLE 16 preparation of PEG-PBLA-containing Mixed micelles of Dihydrotestosterone Isoginsenoside Rg3(Z)

Taking 400mg of PEG-PBLA (the number average molecular weight is 2000), 100mg of isoginsenoside Rg3(Z) and 30mg of dihydrotestosterone, dissolving in 20ml of chloroform, concentrating under reduced pressure at 40 ℃ for rotary film formation, continuing to evaporate to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 30 ℃, and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 46.5nm, and the encapsulation rate is not less than 90%.

Example 17 preparation of PEG-PASP-containing vitamin K3 pseudo-ginsenoside GQ Mixed micelles

Dissolving 400mg of PEG-PASp (with number average molecular weight of 4800) and 100mg of pseudoginsenoside GQ and 30mg of vitamin K3 in 20ml of chloroform, concentrating under reduced pressure at 45 deg.C to form a rotary film, evaporating to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 45 deg.C, and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 32.9nm, and the encapsulation rate is not less than 90%.

EXAMPLE 18 preparation of PEG-PBLG-containing Biphenyl ginsenoside Rp2 Mixed micelle

Taking 400mg of PEG-PBLG (with the number average molecular weight of 4000), 100mg of ginsenoside Rp2 and 30mg of bifendate, dissolving in 20ml of chloroform, concentrating under reduced pressure at 40 ℃ to form a rotary film, continuing to evaporate to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 40 ℃, and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 18.3nm, and the encapsulation rate is not less than 95%.

EXAMPLE 19 preparation of Puerarin pseudoginsenoside HQ Mixed micelle containing PEG-DSPE-NH2

Dissolving 400mg of PEG-DSPE-NH2 (with number average molecular weight of 4000), 100mg of pseudoginsenoside HQ and 30mg of puerarin in 20ml of chloroform, concentrating under reduced pressure at 45 ℃ to form a film by rotation, then continuously evaporating to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 45 ℃, and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 23.6nm, and the encapsulation rate is not less than 90%.

EXAMPLE 20 PEG-PCL-containing Cyclosporin A ginsenoside Rp3 Mixed micelles preparation

Dissolving 400mg of PEG-PCL (number average molecular weight of 2000), 100mg of ginsenoside Rp3 and 30mg of cyclosporine A in 20ml of ethanol, concentrating under reduced pressure at 60 ℃ to form a rotary film, continuously evaporating to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 30 ℃ and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 27nm, and the encapsulation rate is not less than 90%.

Example 21 preparation of PEG-PCL-containing fenofibrate-specific ginsenoside Rh2(Z) Mixed micelles

Taking 400mg of PEG-PCL (the number average molecular weight is 2000), 100mg of isoginsenoside Rh2(Z) and 30mg of fenofibrate, dissolving in 20ml of chloroform, concentrating under reduced pressure at 45 ℃ to form a film by rotation, continuing to evaporate to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 45 ℃, and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 26.2nm, and the encapsulation rate is not less than 90%.

EXAMPLE 22 preparation of PEG-PCL-containing amphotericin B ginsenoside SC-Rp1 Mixed micelles

Dissolving 400mg of PEG-PCL (number average molecular weight of 2000), 200mg of ginsenoside SC-Rp1, 20mg of sodium deoxycholate and 30mg of amphotericin B in 100ml of purified water, concentrating under reduced pressure at 60 ℃ to form a rotary film, continuously evaporating to dryness, adding 20ml of 5% glucose aqueous solution, hydrating at 50 ℃ and dissolving to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 29.6nm, and the encapsulation rate is not less than 90%.

Example 23 preparation of PEG-PCL containing siRNA ginsenoside DC-Rp1 Mixed micelle

Dissolving 400mg of PEG-PCL (number average molecular weight of 2000), 100mg of ginsenoside DC-Rp1 and 30mg of SiRNA in 50ml of physiological saline, stirring at room temperature for 12 hours, filtering with 0.22 μm filter membrane, and freeze-drying to obtain the final product. After redissolution detection, the average particle size is 28.2nm, and the encapsulation rate is not less than 90%.

Example 24: preparation of PEO-PPO-PEO-containing adriamycin ginsenoside Rg2(E) mixed micelle

Dissolving 300mg of poloxamer 188(PEO-PPO-PEO) (the number average molecular weight is 4800) and 100mg of adriamycin in DMSO, adding 10ml of lutidine dissolved with 20mg of DMAP at 30-50 ℃, stirring for 3-4 hours to prepare a conjugate of the micelle and the adriamycin, adding 50ml of PBS buffer solution dissolved with 2mg/ml of isoginsenoside Rg2(E) at pH 7.4, reducing pressure, concentrating to remove the organic solvent, filtering with a 0.22 mu m filter membrane, and freeze-drying to obtain the final product. After redissolution detection, the average particle size is 21.1nm, and the encapsulation rate is not less than 90%. The micelle was found to be stable at pH 7.4, but capable of releasing doxorubicin at pH 6.6-7.2.

Example 25 preparation of all-trans retinoic acid isoginsenoside Rg2(Z) Mixed micelle containing PEG-PBLG

Dissolving 200mg of PEG-PBLG (number average molecular weight of 4000) and 300mg of ginsenoside Rg2(Z) in 20ml of Dimethylformamide (DMF), and stirring at 30 deg.C to dissolve. Then, 50mg of all-trans retinoic acid was added thereto, and the mixture was stirred at room temperature for 12 hours. Then placing the mixed solution into a dialysis bag, dialyzing with purified water for 8 hours, filtering the dialysate through a 0.22 mu m filter membrane, and freeze-drying to obtain the compound. After redissolution detection, the average particle size is 30.7nm, and the encapsulation rate is not less than 90%.

Example 26 preparation of PEG-PASp-containing cisplatin Isoginsenoside Rg3H Mixed micelle

200mg of PEG-PASp (number average molecular weight of 4800) and 30mg of cisplatin were dissolved in 50ml of purified water and stirred at room temperature for 8 hours, so that the cisplatin and the PEG-PASp formed complex micelles. Adding 50mg of isoginsenoside Rg3H, and stirring at 60 deg.C for dissolving. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 42.2nm, and the encapsulation rate is not less than 90%.

Example 27: preparation of PEG-PBLA-containing irinotecan hydrochloride and ginsenoside Rg3E mixed micelle

Dissolving 200mg of PEG-PBLA (number average molecular weight of 2000) and 400mg of ginsenoside Rg3 in 50ml of 50% ethanol water solution to obtain blank micelle solution. 50mg of irinotecan hydrochloride is dissolved in 10ml of chloroform, the mixture is dripped into 50ml of blank micellar solution under the condition of intense stirring at room temperature to form an oil-water mixed solution (or the mixed solution is sheared at high speed for 10 minutes, or is homogenized for 2 to 4 times under high pressure, or is sheared and homogenized together), and the stirring is continued for 2 hours. Then at 60 deg.C, rotary evaporating to remove organic solvent, filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 28.3nm, and the encapsulation rate is not less than 90%.

Example 28: preparation of PEG-PHIS-containing epothilone C ginsenoside Rg2 mixed micelle

Dissolving 400mg of PEG-Phis (number average molecular weight: 4000) and 200mg of ginsenoside Rg2 in 20ml of Dimethylformamide (DMF), and stirring at room temperature to dissolve. An additional 50mg of epothilone C was added and stirred at room temperature for 12 hours. Then placing the mixed solution into a dialysis bag, dialyzing with purified water for 8 hours, filtering the dialysate through a 0.22 mu m filter membrane, and freeze-drying to obtain the compound. After redissolution detection, the average particle size is 24.7nm, and the encapsulation rate is not less than 90%.

Example 29 preparation of homoharringtonine ginsenoside Rg5H1(E) Mixed micelles containing mPEG-PDLLA and Chitosan-cholic acid

500mg of mPEG-PDLLA (with the number average molecular weight of 4000), 100mg of chitosan-cholic acid, 300mg of Rg5H1(E) and 50mg of homoharringtonine are dissolved in 20ml of diethyl ether, and are subjected to reduced pressure rotary concentration at 30 ℃ to form a film, and then the film is continuously evaporated to dryness, 20ml of purified water is added, and the mixture is stirred and hydrated at 30 ℃ to obtain a clear micelle solution after dissolution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 66nm, and the encapsulation rate is not less than 90%.

Example 30 preparation of mPEG-PLA-containing Adriamycin ginsenoside Rg5H1(Z) Mixed micelle

Dissolving 100mg of mPEG-PLA (with the number average molecular weight of 2400), 400mg of ginsenoside Rg5H1(Z) and 50mg of adriamycin in 20ml of methanol, carrying out rotary concentration at 50 ℃ under reduced pressure to form a film, then continuing to evaporate to dryness, adding 20ml of purified water, and carrying out stirring hydration at 40 ℃ to dissolve the purified water to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 27.3nm, and the encapsulation rate is not less than 95%.

Example 31 preparation of PEG-PLGA-containing cisplatin ginsenoside Rk1H Mixed micelles

100mg of PEG-PLGA (the number average molecular weight is 2000), 200mg of ginsenoside Rk1H and 50mg of cisplatin are dissolved in 20ml of THF, and are subjected to rotary concentration at 55 ℃ under reduced pressure to form a film, then the film is continuously evaporated to dryness, 20ml of 5% glucose aqueous solution is added, and the mixture is stirred and hydrated at 55 ℃ to be dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 18.8nm, and the encapsulation rate is not less than 95%.

EXAMPLE 32 preparation of PEG-PHIS-CONTAINING NAPPESSEN GINSOSIDE Rh3H MIXED PELLET

100mg of PEG-Phis (number average molecular weight: 4000) and 200mg of 20(R) -Rh3H and 30mg of naproxen are dissolved in 20ml of methanol, and are subjected to reduced pressure concentration at 50 ℃ to form a rotary film, and then the rotary film is continuously evaporated to dryness, 20ml of 5% glucose aqueous solution is added, and the mixture is hydrated at 30 ℃ to be dissolved to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 22.5nm, and the encapsulation rate is not less than 90%.

Example 33 preparation of homoharringtonine ginsenoside Rg3H1(E) Mixed micelles containing mPEG-PDLLA and Chitosan-cholic acid

500mg of mPEG-PDLLA (with the number average molecular weight of 4000), 100mg of chitosan-cholic acid, 300mg of Rg3H1(E) and 50mg of homoharringtonine are dissolved in 20ml of diethyl ether, and are subjected to reduced pressure rotary concentration at 30 ℃ to form a film, and then the film is continuously evaporated to dryness, 20ml of purified water is added, and the mixture is stirred and hydrated at 30 ℃ to obtain a clear micelle solution after dissolution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 35.5nm, and the encapsulation rate is not less than 90%.

Example 34 preparation of doxorubicin ginsenoside Rg3H1(Z) Mixed micelle containing mPEG-PLA

Dissolving 100mg of mPEG-PLA (with the number average molecular weight of 2400), 400mg of ginsenoside Rg3H1(Z) and 50mg of adriamycin in 20ml of methanol, carrying out rotary concentration at 50 ℃ under reduced pressure to form a film, then continuing to evaporate to dryness, adding 20ml of purified water, and carrying out stirring hydration at 40 ℃ to dissolve the purified water to obtain a clear micelle solution. Filtering with 0.22 μm filter membrane, and freeze drying. After redissolution detection, the average particle size is 24.5nm, and the encapsulation rate is not less than 95%.

Application example 1 hemolytic study of ginsenoside nanomicelle and mixed micelle

Ginsenosides (12 in total): 12 ginsenosides Rg5, 20(R) -Rg5H, 20(S) -Rg5H, Rg5H1(E), Rg5H1(Z), Rk1H, S-Rp1, R-Rp1, R-Rh3H, S-Rh3H, Rh3H1(E), Rh3H1(Z) and the like.

Preparing the ginsenoside nano micelle: referring to the methods of preparation examples 1 to 4 of the mixed micelles, different ginsenosides were replaced without adding an amphiphilic copolymer.

Preparation of mixed micelles: different ginsenosides and amphiphilic polymers are replaced according to the preparation method of the ginsenoside nano-micelle in the application example.

TABLE 2

Numbering	Haemolysis (HD50)	Numbering	Haemolysis (HD50)
				Rg5 nano micelle	45-50ug/ml	Rg5+ PEG-DSPE mixed micelle	1mg/ml shows no hemolysis
Rg5H nano micelle	120-130ug/ml	Rg5H + PEG-DSPE mixed micelle	1mg/ml shows no hemolysis
				Rg5H1(E) nano micelle	70-80ug/ml	Rg5H1(E) + mPEG-PDLLA mixed micelle	1mg/ml shows no hemolysis
Rg5H1(Z) nano micelle	70-80ug/ml	Rg5H1(Z) + mPEG-PLA mixed micelle	1mg/ml shows no hemolysis
				Rk1H nano micelle	70-80ug/ml	Rk1H + PEO-PASp mixed micelle	1mg/ml shows no hemolysis
Rp1 nano-micelle	≤0.5ug/ml	Rp1+ PEG-PCL mixed micelle	1mg/ml shows no hemolysis
				Rh3H nano micelle	35-40ug/ml	Rh3H + PEG-PASp mixed micelle	1mg/ml shows no hemolysis
Rh3H1(E) nano micelle	35-40ug/ml	Rh3H1(E) + PEG-PHIs Mixed micelle	1mg/ml shows no hemolysis
				Rh3H1(Z) nano micelle	35-40ug/ml	Rh3H1(Z) + PEG-PHIs Mixed micelle	1mg/ml shows no hemolysis

And (4) conclusion: the ginsenoside has strong hemolytic property, HD50 is about 50ug/ml generally after the ginsenoside becomes nano gelatin, and Rp1 with strong hemolytic property is even below 0.5ug/ml, so that the ginsenoside is difficult to be applied to a drug delivery system; the mixed micelle is not hemolyzed at 1 mg/ml.

Compared with ginsenoside nano-micelle, the mixed micelle greatly reduces the hemolytic property of the ginsenoside nano-micelle.

Application example 2 nanoparticle size study of Mixed micelles

Preparing nano micelle: referring to the methods of preparation examples 1 to 4 of the mixed micelles, except that ginsenoside or amphiphilic copolymer is not added, the amphiphilic polymer nano-micelle or the ginsenoside nano-micelle is prepared. Each sample was examined 3 times to obtain a particle size range of 3 times.

Mixed micelle: by referring to the preparation method of the nano micelle in the application example, different saponins and amphiphilic polymers are replaced, and each sample is detected for 3 times to obtain a particle size range of 3 times.

TABLE 3

And (4) conclusion: under the same condition, the particle size of the ginsenoside nano micelle is generally 60-100 nm; the amphiphilic copolymer nano micelle is generally 300-800 nm, even reaches the level of mum, and is difficult to be well compatible with organisms; the particle size of the mixed micelle is generally 15-60 nm.

Therefore, compared with ginsenoside nano-micelles and amphiphilic polymer nano-micelles, the particle size of the mixed micelles is greatly reduced.

Application example 3 formulation stability study of Mixed micelles

Preparation of mixed micelles: taking 200mg of amphiphilic copolymer, 400mg of ginsenoside, 50mg of paclitaxel and 50mg of VE, dissolving in 20ml of methanol: chloroform-1: 1 (volume ratio) at 60 ℃, rotary concentrating to form a film, then continuing to evaporate to dryness, adding 20ml of 5% glucose aqueous solution, stirring and hydrating at 60 ℃, and dissolving to obtain a clear micelle solution. The micellar solution was placed in a freezer at 4 ℃ and the time required for the solution to appear cloudy or to precipitate a solid was observed, and the experiment was terminated after 24 hours.

Preparing the ginsenoside nano micelle: referring to the preparation method of mixed micelles in this application example, no amphiphilic copolymer was added.

Preparation of amphiphilic copolymer nano-micelle: referring to the preparation method of the mixed micelle in the application example, ginsenoside was not added.

TABLE 4

And (4) conclusion: under the same condition, the ginsenoside nano-micelle is turbid after being placed for 2-4 hours, the amphiphilic copolymer nano-micelle is turbid after being placed for 3-4 hours, and the mixed micelle can reach 8-12 hours, even the mixed micelle is turbid after more than 24 hours.

Therefore, the preparation stability of the mixed micelle is greatly improved compared with that of the saponin or the polymer nano micelle.

Application example 4 in vitro cell experiment and in vivo animal experiment

Experiments on the drug effect of ginsenoside Rg5+ mPEG-DSPE blank mixed micelle (short for mixed hollow), paclitaxel polymer micelle (short for Genex-PM), paclitaxel ginsenoside Rg5 and PEG-DSPE mixed micelle (short for paclitaxel mixed micelle) on human lung cancer cells (A549)/human lung cancer paclitaxel drug-resistant strains (A549/T).

Remarking: in a preliminary experiment, the paclitaxel ginsenoside Rg5 micelle is unstable in a white mouse, so that the white mouse dies, and therefore, the paclitaxel ginsenoside nano micelle is not included in an in vivo comparison study.

Preparation of paclitaxel mixed micelles: the method of example 5 was prepared with reference to the aforementioned mixed micelles.

Preparation of the mixed blank: referring to the method of the aforementioned mixed micelle preparation example 5, paclitaxel, the active substance, was not added.

Paclitaxel polymer micelles were purchased from Samyang Biopharmaceutics Corporation of Korea (Seoul, Korea).

1. In vitro cell viability assay

According to the in vitro cell experiment method, the cell survival rates of the ginsenoside Rg5+ mPEG-DSPE blank mixed micelle (called mixed hollow for short), the taxol polymer micelle (called Genex-PM for short) produced by Korean Samyang, the taxol ginsenoside Rg5 and the PEG-DSPE mixed micelle (called taxol mixed micelle for short) after the treatment of human lung cancer cells (A549) and human lung cancer taxol-resistant strains (A549/T) are respectively determined.

The drug concentrations were set at 7 as in tables 5 and 6, and the specific experimental data are shown in tables 5 and 6, and fig. 1 and 2.

TABLE 5

As can be seen from table 5 and fig. 1: the activity of the mixed air to human lung cancer cells (A549) is weaker; at high concentration of paclitaxel (200ng/mL), the activity of Genex-PM and paclitaxel mixed micelle on human lung cancer cells (A549) is equivalent; as the concentration of paclitaxel administration is reduced (100, 50, 25, 12.5, 6.5ng/mL), paclitaxel mixed micelle shows better activity on human lung cancer cells (A549) than Genexol-PM.

TABLE 6

As can be seen from table 6 and fig. 2: the activity of the mixed blank and the GenOxol-PM on the paclitaxel-resistant human lung cancer cells (A549/T) is weaker, and the activity of the GenOxol-PM and the paclitaxel mixed micelle on the human lung cancer cells (A549/T) is equivalent when the paclitaxel is at a high concentration (100 mu g/mL); as the concentration of the paclitaxel is reduced (50, 25, 12.5, 6.5, 3.125 and 1.5625 mu g/mL), the paclitaxel mixed micelle shows better activity on a human lung cancer paclitaxel resistant strain (A549/T) than Genexol-PM.

2. In vitro cell IC₅₀Experiment of

According to IC₅₀Experimental method for determining IC of mixed micelle, Genex-PM and paclitaxel mixed micelle on human lung cancer cell (A549) and human lung cancer paclitaxel resistant strain (A549/T) respectively₅₀Values, experimental data are as in table 7:

TABLE 7

Item	A549 cell strain (ng/mL)	A549/T cell line (μ g/mL)
			Mixed air	/	/
Genexol-PM	62.49	22.20
			Paclitaxel mixed micelle	60.66	12.08

As can be seen from Table 7: the IC50 of the paclitaxel mixed micelle to A549 is lower than that of GenOxol-PM, the IC50 of the paclitaxel mixed micelle to a paclitaxel-resistant human lung cancer resistant strain (A549/T) is also lower than that of GenOxol-PM, and compared with a cell strain, the cell drug effects of the paclitaxel mixed micelle group are respectively improved by 3 percent and 46 percent compared with that of the GenOxol-PM group, which shows that the mixed micelle disclosed by the invention is more effective to the drug-resistant strain.

3. In vivo efficacy test

According to the in vivo efficacy test method, 27 subcutaneous tumor-bearing nude mice were randomly divided into 3 groups (9 mice per group), and set as a blank Control group (Control group, 0.9% NaCl), a Genex-PM group, and a paclitaxel mixed micelle group, respectively. The corresponding preparation is injected via tail vein (according to 25 mg. kg)^-1The dosage of (a). Changes in body weight of each group of mice were recorded every two days and the longest and shortest tumor diameters were measured with a vernier caliper and the tumor volume was calculated from the following formula: v ═ dmax × dmin²) /2, where dmin and dmax are the minor and major diameters (mm) of the tumor, respectively; calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows: RTV is Vt/V0. Where V0 is the tumor volume measured at the time of administration and Vt is the tumor volume measured every other day.

3.1 comparison (efficacy) of anti-tumor effect of human lung cancer cells (A549) in Control group, Genex-PM group and paclitaxel mixed micelle group, and the specific experimental data are shown in Table 8 and FIG. 3.

TABLE 8

As can be seen from table 8 and fig. 3: at the same time, tumor volume was greatest in the Control group and smallest in the paclitaxel mixed micelle group, followed by the Genex-PM group. The tumor inhibition rate of the Genex-PM group is 80%, the tumor inhibition rate of the paclitaxel mixed micelle group is 95%, and compared with the tumor inhibition rate, the tumor inhibition rate is improved by 1.19 times.

3.2 comparison (efficacy) of anti-tumor effect of paclitaxel-resistant strain (A549/T) in human lung cancer, controls group, Genex-PM group and paclitaxel mixed micelle group, and the specific experimental data are shown in Table 9 and FIG. 4.

TABLE 9

As can be seen from table 9 and fig. 4: at the same time, tumor volume was greatest in the Control group and smallest in the paclitaxel mixed micelle group, followed by the Genex-PM group. The tumor inhibition rate of the Genex-PM group is 58 percent, and the tumor inhibition rate of the paclitaxel mixed micelle group is 90 percent, compared with the tumor inhibition rate which is improved by 1.55 times.

Claims

1. The active substance loaded mixed micelle is characterized in that one or more of active substances in medicines, cosmetics and substances with health care effect are encapsulated in a blank mixed micelle; the blank mixed micelle comprises an amphiphilic copolymer and ginsenoside shown in a formula I: the mass ratio of the amphiphilic copolymer to the ginsenoside shown in the formula I is 4:1-1: 4;

the ginsenoside is ginsenoside Rk1, protopanaxatriol PPT, damelin A, protopanaxadiol PPD, ginsenoside Rg5, ginsenoside Rg5H, ginsenoside Rh1, 25-methyl-isoginsenoside Rg3, ginsenoside Rg4, ginsenoside Rh4, ginsenoside Rh2, ginsenoside Rp1, ginsenoside Rg3, ginsenoside Rh3, isoginsenoside Rh2(E), isoginsenoside Rg3(Z), pseudoginsenoside GQ, ginsenoside Rp2, pseudoginsenoside HQ, ginsenoside 3, isoginsenoside Rh2(Z), 3-sodium sulfate-ginsenoside Rp1, 3- (N, N-dimethylaminoethyl) -carbamoyl-ginsenoside Rp1, isoginsenoside 2(E), isoginsenoside Rg2(Z), isoginsenoside Rg3, isoginsenoside Rp 56, isoginsenoside Rg 8656 (E) 82695 8653), ginsenoside Rp 8427 (E) and ginsenoside Rg 1, One or more of ginsenoside Rg5H1(Z), ginsenoside Rk1H, ginsenoside Rh3H, ginsenoside Rg3H1(E), ginsenoside Rg3H1(Z), ginsenoside Rh3H1(E) and ginsenoside Rh3H1 (Z);

the amphiphilic copolymer is one or more of mPEG-DSPE, mPEG-PDLLA, mPEG-PLA, PEG-DSPE-NH2, PEG-PASp, PEG-PBLA, PEG-PBLG, PEG-PCL, PEG-Phis, PEG-PLGA, PEO-PASp and PEO-PPO-PEO.

2. The active material-supporting mixed micelle according to claim 1,

the number average molecular weight of the mPEG-DSPE is 2000;

or the mPEG-PDLLA has the number average molecular weight of 2000 or 4000;

or the number average molecular weight of the mPEG-PLA is 2400;

or, the number average molecular weight of the PEG-DSPE is 2000 or 4000;

or, the number average molecular weight of the PEG-DSPE-NH2 is 4000;

or, the number average molecular weight of the PEG-PASp is 4800;

or, the number average molecular weight of the PEG-PBLA is 2000;

or, the PEG-PBLG has the number average molecular weight of 4000;

or, the number average molecular weight of the PEG-PCL is 2000;

or, the number average molecular weight of the PEG-Phis is 4000;

or, the number average molecular weight of the PEG-PLGA is 2000;

or, the PEO-PASp has the number average molecular weight of 4800;

or the number average molecular weight of the PEO-PPO-PEO is 4800.

3. The active material-loaded mixed micelle of claim 1 wherein the blank mixed micelle further comprises one or more of an antioxidant, a lyoprotectant, an emulsifier, and a co-emulsifier.

4. The active agent-loaded mixed micelle of claim 3 wherein the antioxidant is vitamin C and/or vitamin E;

or, the content of the antioxidant in the blank mixed micelle is less than or equal to 25 percent; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank mixed micelle;

or, the lyoprotectant is a sugar and/or a buffer;

or, the content of the freeze-drying protective agent in the blank mixed micelle is less than or equal to 80 percent; the percentage refers to the percentage of the mass of the freeze-drying protective agent in the total mass of the blank mixed micelle;

or, the emulsifier is cholesterol;

or, the content of the emulsifier in the blank mixed micelle is less than or equal to 10 percent; the percentage refers to the percentage of the mass of the emulsifier in the total mass of the blank mixed micelle.

5. The active material-loaded mixed micelle of claim 4 wherein the antioxidant is present in the blank mixed micelle in an amount of 0.001% to 15%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank mixed micelle;

or, when the lyoprotectant is a sugar and/or a buffer, the sugar is a monosaccharide;

or, when the lyoprotectant is a sugar and/or a buffer, the buffer refers to a buffer solution;

or, the content of the freeze-drying protective agent in the blank mixed micelle is 61.73-75.76%; the percentage refers to the percentage of the mass of the freeze-drying protective agent in the total mass of the blank mixed micelle;

or, the content of the emulsifier in the blank mixed micelle is 0.01-10%; the percentage refers to the percentage of the mass of the emulsifier in the total mass of the blank mixed micelle.

6. The active material-loaded mixed micelle of claim 5 wherein the antioxidant is present in the blank mixed micelle in an amount of 14% or 0.01% to 10%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank mixed micelle;

or, when the lyoprotectant is sugar and/or buffer, and the buffer is buffer solution, the buffer solution is buffer solution with pH value between 3-10;

or, the content of the lyoprotectant in the blank mixed micelle is 61.73%, 65.36%, 65.57%, 74.07%, 75.19% or 75.76%; the percentage refers to the percentage of the mass of the freeze-drying protective agent in the total mass of the blank mixed micelle;

or, the content of the emulsifier in the blank mixed micelle is 0.1-5%; the percentage refers to the percentage of the mass of the emulsifier in the total mass of the blank mixed micelle.

7. The active material-loaded mixed micelle of claim 6 wherein the antioxidant is present in the blank mixed micelle in an amount of 6% or 0.01% to 5%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank mixed micelle;

or, the content of the emulsifier in the blank mixed micelle is 1% -5%; the percentage refers to the percentage of the mass of the emulsifier in the total mass of the blank mixed micelle.

8. The active material-loaded mixed micelle of claim 7 wherein the antioxidant is present in the blank mixed micelle in an amount of 3% or 0.1% to 1%; the percentages refer to the mass of antioxidant as a percentage of the total mass of the blank mixed micelles.

9. The active material-loaded mixed micelle of claim 6 wherein when the lyoprotectant is a sugar and/or a buffer, the buffer is a buffered solution having a pH of between 5 and 7.

10. The active material-loaded mixed micelle of claim 3 wherein the lyoprotectant is one or more of a 5% aqueous glucose solution, physiological saline and phosphate buffer solution;

or, the content of the freeze-drying protective agent in the blank mixed micelle is 0.5% -60%; the percentage refers to the percentage of the mass of the freeze-drying protective agent in the total mass of the blank mixed micelle.

11. The active material-loaded mixed micelle of claim 10 wherein the lyoprotectant is present in the blank mixed micelle in an amount of 5% to 60%; the percentage refers to the percentage of the mass of the freeze-drying protective agent in the total mass of the blank mixed micelle.

12. The active material-loaded mixed micelle of claim 11 wherein the lyoprotectant is present in the blank mixed micelle in an amount of 30% to 60%; the percentage refers to the percentage of the mass of the freeze-drying protective agent in the total mass of the blank mixed micelle.

13. The active-material-loaded mixed micelle of claim 1, wherein the mass ratio of the ginsenoside represented by formula I to the amphiphilic copolymer to the drug is 100:1-1: 1;

or the medicine is one or more of antitumor drugs, anti-inflammatory drugs, antibacterial drugs, sedative hypnotic drugs, antipsychotic drugs, hormone drugs, antibiotic drugs, calcium ion antagonists, anesthetics, cardiovascular and cerebrovascular and vasodilator drugs, polynucleotides and oligonucleotides;

or the active substance in the cosmetic is vitamin K3.

14. The active substance-loaded mixed micelle of claim 13, wherein the mass ratio of the ginsenoside represented by formula I to the amphiphilic copolymer to the drug is 20:1, 16.7:1, 16:1, 12:1, 10:1, 8.3:1, 6:1, 4: 1.

15. The active material-supporting mixed micelle according to claim 13,

the mass ratio of the ginsenoside shown in the formula I to the amphiphilic copolymer to the medicine is 25:1-5: 1;

or the anti-tumor drug is one or more of paclitaxel, docetaxel, camptothecin, homoharringtonine, adriamycin, cisplatin, oxaliplatin, epothilone C, irinotecan hydrochloride and all-trans retinoic acid;

or the anti-inflammatory drug is one or more of indometacin, naproxen and bifendate;

or, the antibacterial drug is amphotericin B;

or, the sedative hypnotic drug is clonazepam;

or, the antipsychotic is haloperidol;

or, the hormone medicine is dihydrotestosterone;

or, the antibiotic is cyclosporin a;

or, the calcium ion antagonist is fenofibrate;

or, the anesthetic is propofol;

or, the cardiovascular and cerebrovascular and vasodilatation drugs are puerarin;

or, the polynucleotide or oligonucleotide refers to SiRNA, antisense nucleic acid or RNAi sequence of microglia NLRP3 gene.

16. The active material-supporting mixed micelle according to claim 15,

the polynucleotide or oligonucleotide refers to SiRNA.

17. The active material-supporting mixed micelle according to claim 15,

the mass ratio of the ginsenoside shown in the formula I to the amphiphilic copolymer to the medicine is 15:1-5: 1.

18. A method for preparing an active agent-loaded mixed micelle according to any one of claims 1 to 17, comprising any one of the following methods

Method a comprises the following steps:

the method B comprises the following steps:

method C comprises the following steps:

mixing an active substance and an organic solvent to obtain a mixture A, mixing an amphiphilic copolymer, ginsenoside shown as a formula I and water or a buffer solution to obtain a mixture B, dropwise adding the mixture A into the mixture B to form an oil/water mixed emulsion, optionally adding one or more of an antioxidant, a freeze-drying protective agent, an emulsifier and a co-emulsifier, removing the organic solvent, filtering, and freeze-drying;

method E comprises the following steps:

when the active substance is easily soluble in water, mixing the active substance, amphiphilic copolymer, ginsenoside represented by formula I and water, optionally adding one or more of antioxidant, emulsifier and co-emulsifier, filtering, and freeze drying;

the amphiphilic copolymer of process A, B, C or E is according to claim 1, the ginsenoside of formula I is according to claim 1, and the antioxidant, lyoprotectant, emulsifier and co-emulsifier are according to claims 4-12.

19. The method of claim 18,

in method B, the dialysis procedure comprises the following steps: putting the mixed micelle solution into a glucose aqueous solution or pure water for dialysis; the dialysis time is 5-20 hours;

or the organic solvent is one or more of dichloromethane, trichloromethane, methanol, ethanol, diethyl ether, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide and pyridine;

alternatively, in process A, B, C or E, the active substance is used in the form of an aqueous solution of the active substance or an organic solution of the active substance, depending on the lipid solubility or water solubility of the active substance.

20. The method of claim 19, wherein the step of dialyzing according to method B comprises the steps of: putting the mixed micelle solution into a glucose aqueous solution or pure water for dialysis; the dialysis time is 12 hours;

or, in method A, B, C or E, when the active substance is used in the form of an aqueous solution of the active substance or an organic solution of the active substance, the percentage is the percentage of the mass of the active substance to the total volume of the aqueous solution of the active substance or the organic solution of the active substance, depending on the liposolubility or water solubility of the active substance, the aqueous solution of the active substance or the organic solution of the active substance is 1-20% by mass volume.