CN109833298B

CN109833298B - Novel blank liposome taking ginsenoside derivative as membrane material, preparation method and application thereof

Info

Publication number: CN109833298B
Application number: CN201811447245.3A
Authority: CN
Inventors: 王丹; 李翀; 王亚华; 孟强; 缪鹏飞; 詹华杏
Original assignee: Xiamen Ginposome Pharmaceutical Co ltd
Current assignee: Xiamen Ginposome Pharmaceutical Co ltd
Priority date: 2017-11-29
Filing date: 2018-11-29
Publication date: 2022-12-13
Anticipated expiration: 2038-11-29
Also published as: CN109833298A; WO2019105408A1

Abstract

The invention discloses a novel blank liposome taking ginsenoside derivatives as membrane materials, a preparation method and application thereof. The ginsenoside derivative disclosed by the invention is good in activity and low in hemolysis, can be used as a liposome membrane material to prepare blank liposomes, and meanwhile, part of compounds are novel in structure. The hemolysis of the liposome prepared by the method meets the requirements, and the liposome has higher safety, better film-forming property and better stability, and has important application value.

Description

Novel blank liposome taking ginsenoside derivative as membrane material, preparation method and application thereof

Technical Field

The invention relates to a novel blank liposome taking ginsenoside derivatives as membrane materials, a preparation method and application thereof.

Background

Ginsenoside has complex and various pharmacological activities, and in order to highlight the pharmacological action of each monomer saponin, the current researches on ginsenoside mainly focus on the aspects of development and utilization of monomer saponin. Hemolysis is one of the properties of partial saponins, and is the biggest obstacle to the use of monomeric saponins as injections. The total ginsenoside has no obvious hemolytic effect, and the monomer saponin after separation and purification has opposite hemolytic or anti-hemolytic effect and similar curve relationship between the hemolysis and anti-hemolytic effect and concentration. The research on the hemolysis and anti-hemolysis effects of ginsenoside is less, and the researches on the hemolysis and anti-hemolysis effects of ginsenoside such as Cheng Daren at the 4 th stage of volume 9 of month 4 in 2007 of modern Chinese medicine in China consider that the protopanaxatriol type ginsenosides Re, rg1, 20 (R) -Rg2, 20 (S) -Rg2 and Rh1 all have the anti-hemolysis effect, wherein the 20 (R) -Rg2, 20 (S) -Rg2 and Rhl also show the hemolysis effect at higher concentration; protopanaxadiol type ginsenosides Rb1, rb2, rc and Rd all exhibit anti-hemolytic action, wherein Rd exhibits hemolytic action at higher concentrations. The oleanolic type ginsenoside Ro shows anti-hemolytic effect at a lower concentration, and no hemolytic effect is seen. Some saponins with stronger activity, such as Rg3, rh2, C-K, protopanaxadiol, protopanaxatriol and the like, can not be used as injections because of the stronger hemolytic property. The results of the hemolytic experiments of these ginsenosides are shown in table 1.

TABLE 1

The term,/means no such effect.

Therefore, the research on a monomer saponin with better activity and hemolytic property meeting the requirement of an injection is always the technical problem of the research on ginsenoside medicaments.

According to the method disclosed in the Chinese invention patent CN201610693884.2, amphiphilic saponin represented by ginsenoside Rg5 or Rk1 and the like can be used as a liposome membrane material. However, the saponin should have a lipophilic end and a hydrophilic end, and the lipophilic end should have more than two double bonds. When the saponins such as Rg3, rh2 and the like are used as liposome membrane materials to encapsulate paclitaxel, the obtained liposome has poor appearance, encapsulation efficiency, particle size and stability, particularly particle size and stability, the particle size is more than 1 mu m, precipitates are generated after the liposome is placed for 7 days, and the encapsulation efficiency is less than or equal to 80%.

Therefore, the development of the novel saponin liposome with good activity, high hemolytic property, high safety, high film forming property and high stability has important application value.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects that in the prior art, the appearance, encapsulation efficiency, particle size and stability of the obtained liposome are poor or part of ginsenoside has high hemolytic property and cannot be used as a liposome membrane material when the saponin with only one double bond or no double bond at the oleophilic end of Rg3, rh2 and the like is used as the liposome membrane material, and provides a novel blank liposome taking a ginsenoside derivative as the membrane material, a preparation method and application thereof. The ginsenoside derivative has only one double bond or no double bond at the lipophilic end, and has good activity, low hemolysis and novel structure of partial compounds. The hemolysis of the liposome prepared by the method meets the requirements, and the liposome has higher safety, better film-forming property and better stability, and has important application value.

The present invention solves the above-mentioned technical problems by the following technical solutions.

The invention provides a blank liposome taking ginsenoside derivatives shown in formula I as membrane materials, wherein the blank liposome is provided with a membrane, and the membrane comprises lipid substances and ginsenoside derivatives shown in formula I:

wherein R is ¹ And R ² Each independently is H, -OH, R ¹⁰ 、R ¹¹ 、R ¹² Or R ¹³ However, R is ¹ And R ² Not H or-OH simultaneously;

R ³ is composed of

R ⁴ Is 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);

R ⁵ is H, -OH, ketone (= O), methoxy (-OCH) ₃ ) Or acetoxy (-OAc);

each R ⁶ Independently is-OH, methoxy (-OCH) ₃ ) A hydroperoxy group (-OOH), an acetoxy group (-OAc), or a benzoyl group (-OBz);

R ⁷ 、R ⁹ and R ⁸ Independently is H, -OH, methoxy (-OCH) ₃ ) Formyloxy (-OCHO), acetoxy (-OAc), or benzoyl (-OBz);

R ¹⁰ is 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 numbers indicate carbon positions, the connection relationship is represented on a → basis, the same as above), -O-Glc (6 → 1) Glc, -O-Glc (2 → 1) Rha, -O-Glc (2 → 1) Xyl, -O-Glc (6 → 1)) <xnotran> 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) Glc (2 → 1) Glc, -O-Glc (2 → 1) Glc (2 → 1) Xyl, -O-Glc (6 → 1) Glc (6 → 1) Xyl, -O-Glc (2 → 1) Glc (4 → 1) Xyl, -O-Glc (2 → 1) 5754 zxft 5754, -O-Glc (6 → 1) 3252 zxft 3252, -O-Glc (2 → 1) Glc (2 → 1) Rha, -O-Glc (2 → 1) Glc (2 → 1) 3532 zxft 3532, -O-Glc (2 → 1) Glc (2 → 1) Ara (f), -O-Glc (2 → 1) 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) Xyl, -O-Glc (2 → 1) Glc (6 → 1) 3425 zxft 3425, </xnotran> -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 (6 → 1) Glc, -O-Glc (6 → 1) Glc (6 → 1) Rha, -O-Glc (6 → 1) Glc (6 → 1) Lyx, -O-Glc (6 → 1) Glc (6 → 1) Ara (f), or-O-Glc (6 → 1) Glc (6 → 1) Ara (p);

R ¹¹ is R ¹⁰ With more than one hydroxy group of R ¹⁰ Substituted, each R ¹⁰ (when there are two or more) are each independently the same or different;

R ¹² is 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 ) _a CO-、-NH(CH ₂ ) _a CO-、-NH(CH _2)b X-or-CO-Ar-CH ₂ -; 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)C ₄ -C ₂₂ fatty acyl, phosphate, succinate, n-butyrate, sulfonate, malate, and mixtures thereof,

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;

R ¹³ is any one of the following groups: (N, N-dimethylaminoethyl) -carbamoyl, abbreviated DC, having the structure

(N, N-dimethylaminopropyl) -carbamoyl, DMAPA for short, having the structure

N- (N ', N' -dimethyl) ethylsuccinic acid monoamide with the structure of

N- (N ', N' -dimethyl) propyl succinic acid monoamide with the structure of

In a preferred embodiment of the invention, R ¹ preferably-OH,

In a preferred embodiment of the invention, R ² Preferably H or

In a preferred embodiment of the invention, R ³ Preferably a

More preferably

In a preferred embodiment of the invention, R ⁴ preferably-OH, -OAc or = O.

In a preferred embodiment of the invention, R ⁵ Preferably H or-OH.

In a preferred embodiment of the present invention, more than one hydroxyl group in the ginsenoside derivative represented by formula I is optionally substituted by R ¹¹ Substituted with each R ¹¹ (when there are two or more) are each independently the same or different.

In a preferred embodiment of the present invention, more than one hydroxyl group in the ginsenoside derivative represented by formula I can be optionally substituted by R ¹² Substituted; each R ¹² (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, 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 structural formula:

in the formula of-O-Ara (f), ara (f) has the structural formula

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

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

Mal has a structural formula of

Wherein the molecular weight of the PEG, PEO, PVP or EPEG is preferably 200 to 20000 independently.

Wherein, the fatty acyl can be acyl of naturally occurring saturated or unsaturated fatty acid, and acyl of synthetic saturated or unsaturated fatty acid, preferably stearyl acyl or palmityl.

In a preferred embodiment of the present invention, the ginsenoside derivative represented by formula I is one or more of the compounds in Table 2:

TABLE 2

In the blank liposome, the HPLC purity of the ginsenoside derivative shown in the formula I is preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent, and the percentage refers to the percentage of the mass of the ginsenoside derivative shown in the formula I in the total mass of the blank liposome.

In the blank liposome, the mass ratio of the lipid substance to the ginsenoside derivative represented by the formula I is generally 0.5.

In the blank liposome, the membrane preferably further comprises cholesterol.

When the blank liposome further comprises cholesterol, wherein the mass ratio of the lipid substance to the ginsenoside represented by formula I is preferably 1; the mass ratio of the cholesterol to the ginsenoside represented by formula I is preferably 1.1-1, more preferably 1.5-1.

In the blank liposome, the content of the ginsenoside derivative shown in the formula I in the membrane is preferably 0.01-80% (e.g. 0.03%, 37.5%, 10%, 5%, 15.4%, 20.5%, 19.9%, 18.0%, 6.25%, 13.6%, 33.3% or 17.4%); the phospholipid is preferably present in the membrane in an amount of 5% to 99.9% (e.g., 37.5%, 40%, 35%, 34.6%, 25.6%, 34.8%, 36.0%, 21.875%, 36.3%, 66.7%, 39.1% or 34.8%); the content of the cholesterol in the membrane is preferably 0% -50%; the above percentages (%) all refer to the mass of each component as a percentage of the total mass of the film.

In the blank liposome, the content of the ginsenoside derivative shown in formula I in the membrane is preferably 10% -80%, more preferably 10% -40%, and even more preferably 20% -40% (e.g. 25% -40%, such as 37.5%, preferably 25% -35%). The content of the lipid substance in the membrane is preferably 10% -70%, more preferably 30% -70%, and still more preferably 30% -60%. The content of cholesterol in the membrane is preferably 0% to 50%, more preferably 0% to 20%, and still more preferably 0% to 20% (e.g., 0% to 10%).

In a preferred embodiment of the present invention, the blank liposome may further comprise an antioxidant and is encapsulated in the membrane. The content of the antioxidant in the blank liposome is generally less than or equal to 25%, preferably 0.001% -15%, more preferably 0.01% -10%, and still more preferably 0.01% -5% (e.g., 0.1% -1%); the percentage (%) refers to the percentage of the mass of the antioxidant to the total mass of the blank liposomes.

In a preferred embodiment of the present invention, the blank liposome may further comprise a lyoprotectant and is encapsulated in a membrane. The content of the lyoprotectant in the blank liposome can be the conventional content of the blank liposome in the field, such as less than or equal to 95% or less than or equal to 80%, preferably 0.5% -70%, more preferably 5% -60%, and most preferably 30% -60%; the percentage (%) is the mass of the lyoprotectant in percentage of the total mass of the blank liposome.

In a preferred embodiment of the present invention, the blank liposome may further comprise soybean oil and/or sodium oleate and is encapsulated in a membrane. The soybean oil and/or sodium oleate is preferably present in an amount of 1% to 90%, more preferably 15% to 80%, most preferably 20% to 70% (e.g., 3% to 5%, 25% to 62.5%,20% to 30%, or 60% to 70%); the percentage refers to the percentage of the mass of the soybean oil and/or the sodium oleate in the total mass of the blank liposome. The "soybean oil and/or sodium oleate" and the phospholipid preferably have a mass ratio of 1.

In another preferred embodiment of the present invention, the blank liposome comprises the following components: the lipid material and the ginsenoside derivative shown in the formula I, or the ginsenoside derivative, the lipid material and the antioxidant shown in the formula I, or the ginsenoside derivative, the lipid material and the freeze-drying protective agent shown in the formula I, or the ginsenoside derivative, the soybean oil and/or the sodium oleate and the phospholipid shown in the formula I, or the ginsenoside derivative, the soybean oil and/or the sodium oleate, the lipid material and the antioxidant shown in the formula I, or the ginsenoside derivative, the soybean oil and/or the sodium oleate and the freeze-drying protective agent shown in the formula I, or the ginsenoside derivative, the soybean oil and/or the sodium oleate, the lipid material, the antioxidant and the freeze-drying protective agent shown in the formula I, or the ginsenoside derivative, the lipid material and the cholesterol shown in the formula I, or the ginsenoside derivative, the lipid material, the cholesterol and the antioxidant shown in the formula I, or the ginsenoside derivative, the lipid material, the cholesterol and the freeze-drying protective agent shown in the formula I, or the ginsenoside derivative, the soybean oil and/or the lipid material, the cholesterol protective agent shown in the formula I, or the cholesterol and the antioxidant, or the cholesterol derivative, the soybean oil and/or the lipid material and the freeze-drying protective agent shown in the formula I, or the cholesterol protective agent.

In another preferred embodiment of the present invention, the blank liposome is composed of the above-mentioned components.

In another preferred embodiment of the present invention, the blank liposome comprises the following components: ginsenoside derivatives represented by formula I, lipid substances, cholesterol, soybean oil and/or sodium oleate, antioxidant and lyophilized protectant. The mass ratio of said "soybean oil and/or sodium oleate" and said "cholesterol" is preferably from 1.1 to 1, more preferably from 1, 0.5 to 1:5, most preferably from 1, 0.5 to 1:1. The content of cholesterol is preferably 1% -20%, more preferably 10% -20%; the soybean oil and/or sodium oleate is preferably present in an amount of 1% to 90%, more preferably 15% to 80%, most preferably 20% to 70% (e.g., 25% to 62.5%,20% to 30%, or 60% to 70%).

In a preferred embodiment of the present invention, the blank liposome is composed of the following components: lipid substances, and ginsenoside derivatives represented by formula I.

In a preferred embodiment of the present invention, the blank liposome is composed of the following components: ginsenoside derivatives represented by formula I, lipid substances and cholesterol.

In another preferred embodiment of the present invention, the blank liposome is composed of the following components: ginsenoside derivatives represented by formula I, lipid substances, cholesterol, antioxidant and lyophilized protectant.

In a preferred embodiment of the present invention, the blank liposome is composed of the following components: ginsenoside derivatives represented by formula I, lipid substances, cholesterol, soybean oil and/or sodium oleate, antioxidant and lyophilized protectant.

In the present invention, the lipid material may be a lipid material conventional in the art, and generally refers to a phospholipid, preferably one or more of natural phospholipid, semi-synthetic phospholipid and fully synthetic phospholipid.

In the present invention, the natural phospholipid is generally natural phospholipid derived from soybean, egg yolk, animal brain or viscera, preferably one or more of natural lecithin, sphingomyelin, glycerophospholipid, soybean lecithin, egg yolk lecithin and cephalin.

In the present invention, the semisynthetic or fully synthetic phospholipid may be one or more of phospholipids conventional in the art, preferably phosphatidylcholine, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine-based phospholipids, phosphatidylglycerol (DSPG), dicetyl phosphate (DCP), PEG-modified phospholipids, cholesteryl succinate (CHS), and 2-oleoyl-1-palmitoyl-glycerol-3-phosphocholine (16. Because the semisynthetic or fully synthesized phospholipids such as dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine have heat sensitivity, the phospholipids can be used as heat-sensitive auxiliary materials at the same time.

In the present invention, the phospholipid of phosphatidylcholine may be phospholipid of phosphatidylcholine, which is conventional in the art, and preferably is one or more of hydrogenated soybean lecithin (HSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), phosphatidylcholine (SPC), monopalmitoylphosphatidylcholine (MPPC) and Glycerophosphatidylcholine (GPC).

In the present invention, the phospholipid of phosphatidylethanolamine may be phospholipid of phosphatidylethanolamine which is conventional in the art, and preferably is one or more of 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), dilauroyl phosphatidylethanolamine (DLPE), erucyl phosphatidylethanolamine (DEPE), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE) and dimyristoyl phosphatidylethanolamine (DMPE).

In the present invention, the PEG-modified phospholipid may be a PEG-modified phospholipid which is conventional in the art, preferably phosphatidylethanolamine-polyethylene glycol (DMPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG), C8 Ceramide-polyethylene glycol (C8 Ceramide-PEG), C16 Ceramide-polyethylene glycol (C16 Ceramide-PEG), distearoylphosphatidylethanolamine-polyethylene glycol-Succinyl (DSPE-PEG Succinyl), distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG) Carboxylic Acid, distearoylphosphatidylethanolamine-polyethylene glycol-Maleimide (DSPE-Maleimide), distearoylphosphatidylethanolamine-polyethylene glycol-propionamide dimercaptopyridine (DSPE-PEGPDP), distearoylphosphatidylethanolamine-polyethylene glycol-cyanuric Acid (DSPE-PEGCyanur), distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEGAmine), distearoylphosphatidylethanolamine-biotin (DSPE-DSGBPEG), distearoylphosphatidylethanolamine (DSPE-PEGBPEGBPEO-PEGOlate), distearoylphosphatidylethanolamine (DSPE-PEGBPE-PEGBO-PEGAL), distearoylphosphatidylethanolamine (DSPE-PEGBPE-PEGBO-PEGAL), distearoylphosphatidylethanolamine (DSPE-PEGBO-PEG), DSPE-PEGBL-PEG, PEG, 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-hydroxyl phosphatidyl ethanolamine (DSPE-OH-2-NH-PEG-NH-2-NH-PEG-NHS), and distearoyl phosphatidyl ethanolamine (DSPE-PEG-NH-2-NH-PEG-NH-NHS), dipalmitoylphosphatidylethanolamine-polyethylene glycol-amino (DPPE-PEG-NH 2), dilauroylphosphatidylethanolamine-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-N3), cholesterol-polyethylene glycol (Cholesterol PEG), methoxy-polyethylene glycol-Cholesterol (mPEG-CLS), cholesterol-polyethylene glycol-active ester (Cholesterol PEG NHS), cholesterol-polyethylene glycol-maleimide (CLS-Cholesterol-polyethylene glycol-Maltol), cholesterol-Cholesterol (Cholesterol-Biofluorescein-PEG-fluorescein (Cholesterol-PEG-Biofluorescein), one or more of Cholesterol-polyethylene glycol-carboxyl (Cholesterol PEG COOH), cholesterol-polyethylene glycol-amino (Cholesterol PEG NH 2), and Cholesterol-polyethylene glycol-thio (Cholesterol PEG SH). Wherein the relative 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 DMPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000 or 5000. The DPPE-PEG preferably has a number average molecular weight of 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 DOPE-PEG preferably has a number average molecular weight of 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 DSPE-PEG-NHS preferably has a number average molecular weight of 1000, 2000, 5000, 10000, 20000, 30000 or 40000. The DMPE-PEG-NHS preferably has a number average molecular weight of 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 DPPE-PEG-Maleimide is preferably 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of DLPE-PEG-Maleimid is preferably 1000, 2000, 3400, 5000 or 10000. The DSPE-PEG-Biotin preferably has a number average molecular weight of 1000, 2000, 3400, 5000 or 10000. The DSPE-PEG-FITC preferably has a number average molecular weight of 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 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 DMPE-PEG-COOH preferably has a number average molecular weight of 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 preferably 3400. 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 Cholesterol PEG NHS ester preferably has a number average molecular weight of 1000, 2000, 3400, 5000 or 10000. The number average molecular weight of the CLS-PEG-Mal is preferably 2000, 3400, 5000 or 10000. The CLS-PEG-Biotin preferably has a number average molecular weight of 2000, 3400 or 5000. The number average molecular weight of CLS-PEG-FITC is preferably 2000, 3400 or 5000. The number average molecular weight of the Cholesterol PEG COOH is preferably 3400. The number average molecular weight of the Cholesterol PEG amine is preferably 3400. The Cholesterol PEG Thiol/Sulfhydral preferably has a number average molecular weight of 3400.

In a preferred embodiment of the invention, the lipid material is egg yolk lecithin, soybean lecithin, HSPC, DSPE-PEG (2000), DMPC, POPE, HSPC, DPPC, soybean lecithin S100, mPEG2000-DSPE or DOPE-PEG.

In the present invention, the antioxidant may be an antioxidant conventional in the art, and preferably is one or more of sodium metabisulfite, sodium thiosulfate, propyl gallate, α -tocopherol, α -hydroxy acid, flavonoid, phenylpropanoid phenolic compound, vitamin E, vitamin C, fumaric acid, cysteine, methionine, butylated Hydroxyanisole (BHA), dibutylhydroxytoluene (BHT), thiodipropionic acid, sulfite (such as sodium sulfite), bisulfite (such as sodium bisulfite), dithioaminobenzoic compound, citric acid, malic acid, sorbitol, glycerol, propylene glycol, hydroquinone, hydroxycoumarin, ethanolamine, phosphoric acid, and phosphorous acid.

In a preferred embodiment of the present invention, the antioxidant is vitamin E, vitamin C, sodium thiosulfate or sodium sulfite.

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 an ethanol-acetic acid buffer solution, a tris buffer solution, a barbital 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 (PBS).

In a preferred embodiment of the present invention, the lyoprotectant is glucose, sucrose, mannitol, propylene glycol, glycerol, galactose, trehalose, lactose, xylitol or phosphate buffer solution.

Preferably, the blank liposome can further comprise other auxiliary materials and is encapsulated in a membrane. The other auxiliary materials can be other auxiliary materials which are added conventionally in the field for preparing the liposome except for the antioxidant and the freeze-drying protective agent, such as one or more of a surfactant, a heat-sensitive auxiliary material, a pH-sensitive substance and an ionic additive.

In the present invention, the surfactant is preferably polyethylene glycol and/or polysorbate. Wherein, the number average molecular weight of the polyethylene glycol is preferably 200-8000. The polysorbate is preferably one or more of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan trioleate, polyethylene glycol-phosphatidylethanolamine, polyethylene glycol-polylactic acid, polylysine-polylactide glycolide, polyetherimide-polylactic acid, polyethylene glycol-polycaprolactone, polyethylene glycol-polyglycolide lactide, poloxamer 188, polyoxyethylene fatty acid esters, polyoxyethylene fatty acid ethers, and polyoxyethylene methyl castor oil ethers.

In the present invention, the thermosensitive auxiliary material generally refers to one or more of polymers and/or surfactants that can make the liposome thermosensitive. Wherein, the polymer is preferably one or more of polyacrylamide, polyacrylic acid, polyphosphate ester and poly-phosphoramide copolymer. The surfactant is preferably a tween surfactant (e.g. tween 80) and/or a brij surfactant.

In the present invention, the ionic additive is preferably a cationic additive (e.g., octadecylamine) and/or an anionic additive (e.g., phosphatidic acid and/or phosphatidylserine).

In the invention, the dosage of the other auxiliary materials can be selected according to the dosage of the common liposome containing the auxiliary materials in the field. For example, when the blank liposome comprises a surfactant, the content thereof is 0% to 50%, but not 0%; when the blank liposome contains the ionic additive, the content of the ionic additive is 0-10% but not 0%.

In the present invention, the blank liposome can be prepared by a conventional liposome preparation method in the art, and generally, an injection method, a reverse evaporation method, a freeze-thaw method, a multiple emulsion method, an active encapsulation method, a precursor liposome preparation method, a thin film dispersion method, a freeze-drying method, an ammonium sulfate gradient method or a pH gradient method, or a combination of any two of the above methods can be used. The present invention preferably adopts the following method one or method two, wherein the blank liposome prepared by the method one does not contain the freeze-drying protective agent, and the blank liposome prepared by the method two contains the freeze-drying protective agent:

the first method comprises the following steps:

(1) Mixing lipid substances and ginsenoside derivatives shown in formula I in organic solvent, optionally adding one or more of cholesterol, fat-soluble antioxidant, soybean oil and/or sodium oleate, fat-soluble surfactant, fat-soluble thermosensitive adjuvant, fat-soluble pH sensitive substance and fat-soluble ion additive to obtain a clarified solution;

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with water, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ionic additive, performing ultrasonic treatment, high-pressure homogenization or extrusion pushing through the film, filtering to obtain an aqueous solution containing blank liposomes, and drying to obtain the blank liposomes;

the second method comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with the aqueous solution of a freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, carrying out ultrasonic treatment, high-pressure homogenization or extrusion pushing through the film, filtering to obtain an aqueous solution containing blank liposome, and drying to obtain the blank liposome.

In the first or second method, the lipid material, the ginsenoside derivative represented by formula I, the cholesterol, the antioxidant, the soybean oil and/or sodium oleate, the lyoprotectant, the surfactant, the heat-sensitive auxiliary material, the pH-sensitive material and the ionic additive are defined as above.

In the first method or the second method, in the step (1), the organic solvent may be an organic solvent which is conventional in the liposome preparation method in the art, preferably a nitrile solvent, C ₁ -C ₄ One or more of alcohol solvents, ketone solvents, alkane solvents, ether solvents and halogenated hydrocarbon solvents, preferably C ₁ -C ₄ One or more of the alcohol solvent, the nitrile solvent, the ether solvent and the halogenated hydrocarbon solvent. The nitrile solvent is preferably acetonitrile. Said C ₁ -C ₄ The alcoholic solvent is preferably one or more of methanol, ethanol, isopropanol and n-butanol. The ethereal solvent is preferably tetrahydrofuran. The halogenated hydrocarbon solvent is preferably chloroform and/or dichloromethane. The ketone solvent is preferably acetone and/or butanone. The alkane solvent is preferably petroleum ether. The organic solvent can be used in an amountThe amount of the organic solvent used in the method for preparing liposomes in the art is not particularly limited, and generally requires that a clear solution can be obtained by mixing the organic solvent and all the components, and preferably, the volume-to-mass ratio of the organic solvent to all the components in step (1) of the first method or the second method is 5 to 20mL/g.

In the first or second process, step (1), the mixing temperature may be a temperature conventional in the art, and is generally 0-80 ℃, preferably 10-80 ℃, and more preferably 10-65 ℃. 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 the first or second method, in the step (2), the operation of removing the organic solvent from the clarified solution in the step (1) may be a conventional operation in the art, and a rotary evaporator or a film evaporator is generally used to remove the organic solvent. 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 first or second method, step (2), the operation of ultrasound, high pressure homogenization or extrusion through the membrane may be conventional in the art. After the operation of ultrasonic, high-pressure homogenization or extrusion through a membrane is finished, the particle size of liposome particles is generally 0.05-0.3 micron.

In the first or second method, step (2), the filtration may be performed according to the conventional method for liposome preparation in the art, and the purpose is to remove bacteria, solid particles, particularly large liposomes (in the method for preparing liposome carrying active substance, non-encapsulated free drug may also be removed), and the like. 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 second method, in the step (2), the aqueous solution of the lyoprotectant is an aqueous solution formed by mixing the lyoprotectant and 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 amount of the aqueous solution of the lyoprotectant is not particularly limited as long as it does not affect the formation of blank liposomes, 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 lyoprotectant is a buffer, the lyoprotectant is directly mixed with the lyoprotectant after the film-forming operation in step (2) is completed.

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

In the first method or the second method, in order to facilitate storage, the aqueous solution containing the blank liposome obtained in the step (2) is subpackaged in a penicillin bottle, dried, introduced with protective gas (argon or nitrogen), and sealed for storage.

The blank liposome can be used for preparing liposome loaded with active substances by wrapping the active substances in the membrane, and the active substances are one or more of active substances of medicines and exist in the form of a composition. Therefore, the invention also provides an active substance loaded liposome. The active substance-loaded liposome generally means that one or more active substances (active substances) in a medicament are encapsulated in the blank liposome.

In the liposome carrying the active substance, the mass ratio of the active substance to the ginsenoside derivative shown in the formula I is preferably 1.

In the active substance, the drug may be a conventional drug in the art, preferably one or more of an anti-tumor drug, an antifungal drug, an antiviral drug, an antibiotic, a non-steroidal anti-inflammatory drug, a calcium ion antagonist, an immunosuppressant, an anesthetic, a cardiovascular and cerebrovascular and vasodilator, a gastrointestinal drug, an anti-depression drug, a biological agent, a polynucleotide and an oligonucleotide (including a ribonucleotide and a deoxyribonucleotide).

In the active substance, the anti-tumor drug can be a conventional anti-malignant tumor drug in the field, preferably paclitaxel, docetaxel, cabazitaxel, irinotecan hydrochloride, hydroxycamptothecin, aminocamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan hydrochloride, lurtotecan (Lurtocan), topotecan, belotecan, cisplatin, carboplatin, oxaliplatin, nedaplatin (Nedaplatin), lobaplatin (Lobalatin), satraplatin (Saraplatin), miriplatin, pentaplatin, aroplatin (L-NDDP), carmustine, chlorambucil, melphalan, cephalotaxine, homoharringtonine, triptolide, tacrolimus, daunorubicin, doxycycline, doxorubicin hydrochloride, idarubicin, fluorouracil, cytarabine, methotrexate, etoposide phosphate, podophyllotoxin, huperzine A, 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, decitabine, pemetrexed disodium, sorafenib, recombinant human interferon a2B, arabinoside cytosine, all-trans retinoic acid, interleukin-2, etoposide, thymidylate synthase inhibitor, mitoxantrone, minoxidil, azithromycin, epirubicin hydrochloride, doxorubicin (adriamycin), amrubicin hydrochloride, 5-aminolevulinic acid (5-ALA), one or more of gefitinib, imatinib, erlotinib, sunitinib, dasatinib, lapatinib, axitinib, apatinib, nilotinib, bosutinib, vandetanib, tiratinib, neratinib, canertinib, secatinib, sorafenib, erlotinib, rilatinib, lestatinib, tandatinib, dovirtinib, 3',5' -cyclocytidyldipalmitate, and curcumenol.

Among the active substances mentioned above, the antifungal agent is preferably one or more of amphotericin B, gentamicin, indomethacin, penicillin G, econazole nitrate, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, ravuconazole, caspofungin, micafungin, anidulafungin, cefpiramide sodium, cefotaxime sodium, ceftriaxone, cefoperazone, cefditoren pivoxil, cefoxitin sodium, cephalexin, cefuroxime sodium, cefixime, cefpodoxime, cefepime, cefodizime, cefsulodin, ceftizoxime, cefazolin oxime, cefetamet, cefditoren pivoxil, cefbuntan, cefdinir, cefamandole, cefotiazem, cefonicid, cefprozil, cefazolin sodium, cefalotin, cefathiamidine, cefotiadinin, cefotiazetin, cefetacycline, ceftiofur-eye-down, ceftizoxime, ceftezole, cefpirome sodium, cefepime, and florfenidone.

Among the active substances, the antiviral drug is preferably one or more of ribavirin, acyclovir, cytarabine, idoxuridine, acyclovir laurate, acyclovir palmitate, idoxuridine, cyclocytidine dipalmitate, phosphoformate, phosphoacetate, cimetidine, dipyridamole, rifampin, isoniazid, praziquantel, doxycycline, saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, tipranavir, BMS232632, lamivudine, zidovudine, didanosine (dinosine, 3238 zxfft 3238), zalcitabine (zalabetadine, ddc), stavudine (stavudine, d4 t), abacavir (abacavir), addivovir (aifuvirdipipinir, pmea), valavidine (adefovir, pmea), valcanivir (tenovivir, papovavir), fluridovirdine (feprazine), nevirapine (3262), nevirapine (cuvavir), nevirapine (3262), or nevirapine (cudravir (cuvavir), or cells.

Among the active substances mentioned above, the antibiotic is preferably penicillin, penicillin V, amoxicillin, ampicillin, oxacillin, cloxacillin, procaine penicillin, benzathine penicillin, piperacillin, mezlocillin, ticarcillin, azlocillin, mexacillin, carbenicillin, sulbenicillin, furbenicillin, nafcillin, dicloxacillin, pivampicillin, apacillin, aspoxicillin, pimecrillin, methicillin, lenacilin, fosimicillin, flucloxacillin, kanamycin, natamycin, mitomycin, amikacin, tylosin, verteporfin (Verteporfin), pivalamine sodium, netilmicin sulfate, azithromycin, ofloxacin, ciprofloxacin, enoxacin, lomefloxacin one or more of pefloxacin, rufloxacin, sparfloxacin, fleroxacin, moxifloxacin, grepafloxacin, trovafloxacin, gonoxacin, gemifloxacin, gatifloxacin, tosufloxacin, pazufloxacin, sparfloxacin, clarithromycin, clindamycin, polymyxin, tobramycin, vancomycin, azithromycin, doxycycline, tetracycline, oxytetracycline, minocycline, chlortetracycline, demeclocycline, methacycline, etimicin, netilmicin, sisomicin, amikacin, abekacin, dibekacin, aztreonam, meropenem, imipenem, thienamycin, panipenem, neomycin, paromomycin and spectinomycin.

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

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

In the active substance, the immunosuppressant is preferably one or more of cyclosporine, alprostadil (also called prostate E-1), cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil and mizoribine.

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

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

In the active substance, the gastrointestinal drug is preferably one or more of omeprazole, lansoprazole, ilaprazole, pantoprazole, rabeprazole, terazosin, esomeprazole, tenatoprazole, minoprazole, tenatoprazole, dithioprazole and lafutidine.

In the active substance, the antidepressant is preferably one or more of agomelatine, fluoxetine, paroxetine, duloxetine, sertraline, fluvoxamine, citalopram, escitalopram, venlafaxine, mirtazapine, imipramine, amitriptyline, chlorimipramine, doxepin, remeron, venlafaxine, phenelzine, isocarboxazid and tranylcypromine.

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

In the active substance, the biological agent is preferably one or more of monoclonal antibody drugs, insulin, gamma globulin, antitoxic serum, interferon, interleukin, tumor necrosis factor, skin activity factor, epidermal growth factor, influenza vaccine, hepatitis A vaccine, anti-cancer vaccine, recombinant human acidic fibroblast growth factor and vascular endothelial growth factor 2 monoclonal antibody (VEGFR-2 monoclonal antibody) which are conventional in the field.

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 preferably are one or more of ursolic acid, superoxide dismutase (SOD), bioprotein T4N5, vitamin D2, methyl nicotinate, refined snake oil, hyaluronic acid, essential oils and ceramides.

In the active substance, the substance with health care effect may be a substance with health care effect conventional in the art, and preferably is one or more of glycyrrhizin, glycyrrhizic acid, disodium glycyrrhizinate, methyl glycyrrhizinate, diammonium glycyrrhizinate, vitamin E, resveratrol, coenzyme Q10, silymarin, anthocyanin, procyanidin, lutein, folic acid, folinic acid, curcumin, emodin, tea polyphenol, epigallocatechin gallate (EGCG), catechin, blueberry extract, glutathione and oxymatrine.

In a preferred embodiment of the invention, the active substance is paclitaxel, docetaxel, irinotecan, ribavirin, cytarabine, vincristine sulfate, epidermal growth factor antibody, indomethacin, all-trans retinoic acid, cisplatin, doxorubicin hydrochloride, clarithromycin, cyclosporine, siRNA, amphotericin B, nimodipine, cabazitaxel or erbomycin a.

The invention also provides a preparation method of the liposome loaded with the active substance,

when the liposomes comprise a lyoprotectant, the method of preparing the active agent-loaded liposomes preferably comprises any one of the following methods:

method a comprises the following steps:

(1) Mixing lipid substances, ginsenoside derivatives shown in formula I and the active substances in an organic solvent, optionally adding one or more of cholesterol, fat-soluble antioxidant, soybean oil and/or sodium oleate, fat-soluble surfactant, fat-soluble thermosensitive auxiliary materials, fat-soluble pH-sensitive substances and fat-soluble ion additives to obtain a clarified solution;

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with the aqueous solution of a freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, carrying out ultrasonic treatment, high-pressure homogenization or extrusion pushing through the film, filtering to obtain an aqueous solution of the liposome containing the loaded active substance, and drying to obtain the liposome loaded with the active substance;

the method B comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a membrane, mixing with the aqueous solution of the active substance and the freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ionic additive, performing ultrasonic treatment, high-pressure homogenization or extrusion pushing through the membrane to obtain a liposome solution loaded with the active substance, dialyzing, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

method C comprises the following steps:

(1) Mixing lipid material and ginsenoside derivative shown in formula I in organic solvent, optionally adding one or more of cholesterol, antioxidant, soybean oil and/or sodium oleate, surfactant, thermosensitive adjuvants, pH sensitive material and ion additive to obtain a clear solution;

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a membrane, mixing with an aqueous solution containing ammonium sulfate and a freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, performing ultrasonic treatment, high-pressure homogenization or extrusion on the membrane to obtain a blank liposome solution, dialyzing, mixing with the active substance, filtering to obtain an aqueous solution containing the liposome carrying the active substance, and drying to obtain the liposome carrying the active substance;

method D comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a membrane, mixing with an aqueous solution of citric acid and a freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ionic additive, performing ultrasonic treatment, high-pressure homogenization or extrusion pushing through the membrane to obtain a blank liposome solution, mixing with the active substance and an aqueous solution of disodium hydrogen phosphate, filtering to obtain an aqueous solution of a liposome containing a loaded active substance, and drying to obtain the liposome loaded with the active substance;

when the liposomes do not contain a lyoprotectant, the method for preparing the active agent-loaded liposomes preferably comprises any one of the following methods:

method A1 comprises the following steps:

(1) Mixing lipid material, ginsenoside derivative represented by formula I and the active material in organic solvent, optionally adding one or more of cholesterol, antioxidant, soybean oil and/or sodium oleate, surfactant, thermosensitive adjuvant, pH sensitive material and ionic additive to obtain a clear solution;

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with water, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, carrying out ultrasonic, high-pressure homogenization or extrusion pushing on the film, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

method B1 comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with the active substance, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, carrying out ultrasonic treatment, high-pressure homogenization or extrusion pushing through the film to obtain a liposome solution loaded with the active substance, dialyzing, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

method C1 comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a membrane, mixing with an ammonium sulfate aqueous solution, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, carrying out ultrasonic, high-pressure homogenization or extrusion pushing through the membrane to obtain a blank liposome solution, dialyzing, mixing with the active substance, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

method D1 comprises the following steps:

(2) Removing the organic solvent of the clear solution obtained in the step (1), forming a film, mixing with a citric acid aqueous solution, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ionic additive, carrying out ultrasonic treatment, high-pressure homogenization or extrusion pushing through the film to obtain a blank liposome solution, mixing with the active substance and a disodium hydrogen phosphate aqueous solution, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance.

In method A, B, C, D, A, B1, C1 or D1, the conditions and parameters are as described in the first method or the second method of the blank liposome preparation method.

In A, B, C or D, the lyoprotectant in step (2) may also be added after an aqueous solution containing the active agent-loaded liposomes is obtained and prior to drying.

In the method B, C, B1 or C1, the dialysis can be performed as is conventional in the art of liposome preparation, and the present invention preferably dialyzes the blank liposome solution or the active agent-loaded liposome solution in an aqueous glucose solution (e.g., 0.15 mol/L) or in pure water. The dialysis time may be a time conventional in the art for liposome preparation, preferably 5 to 20 hours, more preferably 12 hours. In the method B, C, B or C1, the dialysis can also be performed prior to sonication, high pressure homogenization, or membrane extrusion.

In method C or C1, the amount of ammonium sulfate in the aqueous solution containing ammonium sulfate and the lyoprotectant or the aqueous solution of ammonium sulfate is not particularly limited, and is an amount conventionally used in the art for preparing liposomal ammonium sulfate gradient. In the aqueous solution containing ammonium sulfate and the freeze-drying protective agent or the aqueous solution of ammonium sulfate, the mass fraction of ammonium sulfate is preferably 1-15%, more preferably 6.6%, and the percentage refers to the mass percentage of ammonium sulfate in the total mass of the aqueous solution.

In the method C or C1, before the filtration operation, it is preferable that an operation of keeping the temperature is further included. The incubation is preferably carried out at 30 ℃ to 80 ℃ (e.g., 37 ℃) for 5 minutes to 1 hour (e.g., 30 minutes).

In method D or D1, the concentration and the amount of the citric acid aqueous solution may not be particularly limited, and are those conventionally used in the art for preparing liposome pH gradient method. In the present invention, the mass fraction of the citric acid aqueous solution is preferably 1% to 15%, more preferably 5.76%, and the percentage refers to the mass percentage of citric acid in the total mass of the citric acid aqueous solution. The concentration and amount of the aqueous solution of disodium hydrogen phosphate may not be particularly limited, and are those conventionally used in the art for preparing liposome pH gradient methods. In the present invention, the mass fraction of the disodium hydrogen phosphate aqueous solution is preferably 5% to 20%, more preferably 7.1%. The aqueous disodium hydrogen phosphate solution is generally used in such an amount that the pH of the aqueous liposome solution (external aqueous phase) containing the loaded active substance is between 6.5 and 7.5 (e.g., 7.3). To achieve the desired pH quickly, pure water is added to bring the pH of the aqueous liposome solution loaded with the active substance to between 6.5 and 7.5 (e.g., 7.3) prior to the filtration operation.

In the method D or D1, before the filtration operation, an operation of keeping the temperature may be further included. The incubation is preferably carried out at 30 ℃ to 80 ℃ (e.g., 60 ℃) for 5 minutes to 1 hour (e.g., 30 minutes).

In each of the above methods, 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 the preparation method of the liposome carrying the active substance, the dosage of the active substance can be the dosage which is conventional in the field, and preferably, the mass ratio of the active substance to the ginsenoside derivative shown in the formula I is 1.1-1, more preferably 1:2-1:6 (for example, 1.

The particle size of the blank liposome or the liposome carrying the active substance can be the conventional particle size in the art, preferably 30-2000nm, more preferably 30-300nm, and most preferably 50-300nm. In the liposome carrying the active substance, the entrapment rate is preferably 80% or more, more preferably 90% or more, and most preferably 95% or more.

When the active substance in the liposome carrying the active substance is a drug, the administration route of the liposome carrying the active substance can be the conventional administration route in the field, preferably injection administration, oral administration or transdermal administration, and is used for the treatment of diseases and/or medical care. Therefore, the liposome loaded with the active substance is generally prepared into the forms of injection, freeze-dried injection, oral solid preparation, oral liquid, liniment, ointment, tincture or aerosol. The injection administration mode is preferably intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. Generally, the liposome loaded with the active substance is added to physiological saline, phosphate buffer solution or 5% glucose aqueous solution to prepare injection solution for injection administration.

In the active substance-loaded liposome, when the active substance is an anti-tumor drug, the active substance-loaded liposome generally has a targeting effect on tumor cells. The invention also provides a ginsenoside derivative shown in the formula I:

wherein R is ¹ 、R ² 、R ⁴ And R ⁵ The definitions of (A) and (B) are the same as described above; r ³ Is composed of

R ⁷ And R ⁸ The definitions of (A) and (B) are the same as described above;

wherein the compound shown in the formula I is not any one of the following compounds:

in a preferred embodiment of the invention, R ³ Preference is given to

More preferably

The ginsenoside derivative shown in the formula I is any one of the following compounds in the following table 3:

TABLE 3

The invention also provides a preparation method of the ginsenoside derivative shown in the formula I, which comprises the following steps: and (2) carrying out deprotection reaction on the acetylation product of the compound shown in the formula I in an organic solvent to obtain the compound shown in the formula I.

The acetylation product of the compound of formula I is: when 20-position of the compound shown as the formula I has hydroxyl, the hydroxyl at the rest positions except the 20-position hydroxyl and acetic anhydride are subjected to esterification reaction to obtain a product; when the compound shown as the formula I has no hydroxyl at the position 20, the hydroxyl at all the positions of the compound and acetic anhydride are subjected to esterification reaction to obtain a product.

The acetyl groups in the acetylation product may also be replaced by hydroxyl protecting groups conventional in the art.

In the deprotection reaction, the organic solvent may be an organic solvent which is conventional for the deprotection reaction, preferably an alcohol solvent and/or an ether solvent, and more preferably a mixed solvent of the alcohol solvent and the ether solvent. In the mixed solvent, the volume ratio of the alcohol solvent to the ether solvent is preferably 1:1. The alcohol solvent is preferably methanol. The ether solvent is preferably dioxane. The amount of the solvent to be used is not particularly limited as long as the reaction proceeds.

The deprotection reaction is preferably carried out under the action of a base. The base is preferably an alkali metal hydroxide, such as potassium hydroxide. The amount of the base to be used is not particularly limited, and it is sufficient to provide a basic environment and remove the hydroxyl protecting group (acetyl group).

The temperature of the deprotection reaction is preferably the solvent reflux temperature at normal pressure, for example, 60 to 110 ℃.

The progress of the deprotection reaction can be monitored according to detection methods conventional in the art, and is preferably used as the end point of the reaction when the acetylation product of the compound shown in formula I disappears. The deprotection reaction time is preferably 8 to 15 hours, for example 10 to 12 hours.

After the completion of the deprotection reaction, the operation preferably includes a post-treatment. The post-treatment preferably comprises the following steps: adjusting pH of the reaction solution to neutral, extracting with organic solvent (such as ester solvent, such as ethyl acetate) preferably for 3 times, mixing organic layers, drying (such as anhydrous sodium sulfate), removing organic solvent, and separating by high pressure chromatography.

In a preferred embodiment of the present invention, the preparation method of ginsenoside Rg5H comprises the following steps: in an organic solvent, the acetylation product of the ginsenoside Rg5H is subjected to deprotection reaction as shown in the specification,

wherein the deprotection reaction conditions are the same as those described above.

The preparation method of the ginsenoside Rg5H acetylation product preferably comprises the following steps: in an organic solvent, carrying out a 20-hydroxyl removal reaction on the acetylation product of the ginsenoside Rg3 to prepare an acetylation product of the ginsenoside Rg 5H;

the dehydroxylation reaction may be carried out by methods and conditions conventional in the art for such dehydroxylation. The following conditions are preferred in the present invention: the organic solvent is preferably a halogenated hydrocarbon solvent, such as methylene chloride. The amount of the organic solvent to be used is not particularly limited as long as the reaction proceeds. The reaction for removing the 20-position hydroxyl is preferably carried out under the action of boron trifluoride diethyl etherate and triethylsilane. The mass ratio of boron trifluoride diethyl etherate to triethylsilane is preferably 1.5 to 1:5, for example 1:1. The mass ratio of triethylsilane to Rg3 acetylation product is preferably 1:5-1, e.g. 1. The temperature for the reaction for removing the 20-hydroxyl is preferably room temperature. The process of the reaction for removing the 20-hydroxyl can be monitored according to a conventional detection method in the field, and the process is preferably used as the end point of the reaction when the acetylation product of the ginsenoside Rg3 disappears. The reaction time for removing the 20-hydroxyl is preferably 5 to 10 minutes.

After the reaction for removing the 20-position hydroxyl group is completed, the operation preferably includes a post-treatment. The post-treatment preferably comprises the following steps: the reaction mixture was mixed with ice water, quenched, washed with saturated sodium bicarbonate solution (three times), dried (e.g., anhydrous sodium sulfate), and the organic solvent was removed.

Similarly, by adopting the method, ginsenoside Rh3H, ginsenoside Rh4H and ginsenoside Rk4H can be prepared and replaced by corresponding raw materials.

In a preferred embodiment of the present invention, the preparation method of the acetylation product of ginsenoside Rg5H comprises the following steps: in an organic solvent, under the action of a catalyst, performing acylation reaction on ginsenoside Rg3 (racemate, R or S type) and acetic anhydride as shown in the specification to obtain an acetylated product of ginsenoside Rg 3;

the acetylation reaction may be any method and conditions conventional in the art for such acetylation reactions. The following conditions are preferred in the present invention: the organic solvent is preferably a basic organic solvent, such as pyridine. The alkaline organic solvent is used as an organic solvent, preferably an acid-binding agent. In a preferred embodiment of the invention, it is also possible to use conventional non-basic organic solvents, with the addition of an acid-binding agent. The amount of the organic solvent to be used is not particularly limited as long as the reaction proceeds. The acetylation reaction is preferably carried out under the action of the catalyst DMAP. The catalyst is generally used in catalytic amounts. The mass-volume ratio of the ginsenoside Rg3 to the acetic anhydride is preferably 0.1g/mL-5g/mL, such as 1g/mL. The temperature of the acetylation reaction is preferably room temperature. The acetylation process can be monitored according to conventional detection methods in the art, and preferably, the end point of the reaction is the time when the ginsenoside Rg3 disappears. The time for the acetylation reaction is preferably 8 to 15 hours, for example 10 hours.

After the acetylation reaction is completed, the operation preferably includes a post-treatment. The post-treatment preferably comprises the following steps: the reaction mixture was concentrated under reduced pressure, the solvent was removed, and the reaction mixture was washed with a saturated sodium bicarbonate solution (three times), dried (for example, anhydrous sodium sulfate), and concentrated to dryness.

Similarly, by adopting the method, the acetylation product of ginsenoside Rh3H, the acetylation product of ginsenoside Rh4H and the acetylation product of ginsenoside Rk4H can be prepared and replaced by corresponding raw materials.

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 lyoprotectant solution or the aqueous active substance solution is calculated as 1g/mL (i.e., the density of water), and thus the total mass m = ρ × V of the aqueous lyoprotectant solution or the aqueous active substance solution.

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 1.1g/mL.

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

The positive progress effects of the invention are as follows:

the ginsenoside derivative disclosed by the invention is good in activity and low in hemolysis, can be used as a liposome membrane material to prepare blank liposomes, and meanwhile, part of compounds are novel in structure. The hemolysis of the liposome prepared by the method meets the requirements, and the liposome has higher safety, better film-forming property and better stability, and has important application value. The blank liposome has the advantages of high efficiency, safety, stability, strong targeting property, good uniformity, stable and reliable quality and simple and convenient preparation process. The blank liposomes of the present invention can be used to prepare active agent loaded liposomes by entrapping the active agent in the membrane. When the active substance is an anti-tumor drug, the liposome loaded with the active substance generally has the targeting effect on tumor cells, the multi-drug resistance effect, the synergy and the attenuation and the drug synergistic effect.

Drawings

Fig. 1 is a graph of cell survival rates of Rg5H empty, rg5 empty, rp1 empty, rg3H empty, and GQ empty for human lung cancer cells (a 549).

FIG. 2 is a graph showing the cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H and Taxol + GQ for human lung cancer cells (A549)

FIG. 3 is a graph showing the cell survival rate of Rg5H null, rg5 null, rp1 null, rg3H null and GQ null human lung cancer paclitaxel resistant strain (A549/T)

FIG. 4 is a graph showing the cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H and Taxol + GQ against human lung cancer Taxol-resistant strain (A549/T).

FIG. 5 is an in vivo profile of control, ginsenoside Rg5H, rg, rp1, rg3H, GQ liposomes at 2, 6 and 10h with a living body imager to record IR783 fluorescence; FIGS. 5-A, 5-B and 5-C are in vivo profiles of IR783 fluorescence recorded by a Living body imager at 2, 6 and 10h for the control group, respectively; FIG. 5-S shows a fluorescence scale, which is red-yellow-green-blue in order of the intensity of fluorescence, red showing the intensity of fluorescence, and blue showing the intensity of fluorescence. FIGS. 5-D to 5-R are in vivo distribution plots of IR783 fluorescence recorded by the in vivo imager in the experimental groups at 2, 6 and 10 h. Wherein, fig. 5-D, fig. 5-E and fig. 5-F are ginsenoside Rg5H experimental groups respectively; FIG. 5-G, FIG. 5-H and FIG. 5-I are ginsenoside Rg5 experimental groups, respectively; FIGS. 5-G, 5-K and 5-L are experimental groups of ginsenoside Rp1, respectively; FIG. 5-M, FIG. 5-N and FIG. 5-O are experimental groups of ginsenoside Rg3H, respectively; FIG. 5-P, FIG. 5-Q and FIG. 5-R are the experimental groups of pseudo-ginsenoside GQ, respectively.

FIG. 6 is a graph showing the ex vivo fluorescence profiles of liposomes after 12h injection into mice; A. b, C, D, E and F are control group, ginsenoside Rg5H, rg, rp1, rg3H, GQ liposome respectively.

FIG. 7 is a tumor-suppressing graph of human lung cancer cell A549 in Control group, taxol + Rg5H group, taxol + Rg5 group, taxol + Rp1 group, taxol + Rg3H group, and Taxol + GQ group.

FIG. 8 is a tumor-suppression curve chart of the Control group, the Taxol + Rg5H group, the Taxol + Rg5 group, the Taxol + Rp1 group, the Taxol + Rg3H group and the Taxol + GQ group for the paclitaxel resistant strain (A549/T) for human lung cancer.

Fig. 9 is a graph of cell viability for human breast cancer cells (MCF-7) for Rg5H empty, rg5 empty, rp1 empty, rg3H empty, and GQ empty.

FIG. 10 is a graph of cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, taxol + GQ for human breast cancer cells (MCF-7).

FIG. 11 is a graph of cell viability of Rg5H null, rg5 null, rp1 null, rg3H null, and GQ null against human mammary-resistant paclitaxel-resistant strains (MCF-7/T).

FIG. 12 is a graph of cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H and Taxol + GQ for human breast paclitaxel-resistant strains (MCF-7/T).

FIG. 13 is a graph showing tumor suppression curves of human breast cancer cells (MCF-7) in the Control group, the Taxol + Rg5H group, the Taxol + Rg5 group, the Taxol + Rp1 group, the Taxol + Rg3H group and the Taxol + GQ group.

FIG. 14 is a tumor suppression graph of paclitaxel-resistant strains (MCF-7/T) in human mammary gland for Control group, taxol + Rg5H group, taxol + Rg5 group, taxol + Rp1 group, taxol + Rg3H group, and Taxol + GQ group.

Detailed Description

1. Experimental drugs: 20 (S) -Panaxadiol PD, 20 (R) -Panaxadiol PD, 20 (S) -Protopanaxadiol PPD, 20 (R) -Protopanaxadiol PPD, pseudoprotopanaxadiol PPD, 20 (S) -panaxatriol PT, 20 (R) -panaxatriol PT, 20 (S) -protopanaxatriol PPT, 20 (R) -Propanaxatriol PPT, 20 (S) -ginsenoside Rg3, 20 (R) -ginsenoside Rg3, isoginsenoside Rg3, 20 (S) -ginsenoside Rh2, ginsenoside Rg5, 20 (S) -ginsenoside Rg2, and pseudoginsenoside PF11 are commercially available in the art, such as Shanghai plain pharmaceuticals, suzhou Xinghai jin pharmaceuticals, shanghai plain leaf biotechnology, etc., or prepared according to methods known in the art.

Some of the ginsenoside derivatives mentioned in the following examples can also be prepared according to methods known in the art, for example, the preparation method of 25-hydroxy-ginsenoside Rg3 (25-OH-Rg 3) can be referred to the method in the thesis of "ingredient research of saponin degradation product of stem and leaf diol group of panax quinquefolius" (2009, yu Zhibo, university of Jilin university); 20 (S) -methyl-protopanaxadiol and 12,20-dimethoxy-protopanaxadiol (12,20-DiMe-PPD) were prepared by the method of example 5 with reference to CN 2010107476.7; the preparation methods of 20-methyl-ginsenoside Rg3 (20-Me-Rg 3), 12-acetyl-20-methyl-ginsenoside Rg3 (12-Ac-20-Me-Rg 3) and 12-acetyl-20-methyl-ginsenoside Rh2 can refer to methods in 2015, university of Chinese science and technology Yu Junbo, the gold catalyzed glycosylation method applied to the glycosylation of oxime and the synthesis of ginsenoside Rb 2.

In table 4, the carbon marked with an x is a chiral carbon, which is in S configuration or R configuration. In addition, when R is ³ When there is a double bond, E-type or Z-type is present.

TABLE 4

2. The instruments used in the following examples are the instruments owned by Shanghai Benzhen medicine science and technology Co., ltd, and the model numbers and source information of the instruments are as follows:

and (3) preparing a chromatogram: HB-DAC-100 separation system, NP 7060X 2 platform, NP 7010X 1 platform, NU 3000X 1 platform, DAC-100mm column one, ontai 3300ELSD detector, jiangsu Hanbang science and technology Limited;

agilent liquid chromatography: agilent 1100, autai 3300ELSD, agilent technologies (China) Inc.;

and (3) rotary evaporation of an evaporator: ZX98-1 5L, shanghai Luyi Industrial trade, inc.;

20L rotary evaporator: R5002K, shanghai xiafeng industries ltd;

a freeze dryer: FD-1D-80, shanghai Bilang instruments manufacturing Co., ltd;

a freeze dryer: PDFD GLZ-1B, pudong Freeze drying Equipment, inc., shanghai;

an electronic balance: CPA2250 (accuracy 0.00001 g), sartorius trade ltd;

an electronic balance: JY3003 (precision 0.001 g), shanghai Shunhui constant-level scientific instruments, inc.

3. The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions. 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. The reflux temperature, unless otherwise specified, is a problem of solvent reflux.

4. The high pressure chromatographic separation equipment in the following examples is a preparative chromatography system for Hanbang DAC-100 listed above; the filler is C18 filler of 10um-120A of Japanese Fuji (Fuji); the detector is an evaporative light diffuser (ELSD); the flow rate is 400ml/min; the working pressure is 8MPa; ultraviolet 203nm; the mobile phase has 2 gradients, 75% methanol water and over 95% methanol water.

The method comprises the following specific operations: the sample to be separated is dissolved in an appropriate amount of 75% methanol water, equilibrated with 75% methanol water for 20 minutes, loaded, eluted with 75% methanol water for about 100 minutes, collected in fractions, and then washed with 95% or more methanol water for about 20 minutes until the TLC spot plate is free of black spots.

Wherein, the X% methanol water is the volume percentage of the methanol volume in the total volume of the methanol water. For example, 75% methanol water means that the volume percent of methanol to the volume of methanol water is 75%.

20L rotary evaporator: R5002K, shanghai xiafeng industries ltd;

a freeze dryer: FD-1D-80, shanghai Bilang instruments manufacturing Co., ltd;

a freeze dryer: PDFD GLZ-1B, pudong Freeze drying Equipment, inc., shanghai;

an electronic balance: CPA2250 (precision 0.00001 g), sartorius trade ltd;

3. 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. 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 a standard atmospheric pressure. The reflux temperature, unless otherwise specified, is a problem of solvent reflux.

4. The high pressure chromatographic separation equipment in the following examples was a preparative chromatography system for the Hanbang DAC-100 listed above; the filler is C18 filler of 10um-120A of Japanese Fuji (Fuji); the detector is an evaporative light diffuser (ELSD); the flow rate is 400ml/min; the working pressure is 8MPa; ultraviolet 203nm; the mobile phase has 2 gradients, 75% methanol water and over 95% methanol water.

Wherein, the X% methanol water is the volume percentage of the methanol volume in the total volume of the methanol water. For example, 75% methanol water means that the volume of methanol is 75% by volume of the volume of methanol water.

1. Preparation of ginsenoside derivatives

Preparation example 1 preparation of Isoginsenoside Rg3 (O)

Dissolving 10g of isoginsenoside Rg3 (E type) in 20mL of pyridine, dropwise adding acetic anhydride in an ice water bath, controlling the reaction temperature to be not more than 5 ℃, reacting for 10 hours, detecting the disappearance of a raw material point by TLC, then pouring the reaction liquid into ice water, extracting for 3 times by 200 mL/time of ethyl acetate, combining organic phases, washing for 3 times by 200 mL/time of saturated NaCl, drying by using a proper amount of anhydrous sodium sulfate, and concentrating under reduced pressure to be dry to obtain an isoRg 3 (E type) acetylation product.

5g of the acetylated product of the iso Rg3 (E type) peroxide is dissolved in 50mL (THF: water 1:1), 3mL of 33% hydrogen peroxide and 2mL of 2M sodium hydroxide are added, the mixture is reacted at room temperature overnight, TLC detection is carried out until the raw material point disappears, the mixture is cooled, the mixture is washed for 3 times by 100 mL/time saturated sodium bicarbonate solution, anhydrous sodium sulfate is dried, and the mixture is concentrated to dryness under reduced pressure, so that the acetylated product of the iso Rg3 peroxide (E type) is obtained.

Dissolving 1g of acetylated product of peroxyisoginsenoside Rg3 (E type) in 10mL of methanol, adding 0.5g of sodium methoxide, stirring and dissolving, reacting at room temperature for 10 hours, detecting by TLC until the raw material point disappears, extracting with 50 mL/n-butanol for 3 times, combining organic phases, drying with appropriate amount of anhydrous sodium sulfate, concentrating under reduced pressure to dryness, crystallizing with appropriate amount of methanol for 2 times, and drying to obtain over 0.3g98% of peroxyisoginsenoside Rg3 (E type).

¹ H NMR(δ,500M):5.35(1H,d,J＝7.5Hz)，5.08(1H,t,J＝7.0Hz),4.88(1H,d,J＝7.5Hz),4.52(1H,m),4.44-4.48(2H,m),4.23-4.33(5H,m),4.11-4.13(2H,m),3.90-3.91(3H,m),3.26(1H,m)，2.27-2.59(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),1.57(3H,s),1.56(3H,s),1.35-1.50(8H,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,0.78(3H,s),0.73(1H,m).

¹³ C NMR(δ,125M):138.7,126.4,106.2,105.2,89.1,83.5,81.3,78.5,78.3,78.1,78.0,71.9,71.2,63.1,63.0,77.4,56.6,55.0,51.9,50.6,49.4,48.8,40.2,39.9,39.4,37.1,35.4,32.3,31.6,30.0,29.8,27.3,26.9,26.9,25.9,23.2,18.7,17.9,17.1,16.8.

ESI:802.31(M+H) ⁺ ,HR-ESI-MS:802.3112.07219(M+H) ⁺ ,(C ₄₂ H ₇₃ O ₁₄ ),Cal 802.3120.

Preparation example 2 preparation of pseudo-ginsenoside GQ, pseudo-ginsenoside HQ and pseudo-ginsenoside DQ (cf. CN201010204031.0 example 1)

Dissolving 10g of 20 (S) -ginsenoside Rg3 in 500mL of 1,4-dioxane, and adding concentrated sulfuric acid (H) ₂ SO ₄ ) The pH was adjusted to =3, 50mL of m-chloroperbenzoic acid was added dropwise with stirring, the mixture was heated at 75 ℃ under reflux for 90 minutes, and TLC was controlled until the starting material spot disappeared. After completion of the reaction, the reaction mixture was cooled to room temperature, adjusted to pH =7.0 with 0.1M NaOH aqueous solution, and filteredAnd concentrating the filtrate to dryness to obtain 8.5g of crude pseudo-ginsenoside GQ product. And eluting with C18 as filler, detecting with methanol water, detecting with Evaporative Light Scattering Device (ELSD), separating by high pressure chromatography, and concentrating product segment to dry to obtain 4.2g (20S, 24S) -ginsenoside GQ with HPLC purity of above 98% and 0.2g (20S, 24R) -ginsenoside GQ.

Respectively replacing 20 (S) -ginsenoside Rg3 raw material with 20 (S) -ginsenoside Rh2 and 20 (S) -protopanaxadiol PPD, and respectively preparing (20S, 24S) -pseudo ginsenoside HQ and (20S, 24R) -pseudo ginsenoside HQ, (20S, 24S) -pseudo ginsenoside DQ and (20S, 24R) -pseudo ginsenoside DQ by the same method.

Preparation example 3 preparation of ginsenoside Rp1

Dissolving 1.5g of ginsenoside Rg5 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 crude product ginsenoside Rp1 is obtained. Separating by high pressure chromatography, eluting with methanol water gradient with C18 as filler, detecting with Evaporative Light Scattering Device (ELSD), and concentrating product segment to dry to obtain ginsenoside Rp1 with purity of 98% or more by HPLC of 0.36g + 0.55g.

The ginsenoside Rp1 is added into the Chinese medicinal composition,

¹ H NMR(δ,500M):5.38(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(4H,m),2.17(2H,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＝2.8Hz),1.35-1.50(8H,m),1.28(3H,s),1.20(1H,m),1.09(6H,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).

¹³ C NMR(δ,125M):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:772.03(M+H) ⁺ ,HR-ESI-MS:772.0312(M+H) ⁺ (C ₄₂ H ₇₅ O ₁₂ ),Cal 772.0315.

preparation example 4 preparation of Isoginsenoside Rg3H

According to the same method of preparation example 3, the acetylation product of the isoginsenoside Rg3 is taken as the initial raw material, and after hydrogenation, post-treatment and high pressure chromatography separation, the refined product of the isoginsenoside Rg3H is obtained.

The content of the isoginsenoside Rg3H,

¹ H 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).

¹³ C 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) ⁺ ,(C ₄₂ H ₇₃ O ₁₃ ),Cal 788.0728.

preparation example 5 preparation of pseudo-ginsenoside GD

Weighing 10g of 20 (S) -ginsenoside Rg3, dissolving in 100mL of dichloromethane and 100mL of trifluoroacetic acid mixed solution, adding 20mL to 30% ₂ O ₂ The solution was reacted at room temperature for 1 hour, and TLC detected until the starting material spot disappeared. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust pH =7.0, ethyl acetate was extracted 3 times (500 mL/time), and the organic phases were combined and concentrated to dryness under reduced pressure. Separating by high pressure chromatography, eluting with C18 as filler and methanol water, evaporating light diffuser (ELSD), and concentrating product segment to dry to obtain 2.7g of 20 (S) -pseudo-ginsenoside GD with purity of 98% or more.

Changing the raw material into 20 (R) -ginsenoside Rg3 according to the same method to obtain 20 (R) -pseudo-ginsenoside GD.

20 (S) -pseudo-ginsenoside GD, which is a saponin,

¹ H NMR(δ,500M)5.25(1H,d,J＝7.8Hz),4.81(1H,m),4.45(1H,m),4.36(2H,m),4.20(1H,m),4.13(1H,m),4.12(1H,m),4.11(1H,m),4.10(1H,m),4.09(1H,m),3.81(2H,m),3.63(1H,m),3.17(1H,m),2.10(1H,m),2.05(1H,m),2.03(2H,m),1.92(1H,m),1.81(3H,m),1.70(2H,m),1.56(3H,s),1.46(1H,m),1.41(3H,s),1.34(5H,m),1.27(4H,m),1.23(4H,m),1.17(3H,s),1.10(1H,m),0.99(3H,s),0.86(4H,m),0.82(3H,s),0.69(3H,s),0.64(1H,m).

¹³ C NMR:106.2,105.2,89.0,83.6,78.6,78.4,78.2,78.1,78.0,77.3,74.5,71.8,71.7,70.6,62.9,62.8,56.5,52.3,52.2,50.5,49.3,39.8,40.2,39.3,37.0,35.2,32.3,33.2,32.0,30.2,28.2,27.8,26.8,26.5,26.0,23.3,18.6,16.7,16.6,15.8.

ESI-MS:m/z 786.03[M+1] ⁺

20 (R) -pseudo-ginsenoside GD,

ESI-MS:m/z 786.03[M+1] ⁺ .

preparation example 6 preparation of pseudo-ginsenoside GHD

The 20 (S) -pseudo-ginsenoside GHD is obtained by replacing the raw material of example 5 with 20 (S) -ginsenoside Rh 2.

ESI-MS:m/z 786.03[M+1] ⁺ .

Preparation example 7 preparation of methoxy-Isoginsenoside Rg3 (refer to CN201010107476.7 example 5)

Weighing 10g of acetylated product of the isoginsenoside Rg3 (E type), dissolving in 500mL of THF, adding 6g of NaH, adding 18mL of iodomethane after the NaH is completely dissolved, heating to 70 ℃ for reaction for 4 hours, monitoring by TLC until the raw materials disappear, filtering after the reaction is finished, and concentrating the filtrate under reduced pressure until the filtrate is dried to obtain the acetylated product of the 25-methoxy-isoginsenoside Rg 3.

Dissolving 5g of acetylated 25-methoxy-isoginsenoside Rg3 product in 50ml of dioxane and 25ml of ethanol solution, adding 10ml of 50% sodium hydroxide aqueous solution, heating in a constant-temperature water bath at 80 ℃ for 2 hours for reaction, performing TLC (thin layer chromatography) control until the raw material point disappears, extracting with 100 ml/n-butanol for 3 times, combining organic phases, washing with 100 ml/saturated NaCl aqueous solution for 3 times, drying with a proper amount of anhydrous sodium sulfate, reducing pressure, concentrating to dryness, and drying to obtain a crude 25-methoxy-isoginsenoside Rg3 product. Purifying with column to obtain 25-methoxyl-isoginsenoside Rg3 (E type).

¹ H 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)，3.21(3H,s),2.28-2.59(2H,m),2.17(1H,m),1.99-2.02(3H,m),1.80-1.89(2H,m),1.57(3H,s),1.56(3H,s),1.41(6H,s),1.35-1.50(8H,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).

¹³ C NMR(δ,125M):138.8,126.5,106.3,105.3,89.2,83.6,82.1,78.6,78.4,78.2,78.0,71.9,71.2,63.1,63.0,77.4,56.6,55.0,51.9,50.6,49.4,48.8,48.0,40.2,39.9,39.4,37.1,35.4,32.3,31.6,30.0,29.8,27.3,26.9,26.9,25.9,23.2,18.7,17.9,17.2.

ESI:800.07(M+H) ⁺ ,HR-ESI-MS:800.0707(M+H) ⁺ ,(C ₄₃ H ₇₅ O ₁₃ ),Cal 800.0712.

Preparation example 8 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%.

¹ H 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).

¹³ C NMR(C ₅ D ₅ N,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 example 9 preparation of ginsenoside Rg5H

Dissolving 10g of 20 (R) -ginsenoside Rg3 in 20mL of pyridine, dropwise adding 10mL of acetic anhydride in ice-water bath, then adding an appropriate amount (generally catalytic amount, for example 1 g) 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 using 200 mL/ethyl acetate for 3 times, combining organic phases, washing by using 2M hydrochloric acid for 3 times, washing by using saturated sodium bicarbonate for 3 times, drying by using anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain an Rg3 acetylation 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, continuously reacting for 5-10min at the room temperature, cooling to 0 ℃, adding ice water to quench the reaction, washing for 3 times by using 100mL of saturated sodium bicarbonate solution, drying by using anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain the Rg5H acetylated product.

10g of Rg5H acetylation product was taken and dissolved in 50mL of methanol and dioxane 1:1, adding 2g of potassium hydroxide, 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 is =7, extracting by using 100 mL/ethyl acetate 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, separating by using C18 chromatography, 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 Rg5H.

Ginsenoside Rg5H:

¹ H 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).

¹³ C 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(C ₄₂ H ₇₂ NaO ₁₂ ),Cal 792.0230.

preparation example 10 preparation of ginsenoside Rg5H

Dissolving 10g of 20 (S) -ginsenoside Rg3 in 20mL of THF, dropwise adding 10mL of acetic anhydride in an ice-water bath, then adding an appropriate amount (catalytic amount, such as 1 g) 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 using 200 mL/ethyl acetate for 3 times, combining organic phases, washing by using 2M hydrochloric acid for 3 times, washing by using saturated sodium bicarbonate for 3 times, drying by using anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain an Rg3 acetylation product.

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

10g of Rg5H acetylation product was taken and dissolved in 50mL of methanol and dioxane 1:1, adding 2g of potassium hydroxide, 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 is =7, extracting by using 100 mL/ethyl acetate for 3 times, combining organic phases, drying by using anhydrous sodium sulfate, concentrating under reduced pressure until the raw material point is dry, separating by high pressure chromatography, performing gradient elution by using C18 as a filler and methanol water, evaporating a light diffuser (ELSD), and concentrating a product section until the product section is dry to obtain the ginsenoside Rg5H.

Preparation example 11 preparation of the ginsenosides 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 a 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 a raw material point disappears, cooling, washing for 3 times by 100 mL/saturated sodium bicarbonate solution, drying by anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain a crude acetylated product mixture (an 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 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) of the product.

Weighing 5g of crude product mixture, separating by high pressure chromatography, eluting with C18 as filler and methanol water gradient, evaporating the diffuser (ELSD), 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%.

Ginsenoside Rg5H1 (Z)

¹ H 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).

¹³ C 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(C ₄₂ H ₇₃ O ₁₂ ),Cal 770.0315.

Ginsenoside Rg5H1 (E)

¹ H 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).

¹³ C 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.

Ginsenoside Rk1H

¹ H 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).

¹³ C 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.

Preparation example 12 preparation of ginsenoside Rg5H1 (E), rg5H1 (Z), rk1H

Weighing 10g of 20 (S) -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 a raw material point disappears, filtering out the palladium-carbon, concentrating under reduced pressure to remove the methanol, and drying to obtain the 20 (S) -Rg3 acetylated hydrogenation product.

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

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 the crude product mixture of the product.

Weighing 5g of the crude product mixture, separating by high pressure chromatography, eluting with C18 as filler and methanol water gradient, evaporating the diffuser (ELSD), concentrating the product section to dryness, and respectively obtaining 1.6g of Rg5H1 (E) with HPLC purity of more than 98%, 0.3g of Rg5H1 (Z) with HPLC purity of more than 98% and 0.9g of Rk1H with HPLC purity of more than 98%.

Preparation example 13 preparation of ginsenoside Rh3H

Ginsenoside Rh3H was obtained by the same method as in preparation examples 9 and 10, using 20 (R) -Rh2 and 20 (S) -Rh2 as the starting materials, respectively.

The ginsenoside Rh3H is added into the ginsenoside,

¹ H 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).

¹³ C 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(C ₃₆ H ₆₇ O ₇ ),[M+H] ⁺ ,Cal 607.8915.

preparation example 14 preparation of ginsenoside Rh3H1 (E), ginsenoside Rh3H1 (Z) and ginsenoside Rk2H

Ginsenoside Rh3H1 (E), ginsenoside Rh3H1 (Z) and ginsenoside Rk2H with HPLC purity of over 98% are obtained by the same method as preparation examples 11 and 12 and by using 20 (R) -Rh2 acetylated product or 20 (S) -Rh2 acetylated product as raw material.

Ginsenoside Rh3H1 (E),

¹ H 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).

¹³ C 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),

¹ H 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

¹ H 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).

¹³ C 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 15 preparation of ginsenoside Rh4H

Ginsenoside Rh4H was obtained by the same method as in preparation examples 9 and 10, using 20 (R) -Rh1 and 20 (S) -Rh1 as the starting materials, respectively.

The ginsenoside Rh4H is added into the ginsenoside Rh4H,

¹ H 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).

¹³ C 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(C ₃₆ H ₆₇ O ₇ ),[M+H] ⁺ ,Cal 770.0328.

preparation example 16 preparation of ginsenoside Rh4H1 (E), ginsenoside Rh4H1 (Z), ginsenoside Rk3H

Ginsenoside Rh4H1E, ginsenoside Rh4H1Z and ginsenoside Rk3H with HPLC purities of more than 98% are respectively obtained by the same methods as examples 11 and 12, wherein the raw materials are 20 (R) -Rh1 acetylation product and 20 (S) -Rh1 acetylation product.

Ginsenoside Rh4H1 (E)

¹ H 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).

¹³ C 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.

Ginsenoside Rh4H1 (Z),

¹ H 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),0.8(3H,d,J＝10.5Hz).

¹³ C 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.

the ginsenoside Rk3H is added into the Chinese medicinal composition,

¹ H 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),2.59(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),1.63(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).

¹³ C 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 17 preparation of ginsenoside Rk4H

Ginsenoside Rk4H was obtained by the same methods as examples 9 and 10 using 20 (R) -Rg2 and 20 (S) -Rg2 as the starting materials, respectively.

The ginsenoside Rk4H is extracted from the ginseng,

preparation example 18 preparation of ginsenoside Rk4H1 (E), ginsenoside Rk4H1 (Z) and ginsenoside Rg6H

By the same method as in examples 11 and 12,20 (R) -Rg2 acetylated product and 20 (S) -Rg2 acetylated product are used as raw materials, and ginsenoside Rk4H1 (E), ginsenoside Rk4H1 (Z) and ginsenoside Rg6H with HPLC purity of more than 98% are respectively obtained.

Ginsenoside Rh4H1E

The ginsenoside Rh4H1Z is added into the raw materials of the ginseng,

¹ H 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).

ginsenoside Rk3H

¹ H 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).

Preparation example 19 preparation of (20S, 24S) -3-sodium sulfate-pseudo-ginsenoside DQ (SC-DQ)

Weighing 10g (20S, 24S) -pseudo-ginsenoside DQ, dissolving in 100mL pyridine, placing the reaction bottle in a saline-ice bath, cooling to 0 ℃, slowly adding 30mL chlorosulfonic acid dropwise, reacting at room temperature for 2 hours, and detecting by TLC until the raw material point disappears. After the reaction is finished, 0.1M NaOH is added to adjust the pH =7.0, n-butanol is extracted for 3 times, organic phases are combined, and the organic phases are concentrated to be dry under reduced pressure. Separating by high pressure chromatography, eluting with methanol water gradient with C18 as filler, evaporating Light Scatterer (ELSD), and concentrating product segment to dry to obtain 11.4g (20S, 24S) -3-sodium sulfate-pseudo-ginsenoside DQ with HPLC purity above 98%.

¹ H NMR(δ,500M):6.76(1H,s),5.48(1H,t,J＝7.0Hz),3.96(1H,m),3.80(1H,m),2.69(1H,m),2.25(2H,m),1.89(3H,m),1.88(1H,m),1.78(2H,m),1.72(3H,s),1.61(4H,m),1.38(2H,m),1.37(2H,m),1.35(1H,m),1.24(6H,s),1.12(2H,m),1.11(3H,s),0.95(1H,m),0.95(3H,s),0.93(3H,s),0.84(3H,s),0.79(3H,s),0.72(1H,m).

¹³ C NMR(δ,125M):139.7,125.7,78.1,72.8,69.7,56.6,51.7,51.1,51.0,44.4,40.4,39.7,37.6,35.6,32.8,32.4,30.2,29.0,28.9,28.4,23.8,18.9,17.2,16.7,16.5,16.0,13.2.

LRMS(ESI):539.7966[C ₃₀ H ₅₁ O ₆ S])；HRMS(ESI):found 539.7958,[C ₃₀ H ₅₁ O ₆ S]).

Preparation example 20 preparation of sodium sulfate-ginsenoside Rg5H (SC-Rg 5H)

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 with the HPLC purity of more than 98%.

By the same method as in example 19, the 3-sodium sulfate-ginsenoside Rg5H was obtained as the raw material Rg5H.

¹ H 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).

¹³ C 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[C ₃₀ H ₅₁ O ₆ S])；HRMS(ESI):found 523.7923,[C ₃₀ H ₅₁ O ₆ S]).

Preparation example 21 preparation of sodium sulfate-Isoprotopanaxadiol PPD (SC-Iso-PPD)

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 is finished, 0.1M NaOH is added to adjust the pH =7.0, n-butanol is extracted for 3 times, organic phases are combined, and the organic phases are concentrated to be dry under reduced pressure. Separating by high pressure chromatography, eluting with C18 as filler and methanol water, 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%.

¹ H 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).

¹³ C 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 of example 22, 6-sodium dithionate-pseudo-ginsenoside TQ (SC-TQ) (20S, 24S)

The same procedure as in preparation example 19 was used, starting from 20 (S) -pseudoginsenoside TQ, to give 3,6-sodium dithioate-pseudoginsenoside TQ (SC-TQ).

¹ H NMR(δ,500M):3.87(1H,m),3.83(1H,m),3.51(1H,m),2.24(1H,m),1.92(2H,m),1.73(1H,m),1.68(1H,m),1.62(2H,m),1.55(1H,m),1.51(1H,m),1.46(2H,m),1.43(1H,m),1.29(1H,m),1.26(3H,s),1.22(1H,m),1.15(1H,m),1.09(3H,s),1.08(1H,m),1.00(4H,m),0.97(3H,s),0.90(3H,s),0.88(3H,s),0.77(3H,s),0.73(1H,m).

¹³ C NMR(δ,125M):87.5,87.2,78.9,78.7,70.6,70.1,56.1,52.2,50.3,49.0,48.9,39.8,39.0,37.3,34.9,32.3,31.7,28.9,28.6,28.0,27.5,25.1,24.3,18.4,17.3,16.3,15.5,15.3.

ESI-Ms:650.84(M-2Na) ^- .

Preparation example 23 preparation of 3- (N, N-dimethylaminoethyl) -carbamoyl-pseudoginsenoside DQ (DC-DQ)

Weighing 10g of pseudo-ginsenoside DQ, dissolving in 200mL of dry dichloromethane, adding 10g of DMAP, cooling to 0 ℃ in an ice bath, dropwise adding 50mL of triphosgene (10 g) 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 stop the reaction, extracting for 3 times by ethyl acetate, combining organic phases, concentrating under reduced pressure to dryness, crystallizing for 2 times by ethanol, and drying to obtain 6.5g of 3-chloroformyl-pseudo-ginsenoside DQ.

Weighing 5g of 3-chloroformyl-pseudo-ginsenoside DQ, dissolving in 50mL of dichloromethane, cooling to 0 ℃ in 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 3- (N, N-dimethylaminoethyl) -carbamoyl-pseudoginsenoside DQ.

¹ H NMR(δ,500M):3.82(1H,m),3.81(1H,m),3.36(2H,t,J＝7.2Hz),2.70(1H,m),2.44(2H,t,J＝7.2Hz),2.26(6H,s),2.24(1H,m),1.92(2H,m),1.73(1H,m),1.68(1H,m),1.62(2H,m),1.55(1H,m),1.51(1H,m),1.46(2H,m),1.43(1H,m),1.29(2H,m),1.26(3H,s),1.22(1H,m),1.15(1H,m),1.09(3H,s),1.08(1H,m),1.00(4H,m),0.97(3H,s),0.90(3H,s),0.88(3H,s),0.77(3H,s),0.73(1H,m).

¹³ C NMR(δ,125M):156.1,87.5,87.2,78.9,70.6,70.1,56.1,52.2,50.3,49.0,48.9,46.7,39.8,39.0,37.3,34.9,32.3,31.7,28.9,28.6,28.0,27.5,25.1,24.3,18.4,17.3,16.3,15.5,15.3.

ESI-Ms:591.90(M+H) ⁺ .

Preparation example 24 preparation of 3- (N, N-dimethylaminoethyl) -carbamoyl-ginsenoside Rg5H

By the same method as in example 23, rg5H was used as the starting material to obtain DC-ginsenoside Rg5H.

¹ H 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).

¹³ C 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(C ₃₅ H ₆₃ N2O ₃ ),Cal 559.9020.

2. Preparation of liposomes

Example 1 preparation of ginsenoside Rg5H blank liposome

Adding 0.9g of egg yolk lecithin, 0.4g of ginsenoside Rg5H and 0.1g of vitamin E into 20mL of chloroform, stirring at room temperature to form a clear solution, removing an organic solvent by rotary evaporation in a constant-temperature water bath at 40-50 ℃, forming a film, adding 20mL of 5% glucose aqueous solution (the percentage refers to the mass of glucose in the total mass of the glucose aqueous solution), carrying out ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a 0.22 micron microporous filter membrane to obtain a blank liposome aqueous solution containing the ginsenoside Rg5H. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rg5H blank liposome. The liposome was detected to have a D10 of 71nm, a D50 of 104nm and a D90 of 156 nm.

Example 2 preparation of pseudoginsenoside GQ blank liposomes

Adding 0.9g of egg yolk lecithin, 0.4g of pseudo-ginsenoside GQ and 0.1g of vitamin E into 20mL of chloroform, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a film, adding 20mL of 5% glucose aqueous solution (the percentage refers to the mass of glucose in the total mass of the glucose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a 0.22 micron microporous filter membrane to obtain a blank liposome aqueous solution containing the pseudo-ginsenoside GQ. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the pseudo-ginsenoside GQ blank liposome. The liposome was detected to have a D10 of 71nm, a D50 of 100nm and a D90 of 151 nm.

Example 3 preparation of Isoginsenoside Rg3H blank liposomes

Adding 0.8g of soybean lecithin and 0.2g of isoginsenoside Rg3H into 20mL of methanol, stirring at room temperature to form a clear solution, performing rotary evaporation in a thermostatic water bath at 50-60 ℃ to remove an organic solvent to form a membrane, adding 20mL of 5% sucrose aqueous solution (the percentage refers to the mass of sucrose in the total mass of the sucrose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer, and passing through a 0.22 micrometer microporous filter membrane to obtain a blank liposome aqueous solution containing the isoginsenoside Rg 3H. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rg3H blank liposome. The D10, D50 and D90 of the liposome are detected to be 69nm, 99nm and 149nm respectively.

Example 4 preparation of pseudoginsenoside HQ blank liposomes

Adding 0.7g hydrogenated soybean lecithin (HSPC), 0.1g pseudo ginsenoside HQ and 0.2g cholesterol into 20mL ethanol, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 60-65 ℃ to remove an organic solvent to form a film, adding 20mL of 5% mannitol aqueous solution (the percentage refers to the mass of the mannitol in the total mass of the mannitol aqueous solution), and performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer to obtain a blank liposome aqueous solution containing the pseudo ginsenoside HQ. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the pseudo ginsenoside HQ blank liposome. The D10, D50 and D90 of the liposome are respectively 68nm, 139nm and 292nm.

Example 5 preparation of pseudo-ginsenoside GD blank liposomes

Adding 0.9g of egg yolk lecithin, 0.4g of pseudo-ginsenoside GD, 0.2g of soybean oil and 0.1g of vitamin C into 20mL of isopropanol, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 60-65 ℃ to remove an organic solvent to form a film, adding 20mL of 5% propylene glycol aqueous solution (the percentage refers to the mass of propylene glycol accounting for the total mass of the propylene glycol aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a 0.22 micron microporous filter membrane to obtain a blank liposome aqueous solution containing the pseudo-ginsenoside GD. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the pseudo ginsenoside GD blank liposome. The D10, D50 and D90 of the liposome are 62nm, 94nm and 141nm respectively.

Example 6 preparation of ginsenoside Rh3H1 (E-type) blank liposomes

Adding 0.5g of egg yolk lecithin, 0.4g of ginsenoside Rh3H1 (E) and 0.05g of ascorbic acid into 20mL of n-butanol, stirring at room temperature to form a clear solution, performing rotary evaporation to remove an organic solvent in a constant-temperature water bath at 65-75 ℃, forming a film, adding 20mL of 5% glycerol aqueous solution (the percentage refers to the mass of glycerol in the total mass of the glycerol aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a 0.45 micron microporous filter membrane to obtain a blank liposome aqueous solution containing the ginsenoside Rh3H 1. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rh3H1 blank liposome. The D10 of the liposome is 76nm, the D50 is 138nm and the D90 is 234nm.

Example 7 preparation of pseudoginsenoside PF11 blank liposomes

Adding 0.7g of egg yolk lecithin, 0.4g of ginsenoside PF11 and 0.01g of sodium thiosulfate into 20mL of Tetrahydrofuran (THF), stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 60-65 ℃ to remove an organic solvent to form a membrane, adding 20mL of a 5% galactose aqueous solution (the percentage refers to the mass of galactose accounting for the total mass of the galactose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer, and passing through a1 micrometer microporous filter membrane to obtain a blank liposome aqueous solution containing the ginsenoside PF 11. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside PF11 blank liposome. The D10 of the liposome is 66nm, the D50 is 130nm, and the D90 is 251nm.

EXAMPLE 8 preparation of ginsenoside Rp1 blank liposomes

Adding 0.7g of egg yolk lecithin, 0.4g of ginsenoside Rp1 and 0.01g of sodium thiosulfate into 20mL of diethyl ether, stirring at room temperature to form a clear solution, removing an organic solvent by rotary evaporation in a constant-temperature water bath at 30-35 ℃, forming a film, adding 20mL of a 5% galactose aqueous solution (the percentage refers to the mass of galactose accounting for the total mass of the galactose aqueous solution), carrying out ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a 0.22 micron microporous membrane to obtain a blank liposome aqueous solution containing the ginsenoside Rp1. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rp1 blank liposome. The D10 of the liposome is 72nm, the D50 is 131nm and the D90 is 225nm.

Example 9 preparation of blank liposomes of 25-methyl-Isoginsenoside Rg3 (IsoRg 3 Me)

Adding 0.9g of soybean lecithin S100, 0.4g of 25-methyl-isoginsenoside Rg3 and 0.01g of sodium sulfite into 20mL of diethyl ether, stirring at 40-45 ℃ to form a clear solution, and evaporating to remove the organic solvent at 40-50 ℃ by using a film evaporator to form a film. Adding 20mL Phosphate Buffered Saline (PBS), rotating for hydration, completely dissolving, homogenizing with a high-pressure homogenizer until liposome particles are 0.1-0.3 micrometer, and passing through 0.22 micrometer microporous membrane to obtain blank liposome water solution containing 25-methyl-isoginsenoside Rg 3. The solution was then dispensed into vials containing 180mg of liposomes per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the blank liposome of the 25-methyl-isoginsenoside Rg 3. The D10 of the liposome is 64nm, the D50 is 123nm and the D90 is 223nm.

Example 10 preparation of Pseudoginsenoside GQ paclitaxel liposomes

Adding 0.8g of egg yolk lecithin, 0.4g of pseudo-ginsenoside GQ, 0.1g of paclitaxel, 0.1g of vitamin E and 0.1g of soybean oil into 20mL of chloroform, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a film, adding 20mL of 5% lactose aqueous solution (the percentage refers to the mass of lactose accounts for the total mass of the lactose aqueous solution), and performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer to obtain an aqueous solution containing pseudo-ginsenoside GQ paclitaxel liposome. The solution was then dispensed into vials containing 30mg of paclitaxel per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the pseudoginsenoside GQ paclitaxel liposome. Through detection, the liposome has the D10 of 72nm, the D50 of 134nm and the D90 of 246nm, and the entrapment rate is more than or equal to 95 percent.

EXAMPLE 11 preparation of Liposome of ginsenoside Rk1H docetaxel

Adding 0.7g of soybean lecithin, 0.2g of ginsenoside Rk1H, 0.1g of docetaxel, 0.1g of cholesterol, 0.1g of sodium oleate and 0.1g of vitamin C into 20mL of acetonitrile, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 50-60 ℃ to remove an organic solvent to form a membrane, adding 20mL of 10% trehalose aqueous solution (the percentage refers to the mass of glucose accounting for the total mass of the trehalose aqueous solution), and performing ultrasonic treatment until liposome particles are 0.1-0.3 micron to obtain an aqueous solution containing ginsenoside Rk1H docetaxel liposome. The solution was then dispensed into vials containing 20mg of docetaxel per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rk1H docetaxel liposome. Through detection, the D10, D50 and D90 of the liposome are 75nm, 125nm and 264nm respectively, and the encapsulation efficiency is more than or equal to 95 percent.

Example 12 preparation of ginsenoside Rg5H1 irinotecan liposomes

Adding 0.9g distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG (2000)), 0.3g ginsenoside Rg5H1, and 0.1g vitamin C into 20mL ethanol, stirring at room temperature to obtain clear solution, removing organic solvent at 50-60 deg.C with rotary evaporator, and forming into film. Adding a 20mL6.6% ammonium sulfate aqueous solution (the percentage refers to the mass percentage of the ammonium sulfate to the total mass of the ammonium sulfate aqueous solution), carrying out ultrasonic treatment until blank liposome particles are 0.1-0.3 micron to obtain a blank liposome solution, dialyzing the blank liposome solution in a 0.15M (0.15 mol/L) glucose solution for 12 hours, and adding trehalose with corresponding mass according to the volume of the blank liposome solution after dialysis to ensure that the mass fraction of the trehalose in the blank liposome solution reaches 10%, wherein the percentage refers to the mass percentage of the trehalose to the total mass of the blank liposome solution. Adding 1mL of 20% irinotecan hydrochloride aqueous solution (0.2 g of irinotecan hydrochloride) by mass fraction, carrying out heat preservation in water bath at 37 ℃ for 30min to obtain an aqueous solution containing ginsenoside Rg5H1 irinotecan hydrochloride liposome, and then filling into penicillin bottles to ensure that each bottle contains 40mg of irinotecan hydrochloride. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rg5H1 irinotecan hydrochloride liposome. The detection shows that the D10 of the liposome is 77nm, the D50 is 117nm, the D90 is 193nm, and the entrapment rate is more than or equal to 95%.

Example 13 preparation of Liposome of Isoginsenoside Rg3 (E) ribavirin

Adding 0.8g of dimyristoyl phosphatidylcholine (DMPC), 0.3g of isoginsenoside Rg3 (E), 0.1g of ribavirin and 0.1g of ascorbic acid into 20mL of ethanol, stirring at 40-50 ℃ to form a clear solution, evaporating at 50-60 ℃ by using a film evaporator to remove an organic solvent to form a film, adding 20mL of a 5% xylitol aqueous solution (the percentage refers to the mass of xylitol accounting for the total mass of the xylitol aqueous solution), homogenizing by using a high-pressure homogenizer until liposome particles are 0.1-0.3 microns, passing through a 0.22-micron microporous filter membrane to obtain an aqueous solution containing isoginsenoside Rg3 (E) ribavirin liposomes, and then subpackaging in penicillin bottles to ensure that each bottle contains 100mg of ribavirin. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rg3 (E) ribavirin liposome. Through detection, the liposome has the D10 of 72nm, the D50 of 129nm and the D90 of 217nm, and the entrapment rate is more than or equal to 95 percent.

Example 14 preparation of Liposome of Isoginsenoside Rg3 (O) Cytarabine

Adding 0.7g of egg yolk lecithin, 0.3g of isoginsenoside Rg3 (O) and 0.5g of vitamin C into 20mL of ethanol, stirring at room temperature to form a clear solution, removing the organic solvent by using a rotary evaporator at the temperature of 50-60 ℃, and forming a film. Adding 20mL of 6.6% ammonium sulfate aqueous solution (the percentage refers to the mass percentage of the ammonium sulfate to the total mass of the ammonium sulfate aqueous solution), performing ultrasonic treatment until blank liposome particles are 0.1-0.3 micron to obtain a blank liposome solution, dialyzing the blank liposome solution in 0.15M (0.15 mol/L) of glucose solution for 12 hours, and adding trehalose with corresponding mass according to the volume of the blank liposome solution after dialysis so that the mass fraction of the trehalose in the blank liposome solution reaches 10%, wherein the percentage refers to the mass percentage of the trehalose to the total mass of the blank liposome solution. Adding 1mL of 20% cytarabine aqueous solution (0.2 g of cytarabine) in mass fraction, keeping the temperature in a water bath at 37 ℃ for 30min, passing through a 0.22-micron microporous filter membrane to obtain an aqueous solution containing the isoginsenoside Rg3 (O) cytarabine liposome, and then subpackaging in penicillin bottles to ensure that each bottle contains 100mg of cytarabine. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rg3 (O) cytarabine liposome. Through detection, the D10, D50 and D90 of the liposome are 72nm, 130nm and 222nm respectively, and the entrapment rate is more than or equal to 95 percent.

Example 15 preparation of Liposome of Isoginsenoside Rg3 (Z) vincristine sulfate

Adding 0.6g of 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) and 0.3g of isoginsenoside Rg3 (Z) into 20mL of ethanol and chloroform at a volume ratio of =1:1 at room temperature, stirring to form a clear solution, and removing the organic solvent by using a rotary evaporator at the temperature of 50-55 ℃ to form a film. Adding 20mL of 6.6% ammonium sulfate aqueous solution (the percentage refers to the mass percentage of the ammonium sulfate to the total mass of the ammonium sulfate aqueous solution), performing ultrasonic treatment until blank liposome particles are 0.1-0.3 micron to obtain a blank liposome solution, dialyzing the blank liposome solution in 0.15M (0.15 mol/L) of glucose solution for 12 hours, and adding trehalose with corresponding mass according to the volume of the blank liposome solution after dialysis so that the mass fraction of the trehalose in the blank liposome solution reaches 10%, wherein the percentage refers to the mass percentage of the trehalose to the total mass of the blank liposome solution. Adding 1mL of vincristine sulfate aqueous solution (vincristine sulfate 0.2 g) with mass fraction of 20%, maintaining in water bath at 37 deg.C for 30min to obtain an aqueous solution containing ginsenoside Rg3 (Z) vincristine sulfate liposome, and subpackaging in penicillin bottles to make each bottle contain 1mg of vincristine sulfate. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rg3 (Z) vincristine sulfate liposome. Through detection, the D10 of the liposome is 73nm, the D50 is 121nm, the D90 is 245nm, and the entrapment rate is more than or equal to 95%.

Example 16 preparation of ginsenoside Rh3H epidermal growth factor antibody liposome

Adding 0.6g hydrogenated soybean lecithin (HSPC) and 0.3g ginsenoside Rh3H into 20mL ethanol solution, stirring at room temperature to obtain clear solution, removing organic solvent at 50-60 deg.C with rotary evaporator, and forming film. Adding 20mL of purified water, and performing ultrasonic treatment until the blank liposome particles are 0.1-0.3 micrometer to obtain a blank liposome solution. Adding 10mL of mixed solution of epidermal growth factor antibody (10 mg) and water, keeping the temperature in water bath at 37 deg.C for 30min, performing ultrasonic treatment to obtain aqueous solution containing ginsenoside Rh3H epidermal growth factor antibody liposome, and subpackaging in penicillin bottles to make each bottle contain 1mg of epidermal growth factor antibody. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rh3H epidermal growth factor antibody liposome. The detection shows that the liposome has D10 of 70nm, D50 of 147nm, D90 of 330nm and entrapment rate of more than or equal to 95%.

Example 17 preparation of pseudo-ginsenoside GP Indometacin liposomes

Adding 0.9g of soybean lecithin, 0.2g of pseudo-ginsenoside GP, 0.1g of indomethacin and 0.1g of sodium oleate into 20mL of acetonitrile, stirring at room temperature to form a clear solution, performing rotary evaporation to remove an organic solvent in a thermostatic water bath at 50-60 ℃, forming a film, adding 20mL of 10% trehalose aqueous solution (the percentage refers to the mass of glucose accounting for the total mass of the glucose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer, and passing through a1 micrometer microporous filter membrane to obtain an aqueous solution containing the pseudo-ginsenoside GP indomethacin liposome. The solution was then dispensed into vials containing 10mg of indomethacin per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the pseudo-ginsenoside GP indomethacin liposome. The detection shows that the liposome has D10 of 66nm, D50 of 128nm, D90 of 247nm, and entrapment rate of 95% or more.

Example 18 preparation of ginsenoside Rg5H all-trans retinoic acid liposomes

Adding 0.9g dipalmitoyl phosphatidylcholine (DPPC), 0.4g ginsenoside Rg5H and 0.1g all-trans retinoic acid into 20mL chloroform, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a membrane, adding 20mL 5% lactose aqueous solution (the percentage refers to the percentage of the mass of lactose to the total mass of the lactose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer, and passing through a 0.22 micrometer microporous filter membrane to obtain an aqueous solution containing ginsenoside Rg5H all-trans retinoic acid liposome. Then the solution is subpackaged in penicillin bottles, so that each bottle contains 10mg of all-trans retinoic acid. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rg5H all-trans retinoic acid liposome. The detection shows that the liposome has D10 of 76nm, D50 of 137nm, D90 of 232nm, and entrapment rate of 95% or more.

EXAMPLE 19 preparation of Isoginsenoside Rh2 (E) cisplatin Liposome

Adding 0.8g of egg yolk lecithin, 0.4g of isoginsenoside Rh2 (E), 0.1g of cisplatin, 0.1g of vitamin E into 20mL of methanol, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a membrane, adding 20mL of a 5% lactose aqueous solution (the percentage refers to the percentage of the mass of lactose to the total mass of the lactose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a1 micron microporous filter membrane to obtain the aqueous solution containing the isoginsenoside Rh2 (E) cisplatin liposome. The solution was then dispensed into vials containing 10mg of cisplatin per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rh2 (E) cisplatin liposome. Through detection, the D10 of the liposome is 70nm, the D50 is 144nm, the D90 is 315nm, and the entrapment rate is more than or equal to 95%.

EXAMPLE 20 preparation of Liposome of ginsenoside Rk4H1 (type E) Doxil hydrochloride

Adding 0.9g soybean lecithin S100, 0.3g ginsenoside Rk4H1 and 0.1g vitamin E into 20mL ethanol, stirring at 40-50 deg.C to obtain clear solution, evaporating at 50-55 deg.C with a film evaporator to remove organic solvent, and forming film. Adding 20mL of Phosphate Buffered Saline (PBS), carrying out rotary hydration, completely dissolving, homogenizing by a high-pressure homogenizer until liposome particles are 0.1-0.3 micron to obtain a blank liposome aqueous solution containing ginsenoside Rk4H1, adding 1mL of doxorubicin hydrochloride aqueous solution (doxorubicin hydrochloride 0.2 g) with the mass fraction of 20% and 6mL of disodium hydrogen phosphate aqueous solution with the mass fraction of 7.1%, adding pure water to enable the pH value of an external water phase to be 7.30, and carrying out water bath heat preservation at 60 ℃ for 30min to obtain the doxorubicin hydrochloride liposome aqueous solution containing ginsenoside Rk4H 1. The solution was then dispensed into vials containing 20mg of doxorubicin hydrochloride per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rk4H1 doxorubicin hydrochloride liposome. The detection shows that the liposome has D10 of 66nm, D50 of 129nm, D90 of 255nm and entrapment rate of more than or equal to 95%.

Example 21 preparation of liposomes of Isoginsenoside Rg2 Clarithromycin

Adding 0.8g of egg yolk lecithin, 0.4g of isoginsenoside Rg2, 0.1g of clarithromycin and 0.1g of vitamin E into 20mL of methanol, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a membrane, adding 20mL of a 5% lactose aqueous solution (the percentage refers to the mass of lactose accounting for the total mass of the lactose aqueous solution), and performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer to obtain an aqueous solution containing isoginsenoside Rg2 clarithromycin liposome. The solution was then dispensed into vials containing 100mg of clarithromycin per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rg2 clarithromycin liposome. Through detection, the D10 of the liposome is 73nm, the D50 is 121nm, the D90 is 256nm, and the entrapment rate is more than or equal to 95%.

EXAMPLE 22 preparation of ginsenoside Rk4H Cyclosporin liposomes

Adding 0.8g of egg yolk lecithin, 0.4g of ginsenoside Rk4H, 0.1g of cyclosporine and 0.1g of vitamin E into 20mL of methanol, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a film, adding 20mL of a 5% lactose aqueous solution (the percentage refers to the mass of lactose accounts for the total mass of the lactose aqueous solution), and performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer to obtain an aqueous solution containing ginsenoside Rk4H cyclosporine liposome. The solution was then dispensed into vials containing 50mg of cyclosporin per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside Rk4H cyclosporine liposome. Through detection, the liposome has D10 of 67nm, D50 of 134nm, D90 of 277nm, and the entrapment rate is more than or equal to 95 percent.

Example 23 preparation of 3- (N, N-dimethylaminoethyl) -carbamoyl-pseudopanoxadiol DQ (DC-DQ) siRNA liposomes

Adding 0.9g soybean lecithin S100, 0.3g ginsenoside DC-DQ and 0.1g vitamin E into 20mL chloroform, stirring at 40-50 deg.C to obtain clear solution, concentrating at 40-50 deg.C under reduced pressure with rotary evaporator to remove organic solvent, forming film, and vacuumizing overnight to completely remove trace chloroform. Adding 20mL Phosphate Buffered Saline (PBS), rotating for hydration, completely dissolving, homogenizing with a high-pressure homogenizer until liposome particles are 0.1-0.3 micrometer, passing through 0.22 micrometer microporous membrane to obtain blank liposome aqueous solution containing ginsenoside DC-DQ, adding 1mL siRNA aqueous solution with mass fraction of 20%, stirring in 60 deg.C water bath, and maintaining the temperature for 30min. Passing through 0.22 micrometer microporous membrane to obtain water solution containing ginsenoside DC-DQ siRNA. The solution was then dispensed into vials containing 2mg of siRNA per vial. And putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside DC-DQ siRNA liposome. Through detection, the liposome has the D10 of 72nm, the D50 of 130nm, the D90 of 223nm and the entrapment rate of more than or equal to 95 percent.

EXAMPLE 24 preparation of Liposome of Pseudoginsenoside DQ sodium sulfate ester (SC-DQ) amphotericin B

Dissolving 0.4g ginsenoside SC-DQ in 20mL chloroform, keeping the temperature in 45 ℃ water bath, and continuously stirring and dissolving until the ginsenoside SC-DQ is completely dissolved. Then 0.5g of mPEG2000-DSPE is dissolved in 10mL of ethanol, slowly added into the medicine solution, and stirred and mixed uniformly. The organic solvent was removed by concentration under reduced pressure at 60 to 65 ℃, dried under vacuum for 24 hours, added with 100mL of trehalose (100 mg/mL), tris buffer solution with pH =7.4, and 5mL of dimethyl sulfoxide (DMSO) solution in which 0.6g of amphotericin B was dissolved, and subjected to rotational hydration, after complete dissolution, dialyzed against water at room temperature for 24 hours. The solution is processed by a high-pressure homogenizer, and then the solution is subpackaged in penicillin bottles, so that each bottle contains 10mg of amphotericin B. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside SC-DQ amphotericin B liposome. Through detection, the liposome has D10 of 67nm, D50 of 132nm, D90 of 266nm and entrapment rate of more than or equal to 95%.

Example 25 preparation of ginsenoside SC-TQ nimodipine liposomes

Adding 0.9g dioleoyl phosphatidylethanolamine-polyethylene glycol (DOPE-PEG), 0.4g ginsenoside SC-TQ and 0.1g nimodipine into 20mL of chloroform, stirring at room temperature to form a clear solution, performing rotary evaporation in a constant-temperature water bath at 40-50 ℃ to remove an organic solvent to form a membrane, adding 20mL of a 5% lactose aqueous solution (the percentage refers to the mass of lactose accounting for the total mass of the lactose aqueous solution), and performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer to obtain an aqueous solution containing ginsenoside SC-TQ nimodipine liposome. The solution was then dispensed into vials containing 2mg of nimodipine per vial. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the ginsenoside SC-TQ nimodipine liposome. Through detection, the D10, the D50 and the D90 of the liposome are 75nm, 124nm and 253nm respectively, and the encapsulation efficiency is more than or equal to 95 percent.

EXAMPLE 26 preparation of sodium sulfate-Isoprotopanaxadiol PPD (form E) (SC-Iso-PPD (E)) cabazitaxel liposomes

Adding 0.9g of soybean lecithin, 0.3g of SC-Iso-PPD (E) and 0.1g of cabazitaxel into 50mL of DMF, stirring at room temperature to form a clear solution, performing rotary evaporation to remove an organic solvent in a constant-temperature water bath at 60-70 ℃ to form a film, adding 20mL of 5% glucose aqueous solution (the percentage refers to the mass of glucose in the total mass of the glucose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micrometer, and passing through a 1-micrometer microporous filter membrane to obtain an aqueous solution containing pseudo-ginsenoside SC-Iso-PPD (E) cabazitaxel liposome. The solution was then dispensed into penicillin bottles, each containing 20mg of cabazitaxel. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the pseudoginsenoside SC-Iso-PPD (E) cabazitaxel liposome. Through detection, the liposome has D10 of 69nm, D50 of 134nm, D90 of 257nm and encapsulation efficiency of more than or equal to 95 percent.

EXAMPLE 27 preparation of Isoginsenoside Rh2 (Z) Epithromycin A liposomes

Adding 0.9g of soybean lecithin, 0.3g of isoginsenoside Rh2 (Z), 0.1g of epothilone A and 0.1g of sodium oleate into 20mL of methanol, stirring at room temperature to form a clear solution, performing rotary evaporation to remove an organic solvent in a constant-temperature water bath at 45-50 ℃ to form a membrane, adding 20mL of 5% glucose aqueous solution (the percentage refers to the mass of glucose in the total mass of the glucose aqueous solution), performing ultrasonic treatment until liposome particles are 0.1-0.3 micron, and passing through a 1-micron microporous filter membrane to obtain the aqueous solution of the liposome containing the isoginsenoside Rh2 (Z) epothilone A. The solution was then dispensed into penicillin bottles, each containing 10mg of epothilone A. And (3) putting the aqueous solution into a freeze dryer for freeze drying for 72 hours, introducing protective gas (argon or nitrogen), and sealing to obtain the isoginsenoside Rh2 (Z) epothilone A liposome. The detection shows that the liposome has D10 of 97nm, D50 of 237nm, D90 of 482nm, and entrapment rate of 95% or more.

3. Application examples

1. Experimental drugs:

ginsenoside Rg3 (Rg 3 for short), iso Rg3 (E), iso Rg3 (Z), iso Rg3 (O), iso Rg3H, 25-OH-Rg3, 20-Me-Rg3, 12-Ac-20-Me-Rg3, pseudo ginsenoside GQ (pseudo GQ for short), pseudo ginsenoside GD (pseudo GD for short), pseudo ginsenoside GP (pseudo GP for short), ginsenoside Rg5 (Rg 5 for short), rg5H, rg5H1 (E), rk1H, rp1, ginsenoside Rh2 (Rh 2 for short), iso Rh2 (E), iso Rh2 (Z), 12-Ac-20-Me-Rh2, pseudo ginsenoside GHD (pseudo GHD for short), pseudo ginsenoside HQ (pseudo HQ for short), rh3 3532 zxft 3H1 (E), rh3H1 (Z), and mixtures thereof protopanaxadiol PPD (PPD for short) (type E), isopanaxadiol PPD (isopodD for short), panaxadiol PD (PD for short), pseudo-ginsenoside DQ (pseudo-DQ for short), 12,20-DiMe-PPD, ginsenoside Rk4 (Rk 4 for short), isoginsenoside Rg2 (iso-Rg 2 (type E), rk4H, ginsenoside Rk4H1 (type E), ginsenoside Rh4 (Rh 4 for short), ginsenoside Rh4H (Rh 4H), ginsenoside Rh4H1 (Rh 4H1 (E), SC-PPD, SC-DQ, SC-TQ, DC-DQ, pseudo-ginsenoside PF11 (pseudo-PF 11 for short), paclitaxel, docetaxel, cabazitaxel, irinotecan hydrochloride, camptothecin, cytarabine, vincristine sulfate, all-trans retinoic acid, cisplatin, doxorubicin hydrochloride, indomethacin, ribavirin, epidermal growth factor antibodies, clarithromycin, nimodipine, cyclosporine, siRNA, amphotericin B, and epothilones a are commercially available or synthesized according to the examples of the present invention.

Unless otherwise specified, the preparation method of each ginsenoside blank liposome is described in the section of "preparation of liposome", or the corresponding ginsenoside is replaced with the target ginsenoside with reference to example 1 in the preparation of liposome.

2. The instrument used in the following application examples is owned instrument equipment of the medical college of southwest university, and the equipment model and source information are as follows:

high performance liquid chromatography (Agilent 1100);

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

ultrasonic cleaning machine (SB 3200DT, 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 disruptor (JY 92-II, ningbo Xinzhi Biotechnology 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 Instrument Co., ltd.);

clean bench (SW-CJ-1 FD, 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);

A549/T human lung cancer paclitaxel resistant strain (Nanjing Kaikyi organism);

MCF-7 human breast cancer cells (south kyakyl organism);

an MCF-7/T human breast cancer taxol resistant strain establishment method;

BGC-823 human gastric cancer cells (Nanjing Kaikyi organism);

a BGC-823/T human gastric cancer paclitaxel drug-resistant strain establishment method;

and inducing parent BGC-823 cells to establish a human gastric cancer drug-resistant cell line BGC-823/Taxol by adopting a low-concentration dosage continuous induction method. And culturing the newly recovered BGC-823 cells for 2 or 3 generations under conventional conditions to stabilize the growth of the cells. When the culture medium is renewed the next day after the cells are digested and passaged, the parent BGC-823IC is subjected to Taxol ₅₀ 1/10 of the initial concentration was added with paclitaxel. The culture medium is renewed the next day after adding the medicine, and the routine subculture of the concentration of the paclitaxel is maintained. 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.

The establishment method of the MCF-7/T human breast cancer taxol resistant strain comprises the following steps:

and inducing parent MCF7 cells to establish a human breast cancer drug-resistant cell line MCF7/Taxol by adopting a low-concentration and dosage continuous induction method. The newly recovered MCF7 cells are cultured for 2 or 3 generations under the conventional condition, so that the cells grow stably. When the culture medium is renewed the next day after the cell digestion passage, paclitaxel is added at a concentration starting with 1/10 of the IC50 of Taxol to the parent MCF 7. The culture medium is renewed the next day after adding the medicine, and the routine subculture of the concentration of the paclitaxel is maintained. After the cells stably grow at each paclitaxel concentration, the drug concentration is increased to continue the culture until the cells can stably grow in the 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, washing the precipitated red blood cells with 0.9% sodium chloride solution for 2-3 times according to the method until the supernatant does not show 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.

TABLE 5

Test tube numbering	1	2	3	4	5
						2% erythrocyte suspension/mL	2.5	2.5	2.5	2.5	/
0.9% sodium chloride solution/mL	2.2	2.2	2.5	/	4.7
						Purified water/mL	/	/	/	2.5	/
Test solution/mL	0.3	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 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: placing the cell line concerned in a medium containing 5% CO ₂ In 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 blank control group (normal saline group), a positive control group and a drug-loaded ginsenoside liposome group are set. More than 3-6 concentration gradients, one-half or five-fold dilutions, each concentration in triplicate wells, are set.

8. Tumor cell inhibitory concentration IC ₅₀ The experimental method comprises the following steps: the tumor cells in logarithmic growth phase are digested by 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 each well is added with 100 mu L. The next day, fresh medium containing samples at different concentrations and corresponding solvent controls was added, 100. Mu.L per well (final DMSO 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 PBS and 10 mu L 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 equation ₅₀ 。

9. In vitro cell assay methods: tumor cells were collected at logarithmic growth phase and resuspended in DMEM complete medium (containing 10% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin) to a final concentration of 4X 10 ⁴ one/mL. In a 96-well cell culture plate, 200. Mu.L of the above cell suspension (8X 10) was added per well ³ Individual cells/well), at 37 ℃ and 5% CO ₂ The cell culture box is used for culturing for 48 hours, 200 mu L of DMEM complete culture medium is respectively replaced by 200 mu L 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, 4 multiple holes are arranged at each concentration, and the experiment is repeated for 3 times. Cells were assayed at 37 ℃ and 5% CO ₂ After culturing for 72h in the cell culture box, 20 mul of MTT solution with the concentration of 5mg/mL is added into each hole, the cell culture box is placed for continuous culture for 4h, the supernatant is discarded, and 150 mul of DMS is added into each holeO, after shaking for 10min, OD at 490nm was measured with a continuous spectrum multifunctional microplate reader (Tecan infinite M200, TECAN, switzerland) and the cell viability was calculated according to the following formula: (cell survival rate (%) = OD _Medicine /OD _Control ×100％)。

Cell viability (%) = 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 10 ⁷ -10×10 ⁷ Injecting the tumor cells of logarithmic growth phase into the right axilla of 18-20g nude mice slowly by using a 1mL injector, injecting 100 mu L of the tumor cells into each nude mouse, and observing the growth of tumor mass until the volume of the tumor mass is about 100mm ³ . 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: v = 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 = Vt/V0. Where Vt is the tumor volume at a time and V0 is the tumor volume at the beginning of the dose.

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: percent tumor inhibition = (tumor weight of solvent control group-tumor weight of administration group)/tumor weight of solvent control group x 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 Taxol + Rg5 in the ginsenoside Rg5 paclitaxel liposome, the concentration of paclitaxel and the concentration of ginsenoside Rg5, for example, 5+30 refers to the concentration of paclitaxel in the ginsenoside Rg5 paclitaxel liposome is 5 μ M, and the concentration of ginsenoside Rg5 is 30 μ M; time (d) refers to Time (days).

Application example 1 hemolytic study

The results are shown in Table 6.

TABLE 6

As can be seen from the data in the above table, (1) esterification at

positions

12 and 20 did not change the hemolytic properties of Rg3 and Rh 2. And (2) the hemolysis of the iso-Rg 3 is reduced by more than 50 times compared with that of the Rg 3. (3) The hemolysis of the pseudo-GQ, pseudo-GD and pseudo-GP are all reduced by more than 50 times compared with Rg 3. (4) The hemolysis of ginsenoside Rp1 is higher compared to ginsenoside Rg 5. (5) Compared with ginsenoside Rg5, the hemolysis of ginsenoside Rg5H, rg H1E, rk H, iso Rg3 (O), iso Rg3 (H) and the like is greatly reduced. (6) Rg3 blank liposomes were severely hemolyzed. (7) Blank liposomes such as GQ, rg5H, and iso-Rg 3 are less hemolytic than Rg5 blank liposomes. (8) The HD50 of the ginsenoside Rg5 paclitaxel liposome is 450-500 mug/mL. The safe dose of HD5 is around 400 μ g/mL. (9) The hemolysis safe concentration of the ginsenoside Rg5H paclitaxel liposome reaches about 1600 mug/mL (paclitaxel concentration is 400 mug/mL), and the hemolysis is reduced by more than 3 times compared with Rg 5.

Application example 2 in vitro cell experiment and in vivo animal experiment

1. In vitro cell experiments

According to the in vitro cell experiment method, the survival rates of ginsenoside Rg5H blank liposome (Rg 5H empty), ginsenoside Rg5 blank liposome (Rg 5 empty), ginsenoside Rp1 blank liposome (Rp 1 empty), ginsenoside Rg3H blank liposome (Rg 3H empty), pseudo-ginsenoside GQ blank liposome (GQ empty), ginsenoside Rg5H paclitaxel liposome (Taxol + Rg 5H), ginsenoside Rg5 paclitaxel liposome (Taxol + Rg 5), ginsenoside Rp1 paclitaxel liposome (Taxol + Rp 1), pseudo-ginsenoside Rg3H paclitaxel liposome (Taxol + Rg 3H), pseudo-ginsenoside GQ paclitaxel liposome (Taxol + GQ), on human lung cancer cell (a 549) and human lung cancer paclitaxel resistant strain (a 549/T) cell are respectively determined, 7 drug concentrations in table 7 and table 9 are set, and the specific experimental data are shown in tables 8 and 10 and fig. 1-4: fig. 1 is a graph of cell viability of Rg5H empty, rg5 empty, rp1 empty, rg3H empty, and GQ empty for human lung cancer cell (a 549), and fig. 2 is a graph of cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, and Taxol + GQ for human lung cancer cell (a 549); fig. 3 is a graph of cell survival rates of Rg5H null, rg5 null, rp1 null, rg3H null, and GQ null against human lung cancer paclitaxel-resistant strains (a 549/T), and fig. 4 is a graph of cell survival rates of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, and Taxol + GQ against human lung cancer paclitaxel-resistant strains (a 549/T).

TABLE 7 concentration of liposomes in human lung carcinoma cells (A549)

TABLE 8 survival of human lung cancer cells (A549) with liposomes

As can be seen from Table 8 and FIGS. 1-2: the ginsenoside Rg5H, rg, rp1 and Rg3H, GQ blank liposome has weaker activity on human lung cancer cells (A549), and the activities of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, taxol + GQ on human lung cancer cells (A549) are basically consistent.

TABLE 9 concentration of liposomes to paclitaxel resistant human Lung cancer cells (A549/T)

TABLE 10 survival of liposomes against paclitaxel-resistant human lung carcinoma cells (A549/T)

From table 10 and fig. 3-4 it can be seen that: taxol + Rg5H and Taxol + Rg5 have stronger activity than Taxol + Rp1, taxol + Rg3H, taxol + GQ on human lung cancer Taxol resistant strains (A549/T).

2. IC of liposomes to human Lung cancer cells (A549) and human Lung cancer Taxol-resistant strains (A549/T) ₅₀ Value of

According to IC ₅₀ An experimental method for determining IC of Rg5H blank, rg5 blank, rp1 blank, rg3H blank, GQ blank, taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, taxol + GQ on human lung cancer cell (A549) and human lung cancer Taxol-resistant strain (A549/T) respectively ₅₀ The values and experimental results are shown in Table 11.

Table 11 (Unit. Mu.M)

Remarking: IC corresponding to Taxol + Rg5H ₅₀ Of the values 0.07 (0.42. Mu.M), 0.07. Mu.M refers to the IC of Taxol in Taxol + Rg5H ₅₀ The value, 0.42. Mu.M, is the IC for Rg5H in Taxol + Rg5H ₅₀ The value is obtained.

3. Live animal imaging

The size of the subcutaneous tumor of the right anterior limb is 100mm ³ Left and right BALB/C-nu/nu mice with uniform size and no bleeding necrosis are respectively caucasized and calmedInjecting Rg5H, rg, rp1, rg3H and GQ liposome containing 10% of loaded near infrared fluorescent probe (IR 783) in a pulse manner (hereinafter referred to as experimental group, namely ginsenoside Rg5H, rg, rp1, rg3H, GQ blank liposome encapsulating near infrared fluorescent probe (IR 783) and loaded near infrared fluorescent probe (IR 783) Rg5H liposome (hereinafter referred to as control group, namely Rg5H blank liposome encapsulating near infrared fluorescent probe (IR 783), wherein the preparation method is a preparation method of the loaded near infrared fluorescent probe (IR 783) liposome which is conventional in the art), and then recording the in vivo distribution of 78IR 3 fluorescence by a living body imager at 2, 6 and 10H, and particularly shown in figures 5 and 6.

Wherein, FIGS. 5-A, 5-B and 5-C are in vivo profiles of IR783 fluorescence recorded by the in vivo imager at 2, 6 and 10h for the control group, respectively; FIG. 5-S shows a fluorescence scale, which is red, yellow, green and blue in order of the intensity of fluorescence, red showing the strongest fluorescence and blue showing the weak fluorescence. FIGS. 5-D to 5-R are in vivo distribution plots of IR783 fluorescence recorded by the in vivo imager at 2, 6 and 10h for the experimental groups. Wherein, fig. 5-D, fig. 5-E and fig. 5-F are ginsenoside Rg5H experimental groups respectively; FIGS. 5-G, 5-H and 5-I are experimental groups of ginsenoside Rg5, respectively; FIG. 5-G, FIG. 5-K and FIG. 5-L are the experimental groups of ginsenoside R-Rp1, respectively; FIG. 5-M, FIG. 5-N and FIG. 5-O are experimental groups of ginsenoside Rg3H, respectively; FIG. 5-P, FIG. 5-Q and FIG. 5-R are the experimental groups of pseudo-ginsenoside GQ, respectively.

As can be seen from FIG. 5, the right forelimb of the control group mice has no fluorescence, while the right forelimb of the experimental group mice has strong fluorescence, which indicates that the ginsenoside Rg5H, rg, rp1 and Rg3H, GQ blank liposome have strong targeting to tumor cells.

FIGS. 6-A-6-F are the fluorescence profiles of isolated organs of control group and ginsenoside Rg5H, rg, rp1, rg3H, GQ liposome injected into mice for 12h, wherein the top is tumor tissue.

As can be seen from FIG. 6, the tumor tissue of the control group mice has no fluorescence, while the tumor tissue of the experimental group mice has strong fluorescence, which indicates that the ginsenoside Rg5H, rg, rp1, rg3H, GQ blank liposome has strong targeting property to tumor cells.

4. In vivo efficacy test

According to the in vivo efficacy experiment method, 27 subcutaneous tumor loadsNude mice were randomly divided into 3 groups (9 mice per group) and designated as a blank Control group (Control group, 0.9% nacl), a Taxol + Rg5H group, a Taxol + Rg5 group, a Taxol + Rp1 group, a Taxol + Rg3H group, and a Taxol + GQ group, respectively. The corresponding preparation is injected via tail vein (according to 25 mg. Kg) ^-1 The 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 × dmin) ² ) /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 = Vt/V0. Where V0 is the tumor volume measured at the time of administration and Vt is the tumor volume measured every other day.

4.1 comparison of the antitumor action of human Lung cancer cell A549 in control group and experiment group

Specific experimental data and results are shown in table 12 and fig. 7, wherein fig. 7 is a tumor inhibition curve graph of a Control group, a Taxol + Rg5H group, a Taxol + Rg5 group, a Taxol + Rp1 group, a Taxol + Rg3H group, and a Taxol + GQ group on human lung cancer cells a 549.

TABLE 12 antitumor effect of each group on human lung cancer cell A549

As can be seen from table 12 and fig. 7: at the same time, the tumor volume of the Control group was the largest, while the Taxol + Rg5H group was the smallest, followed by Taxol + Rg5 group, and the relative tumor volumes of the remaining other groups were also smaller, and the final values were all less than 3.

4.2 comparison of antitumor action of paclitaxel-resistant strain (A549/T) for human lung cancer with control group and experimental group

Specific experimental data and results are shown in table 13 and fig. 8, wherein fig. 8 is a tumor inhibition curve graph of Control group, taxol + Rg5H group, taxol + Rg5 group, taxol + Rp1 group, taxol + Rg3H group, taxol + GQ group on human lung cancer paclitaxel resistant strain (a 549/T).

TABLE 13 antitumor effect of each group on human Taxol-resistant strain A549/T

As can be seen from table 13 and fig. 8: at the same time, the tumor volume of the Control group is the largest, and the other groups all show obvious effects on tumor inhibition.

Application example 3 in vitro cell experiments and in vivo animal experiments

1. In vitro efficacy test method

The 7 concentrations in tables 14 and 16 were set according to the in vitro cell assay method, and the specific viability data and graphs are shown in tables 15 and 17 and FIGS. 9-12: FIG. 9 is a graph of cell viability of Rg5H null, rg5 null, rp1 null, rg3H null, and GQ null on human breast cancer cells (MCF-7); FIG. 10 is a graph of cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, taxol + GQ for human breast cancer cells (MCF-7); FIG. 11 is a graph of cell viability of Rg5H null, rg5 null, rp1 null, rg3H null, and GQ null against human mammary paclitaxel-resistant strains (MCF-7/T); FIG. 12 is a graph of cell viability of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H and Taxol + GQ for human breast paclitaxel-resistant strains (MCF-7/T).

TABLE 14 concentration of liposomes in human breast cancer cells (MCF-7)

TABLE 15 survival of liposomes to human breast cancer cells (MCF-7)

As can be seen from table 15 and fig. 9-10: the activities of Taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, taxol + GQ on MCF-7 are stronger than the activities of Rg5H empty, rg5 empty, rp1 empty, rg3H empty and GQ empty on MCF-7.

TABLE 16 concentration of liposomes to paclitaxel resistant strains of the human mammary gland (MCF-7/T)

TABLE 16 survival of liposomes against paclitaxel-resistant strains of human mammary gland (MCF-7/T)

From table 16 and fig. 11-12, it can be seen that: taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H and Taxol + GQ have higher cytotoxicity to human mammary gland paclitaxel-resistant drug-resistant strain (MCF-7/T).

2. IC of liposomes to human breast cancer cells (MCF-7) and to human breast paclitaxel-resistant strains (MCF-7/T) ₅₀ Value of

According to IC ₅₀ An experimental method for determining IC of Rg5H blank, rg5 blank, rp1 blank, rg3H blank, GQ blank, taxol + Rg5H, taxol + Rg5, taxol + Rp1, taxol + Rg3H, taxol + GQ on human breast cancer cell (MCF-7) and human mammary gland paclitaxel-resistant strain (MCF-7/T) respectively ₅₀ The values and experimental results are shown in Table 17.

TABLE 17

Item	MCF-7 cell line	MCF-7/T cell line
			Rg5H hollow	7.51μM	80.94μM
Rg5 hollow	16.22μM	169.40μM
			Rp1 null	37.13μM	338.70μM
Rg3H hollow	39.14μM	328.60μM
			GQ air gap	35.71μM	315.60μM
Taxol+Rg5H	0.07μM+0.42μm	0.70μM+4.20μM
			Taxol+Rg5	0.13μM+0.76μm	1.31μM+7.86μM
Taxol+Rp1	0.28μM+1.70μm	2.77μM+16.59μM
			Taxol+Rg3H	0.28μM+1.67μm	2.80μM+16.81μM
Taxol+GQ	0.31μM+1.83μm	2.71μM+16.25μM

3. In vivo efficacy test

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 Taxol + Rg5H group, a Taxol + Rp1H group, a Taxol + Rg3H group, a Taxol + GQ group, and a Taxol + Rg5 group, respectively. The corresponding preparation is injected via tail vein (according to 25 mg. Kg) ^-1 The dosage of (a). The change in body weight of each group of mice was 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 × dmin) ² ) /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 = 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 of the tumor-inhibiting action of human Breast cancer cells (MCF-7) in control and Experimental groups

Specific experimental data and results are shown in table 18 and fig. 13, wherein fig. 13 is a tumor inhibition curve graph of a Control group, a Taxol + Rg5H group, a Taxol + Rg5 group, a Taxol + Rp1 group, a Taxol + Rg3H group, a Taxol + GQ group on human breast cancer cells (MCF-7).

TABLE 18 antitumor Effect of groups on human Breast cancer cells (MCF-7)

As can be seen from table 18 and fig. 13: at the same time, the tumor volume of the Control group was the largest, while the Taxol + Rg5H group was the smallest, and as time extended, by day 21, the relative tumor volume of the Control group reached 11.238; while the other sets are smaller in final relative volume.

3.2 comparison of antitumor Effect of human Breast paclitaxel-resistant Strain (MCF-7/T) on control and Experimental groups

Specific experimental data and results are shown in table 19 and fig. 14. Wherein, fig. 14 is a tumor inhibition curve chart of a Control group, a Taxol + Rg5H group, a Taxol + Rg5 group, a Taxol + Rp1 group, a Taxol + Rg3H group, and a Taxol + GQ group on a paclitaxel-resistant drug-resistant strain (MCF-7/T) of human mammary gland.

TABLE 19 antitumor Effect of each group on human breast paclitaxel-resistant drug-resistant strains (MCF-7/T)

As can be seen from table 19 and fig. 14: at the same time, the tumor volume was maximal in the Control group and minimal in the Taxol + Rg5H group, and as time extended, the relative tumor volume reached 11.438 in the Control group by day 21; the other groups showed very significant tumor suppression effects.

Claims

1. A blank liposome taking ginsenoside derivatives shown in formula I as membrane materials, wherein the blank liposome is provided with a membrane, and the membrane comprises lipid substances and ginsenoside derivatives shown in formula I:

wherein the lipid substance is phospholipid;

the ginsenoside derivative shown in the formula I is one or more of the following compounds:

2. the blank liposome of claim 1, wherein the mass ratio of the lipid substance to the ginsenoside derivative represented by formula I in the blank liposome is 0.5.

3. The blank liposome of claim 2, wherein the mass ratio of the lipid substance to the ginsenoside derivative represented by formula I in the blank liposome is 0.5.

4. The blank liposome of claim 3, wherein the mass ratio of the lipid substance to the ginsenoside derivative represented by formula I in the blank liposome is 0.5-4:1.

5. The blank liposome of claim 1, wherein the membrane further comprises cholesterol in the blank liposome.

6. The blank liposome of claim 5, wherein when the blank liposome further comprises cholesterol, the mass ratio of the lipid substance to the ginsenoside represented by formula I is 1.01-1:3.

7. The blank liposome of claim 6, wherein when the blank liposome further comprises cholesterol, the mass ratio of the lipid substance to the ginsenoside represented by formula I is 1.

8. The blank liposome of claim 7, wherein when the blank liposome further comprises cholesterol, the mass ratio of the lipid substance to the ginsenoside represented by formula I is 1.

9. The blank liposome of claim 5, wherein when the blank liposome further comprises cholesterol, the mass ratio of the cholesterol to the ginsenoside represented by formula I is 1.

10. The blank liposome of claim 9, wherein when the blank liposome further comprises cholesterol, the mass ratio of the cholesterol to the ginsenoside represented by formula I is 1.

11. The blank liposome of claim 10, wherein, when the blank liposome further comprises cholesterol, the mass ratio of the cholesterol to the ginsenoside represented by formula I is 1.

12. The blank liposome of claim 5, wherein the ginsenoside derivative represented by formula I is contained in the membrane at a content of 0.01% -80%; the content of lipid substances in the membrane is 5-99.9%; the content of the cholesterol in the membrane is 0-50%; the above percentages all refer to the mass of each component as a percentage of the total mass of the film.

13. The blank liposome of claim 12, wherein,

the content of the ginsenoside derivative shown in the formula I in the membrane is 10-80%;

and/or the content of the lipid substance in the membrane is 10% -70%;

and/or the content of cholesterol in the membrane is 0-50%.

14. The blank liposome of claim 13, wherein,

the content of the ginsenoside derivative shown in the formula I in the membrane is 10-40%;

and/or the content of the lipid substance in the membrane is 30-70%;

and/or the content of cholesterol in the membrane is 0-20%.

15. The blank liposome of claim 14, wherein,

the content of the ginsenoside derivative shown in the formula I in the membrane is 20-40%;

and/or the content of the lipid substances in the membrane is 30-60%.

16. The blank liposome of claim 1, wherein,

the blank liposome further comprises an antioxidant and is encapsulated in the membrane; the content of the antioxidant in the blank liposome 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 liposome;

and/or, the blank liposome further comprises a freeze-drying protective agent and is encapsulated in a membrane; the content of the freeze-drying protective agent in the blank liposome is less than or equal to 95 percent or less than or equal to 80 percent; the percentage refers to the mass of the lyoprotectant accounting for the total mass of the blank liposome;

and/or, the blank liposome further comprises soybean oil and/or sodium oleate and is encapsulated in a membrane;

and/or, the blank liposome further comprises other auxiliary materials and is encapsulated in a membrane; the other auxiliary materials are one or more of a surfactant, a thermosensitive auxiliary material, a pH sensitive substance and an ionic additive.

17. The blank liposome of claim 16, wherein,

the content of the antioxidant in the blank liposome is 0.001% -15%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank liposome;

and/or, the content of the freeze-drying protective agent in the blank liposome is 0.5% -70%; the percentage refers to the mass percentage of the freeze-drying protective agent in the total mass of the blank liposome;

and/or, the content of the soybean oil and/or the sodium oleate is 1% -90%; the percentage refers to the percentage of the mass of the soybean oil and/or the sodium oleate to the total mass of the blank liposome;

and/or the mass ratio of the soybean oil and/or the sodium oleate to the phospholipid is 1.

18. The blank liposome of claim 17, wherein,

the content of the antioxidant in the blank liposome is 0.01-10%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank liposome;

and/or, the content of the freeze-drying protective agent in the blank liposome is 3% -5% or 5% -60%; the percentage refers to the mass percentage of the freeze-drying protective agent in the total mass of the blank liposome;

and/or, the content of the soybean oil and/or the sodium oleate is 15-80 percent; the percentage refers to the percentage of the mass of the soybean oil and/or the sodium oleate to the total mass of the blank liposome;

and/or the mass ratio of the soybean oil and/or the sodium oleate to the phospholipid is 1.5-1:5.

19. The blank liposome of claim 18, wherein,

the content of the antioxidant in the blank liposome is 0.01-5%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank liposome;

and/or, the content of the lyoprotectant in the blank liposome is 30% -60%; the percentage refers to the mass percentage of the freeze-drying protective agent in the total mass of the blank liposome;

and/or, the content of the soybean oil and/or the sodium oleate is 20-70%; the percentage refers to the percentage of the mass of the soybean oil and/or the sodium oleate to the total mass of the blank liposome;

and/or the mass ratio of the soybean oil and/or the sodium oleate to the phospholipid is 1.5-1:4.

20. The blank liposome of claim 19, wherein,

the content of the antioxidant in the blank liposome is 0.1-1%; the percentage refers to the percentage of the mass of the antioxidant to the total mass of the blank liposome;

and/or the soybean oil and/or the sodium oleate is 25-62.5%, 20-30%, or 60-70%; the percentage refers to the percentage of the mass of the soybean oil and/or the sodium oleate to the total mass of the blank liposome;

and/or the mass ratio of the soybean oil and/or the sodium oleate to the phospholipid is 1:9 or 1:1-1:2.

21. The blank liposome of claim 16, wherein,

the antioxidant is one or more of sodium pyrosulfite, sodium thiosulfate, propyl gallate, alpha-tocopherol, alpha-hydroxy acid, flavonoid compound, phenylpropanoid phenolic compound, vitamin E, vitamin C, fumaric acid, cysteine, methionine, butylated hydroxyanisole, dibutylhydroxytoluene, thiodipropionic acid, sulfite, bisulfite, dithioaminobenzoic acid compound, citric acid, malic acid, sorbitol, glycerol, propylene glycol, hydroquinone, hydroxycoumarin, ethanolamine, phosphoric acid and phosphorous acid;

and/or the freeze-drying protective agent is one or more of sugar, polyalcohol, amino acid and buffering agent;

and/or the surfactant is one or more of polyethylene glycol, polysorbate, poloxamer 188, polyoxyethylene fatty acid esters, polyoxyethylene fatty acid ether and polyoxyethylene methyl castor oil ether;

and/or, the thermosensitive auxiliary material is one or more of thermosensitive polymer and/or thermosensitive surfactant which is added to the liposome;

and/or the ionic additive is a cationic additive and/or an anionic additive.

22. The blank liposome of claim 21, wherein the lipid material is one or more of natural phospholipids, semisynthetic phospholipids, and fully synthetic phospholipids;

and/or the sugar is one or more of monosaccharide, disaccharide and polysaccharide;

and/or, the polyalcohol is propylene glycol and/or glycerol;

and/or, the amino acid is alpha-amino acid, and is selected from one or more of threonine, glycine, glutamic acid, arginine and histidine;

and/or, the buffer refers to a buffer solution;

and/or the number average molecular weight of the polyethylene glycol is 200-8000;

and/or the polysorbate is one or more of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan trioleate;

and/or the polyethylene glycol is one or more of polyethylene glycol-phosphatidylethanolamine, polyethylene glycol-polylactic acid, polylysine-polylactide glycolide, polyetherimide-polylactic acid, polyethylene glycol-polycaprolactone and polyethylene glycol-polyglycolide lactide;

and/or the thermosensitive polymer is one or more of polyacrylamide, polyacrylic acid, polyphosphate ester and polyphosphazene amide copolymer;

and/or the heat-sensitive surfactant is a Tween surfactant and/or a Brij surfactant;

and/or the cationic additive is octadecylamine;

and/or the anionic additive is phosphatidic acid and/or phosphatidyl serine.

23. The blank liposome of claim 22, wherein the natural phospholipid is one or more of natural lecithin, sphingomyelin, glycerophospholipid, and cephalin;

and/or the semi-synthetic or fully synthetic phospholipid is one or more of phospholipid of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phospholipid of phosphatidylethanolamine, phosphatidylglycerol, dicetyl phosphate, phospholipid modified by PEG, cholesteryl succinate and 2-oleoyl-1-palmitoyl-glycerol-3-phosphorylcholine;

and/or the monosaccharide is one or more of glucose, mannitol, galactose, xylitol and sorbitol;

and/or the disaccharide is one or more of sucrose, lactose and maltose;

and/or the polysaccharide is trehalose;

and/or the pH value of the buffer solution is between 3 and 10;

and/or the buffer solution is ethanol-acetic acid buffer solution, tris buffer solution, barbital buffer solution, sodium formate buffer solution, phthalate buffer solution, citrate buffer solution, citric acid-disodium hydrogen phosphate buffer solution, ammonia-ammonium chloride buffer solution, borax-calcium chloride buffer solution, acetate buffer solution, acetic acid-lithium salt buffer solution, acetic acid-sodium acetate buffer solution, acetic acid-ammonium acetate buffer solution, phosphoric acid-triethylamine buffer solution or phosphate buffer solution.

24. The blank liposome of claim 23, wherein the phospholipid of a phosphatidylcholine species is one or more of hydrogenated soy lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylcholine, monopalmitoylphosphatidylcholine, and glycerophosphatidylcholine;

and/or the phospholipid of the phosphatidyl ethanolamine is one or more of 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine, dilauroyl phosphatidyl ethanolamine, erucyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine and dimyristoyl phosphatidyl ethanolamine

And/or the PEG-modified phospholipid is phosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dioleoylphosphatidylethanolamine-polyethylene glycol, C8 ceramide-polyethylene glycol, C16 ceramide-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol-succinyl, distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, distearoylphosphatidylethanolamine-polyethylene glycol-maleimide, distearoylphosphatidylethanolamine-polyethylene glycol-propionamide dimercaptopyridine, distearoylphosphatidylethanolamine-polyethylene glycol-cyanuric chloride, distearoylphosphatidylethanolamine-polyethylene glycol-amino, distearoylphosphatidylethanolamine-polyethylene glycol-biotin, distearoylphosphatidylethanolamine-polyethylene glycol-folic acid, dilauroylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol-active ester, phosphatidylethanolamine-polyethylene glycol-active ester, dipalmitoylphosphatidylethanolamine-polyethylene glycol-active ester, dilauroylphosphatidylethanolamine-polyethylene glycol-maleimide, phosphatidylethanolamine-polyethylene glycol-maleimide, palmitoylphosphatidylethanolamine-polyethylene glycol-maleimide, one or more of dilauroyl phosphatidyl ethanolamine-polyethylene glycol-maleimide, distearoyl phosphatidyl ethanolamine-polyethylene glycol-biotin, distearoyl phosphatidyl ethanolamine-polyethylene glycol-fluorescein, distearoyl phosphatidyl ethanolamine-polyethylene glycol-hydroxyl, distearoyl phosphatidyl ethanolamine-polyethylene glycol-amino, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-amino, dilauroyl phosphatidyl ethanolamine-polyethylene glycol-amino, distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl, dilauroyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl, distearoyl phosphatidyl ethanolamine-polyethylene glycol-thio, distearoyl phosphatidyl ethanolamine-polyethylene glycol-silane, distearoyl phosphatidyl ethanolamine-polyethylene glycol-azide, cholesterol-polyethylene glycol, methoxy-polyethylene glycol-cholesterol, cholesterol-polyethylene glycol-active ester, cholesterol-polyethylene glycol-maleimide, cholesterol-polyethylene glycol-biotin, cholesterol-polyethylene glycol-fluorescein, sterol-polyethylene glycol-carboxyl, sterol-polyethylene glycol-amino, and cholesterol-polyethylene glycol-thio;

and/or the pH value of the buffer solution is between 5 and 7.

25. A method for preparing blank liposomes according to any one of claims 1 to 24, comprising the following method one or method two, wherein the blank liposomes prepared by method one do not contain lyoprotectant, and the blank liposomes prepared by method two contain lyoprotectant:

the first method comprises the following steps:

the second method comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with the aqueous solution of a freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, performing ultrasonic treatment, high-pressure homogenization or extrusion to pass through the film, filtering to obtain an aqueous solution containing blank liposome, and drying to obtain the blank liposome;

in the first or second method, the lipid material, the ginsenoside derivative of formula I, the cholesterol, the antioxidant, the soybean oil and/or sodium oleate, the lyoprotectant, the surfactant, the heat-sensitive excipient, the pH-sensitive material, and the ionic additive are as defined in any one of claims 1-24.

26. The preparation process according to claim 25, wherein,

in the first method or the second method, in the step (1), the organic solvent is nitrile solvent and C ₁ ～C ₄ Alcohol solvent, ketone solvent, and alkane solventOne or more of an agent, an ether solvent and a halogenated hydrocarbon solvent, wherein the volume mass ratio of the organic solvent to all the components in the step (1) of the first method or the second method is 5-20mL/g;

and/or, in the first method or the second method, in the step (1), the temperature of the mixing is 0-80 ℃;

and/or, in the first method or the second method, in the step (2), the operation of removing the organic solvent of the clarified solution in the step (1) is to remove the organic solvent by using a rotary evaporator or a film evaporator;

and/or, in the first method or the second method, in the step (2), the filtration is microfiltration membrane filtration;

and/or, in the second method, in the step (2), the aqueous solution of the lyoprotectant is 5% -10% of the aqueous solution of the lyoprotectant; the percentage is the percentage of the mass of the freeze-drying protective agent in the total mass of the freeze-drying protective agent aqueous solution;

and/or, in the first method or the second method, in the step (2), the drying is freeze-drying by using a freeze-dryer.

27. The preparation process according to claim 26, wherein,

in the first method or the second method, in the step (1), the organic solvent is C ₁ ～C ₄ One or more of the alcohol solvents, nitrile solvents, ether solvents and halogenated hydrocarbon solvents of (a);

and/or, in the first method or the second method, in the step (1), the temperature of the mixing is 10-80 ℃;

and/or, in the first method or the second method, in the step (2), the operation of removing the organic solvent of the clarified solution in the step (1) is to remove the organic solvent by using a rotary evaporator or a film evaporator; the temperature for removing the organic solvent is 25-80 ℃;

and/or, in the first method or the second method, in the step (2), the pore size of the microporous filter membrane is 0.22 micron.

28. The production method according to claim 27, wherein,

the nitrile solvent is acetonitrile;

and/or, said C ₁ ～C ₄ The alcohol solvent is one or more of methanol, ethanol, isopropanol and n-butanol;

and/or the ether solvent is tetrahydrofuran and/or diethyl ether;

and/or the halogenated hydrocarbon solvent is chloroform and/or dichloromethane;

and/or the ketone solvent is acetone and/or butanone;

and/or the alkane solvent is petroleum ether;

and/or, in the first method or the second method, in the step (1), the mixing temperature is 10-65 ℃.

29. Use of a ginsenoside derivative of formula I as defined in claim 1 as a liposome membrane material.

30. An active substance-loaded liposome, wherein the active substance-loaded liposome is prepared by encapsulating one or more of a drug, an active substance in cosmetics and a substance having a health-care effect in the blank liposome according to any one of claims 1 to 24; the medicament further comprises a polynucleotide or oligonucleotide, or the medicament further comprises a biological agent.

31. The active agent-loaded liposome of claim 30,

<xnotran> , , , , , , 7- -10- , , , , , , , , , , , , , aroplatin, , , , , , , , , , , , , , , , , , , , , , , , , , , A, B, C, D, E, F, , , , , , , a2b, , , -2, , , , , , 5- , , , , , , , , , , , , , , , , , , , , , , , </xnotran> <xnotran> , 3',5' - , , B, , , G, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , BMS232632, , , , , , , , , , , , 8978 zxft 8978, , , , V, , , </xnotran> Oxacillin, cloxacillin, procaine penicillin, benzathine penicillin, piperacillin, mezlocillin, ticarcillin, azlocillin, mecillin, carbenicillin, sulbenicillin, furacilin, nafcillin, dicloxacillin, pivampicillin, apacillin, aspoxicillin, pimecrillin, methicillin, lenamicillin, fosimicin, flucloxacillin, kanamycin, natamycin, mitomycin, amikacin, tylosin, verteporfin, cefpiramide sodium, netilmicin sulfate, ofloxacin, ciprofloxacin, enoxacin, lomefloxacin, pefloxacin, rufloxacin, sarfloxacin, fleroxacin, moxifloxacin, glafloxacin, trovafloxacin, gonofloxacin, gemifloxacin, gatifloxacin, tushutfloxacin, pazufloxacin, sparfloxacin, clarithromycin, clindamycin, polymyxin, doxycycline tobramycin, vancomycin, azithromycin, doxycycline, tetracycline, oxytetracycline, minocycline, chlortetracycline, guanmecycline, demeclocycline, metacycline, etimicin, netilmicin, sisomicin, amikacin, arbekacin, dibekacin, aztreonam, meropenem, imipenem, thiomycin, panipenem, ertapenem, neomycin, paromomycin, spectinomycin, nimodipine, nifedipine, nicardipine, nitrendipine, verapamil, amlodipine, diltiazem, flunarizine, prenylamine, galapamide, tiapamil, aspirin, acetaminophen, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib, cyclosporine, alprostadil, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, risperidone, desflurane, doxycycline, tetracycline, clotrimazole, timid, ritin, desflurane, and doxycycline, sevoflurane, isoflurane, enflurane, propofol, fentanyl, uramusan, lidocaine, procaine, tetracaine, bupivacaine, pentobarbital sodium, chloral hydrate, ketamine, aldose chloride, morphine, dabigatran etexilate, alogliptin, sodium alginate diester, bilobalide, ginkgetin, ginkgo biloba extract, asarone, olmesartan medoxomil, repaglinide, lipoic acid, breviscapine, urapidil, nicotinic acid, captopril, losartan, puerarin, tanshinone IIA, sarpogrelate hydrochloride, fluvastatin, pravastatin, lovastatin, simvastatin, mevastatin, cerivastatin, fluvastatin, lovastatin, simvastatin, and the like one or more of rosuvastatin, atorvastatin calcium, rosuvastatin calcium, omeprazole, lansoprazole, ilaprazole, pantoprazole, rabeprazole, terazosin, esomeprazole, tenatoprazole, lyminoprazole, tenatoprazole, dithioprazole, lafutidine, agomelatine, fluoxetine, paroxetine, duloxetine, sertraline, fluvoxamine, citalopram, escitalopram, venlafaxine, mirtazapine, imipramine, amitriptyline, clomipramine, doxepin, remelone, venlafaxine, phenelzine, isocarboxazid, tranylcypromine;

and/or, the polynucleotide or oligonucleotide refers to a segment with genetic function, which is composed of A, T, C, G, U bases and the like;

and/or the biological agent is one or more of monoclonal antibody medicines, insulin, gamma globulin, antitoxic serum, interferon, interleukin, tumor necrosis factor, skin activity factors, epidermal growth factors, influenza vaccines, hepatitis A vaccines, anti-cancer vaccines and recombinant human acidic fibroblast growth factors;

and/or the active substances in the cosmetics are one or more of ursolic acid, superoxide dismutase, bioprotein T4N5, vitamin D2, methyl nicotinate, refined snake oil, hyaluronic acid, essential oil and ceramide;

and/or the substance with health care effect is one or more of glycyrrhizin, glycyrrhizic acid, disodium glycyrrhizinate, methyl glycyrrhizinate, diammonium glycyrrhizinate, vitamin E, resveratrol, coenzyme Q10, silymarin, anthocyanin, procyanidine, lutein, folic acid, folinic acid, curcumin, emodin, tea polyphenol, epigallocatechin gallate, catechin, blueberry extract, glutathione and oxymatrine.

32. The active substance-loaded liposome according to claim 30 or 31, wherein when the active substance in the active substance-loaded liposome is a drug and/or a substance having a health-care function, the active substance-loaded liposome is prepared for use in the form of injection, lyophilized injection, oral solid preparation, oral liquid, liniment, ointment, tincture or aerosol.

33. A process for the preparation of active substance loaded liposomes according to claim 30 or 31 wherein,

when the liposome contains the freeze-drying protective agent, the preparation method of the liposome loaded with the active substance comprises any one of the following methods:

method a comprises the following steps:

(1) Mixing lipid substances, ginsenoside shown in formula I and the active substances in an organic solvent, optionally adding one or more of cholesterol, fat-soluble antioxidant, soybean oil and/or sodium oleate, fat-soluble surfactant, fat-soluble thermosensitive auxiliary materials, fat-soluble pH sensitive substances and fat-soluble ion additives to obtain a clarified solution;

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing the film with the aqueous solution of a freeze-drying protective agent, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, performing ultrasonic treatment, high-pressure homogenization or extrusion to pass through the film, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

the method B comprises the following steps:

(1) Mixing lipid substances and ginsenoside represented by formula I in organic solvent, optionally adding one or more of cholesterol, fat-soluble antioxidant, soybean oil and/or sodium oleate, fat-soluble surfactant, fat-soluble thermosensitive adjuvant, fat-soluble pH sensitive substance and fat-soluble ion additive to obtain a clarified solution;

method C comprises the steps of:

(1) Mixing lipid substances and ginsenoside shown in formula I in organic solvent, optionally adding one or more of cholesterol, fat-soluble antioxidant, soybean oil and/or sodium oleate, fat-soluble surfactant, fat-soluble thermosensitive adjuvant, fat-soluble pH sensitive substance and fat-soluble ion additive to obtain a clarified solution;

method D comprises the following steps:

when the liposome does not contain the freeze-drying protective agent, the preparation method of the liposome loaded with the active substance comprises any one of the following methods:

method A1 comprises the following steps:

(1) Mixing lipid substances, ginsenoside shown in formula I and the active substances in an organic solvent, optionally adding one or more of cholesterol, fat-soluble antioxidant, soybean oil and/or sodium oleate, fat-soluble surfactant, fat-soluble thermosensitive auxiliary materials, fat-soluble pH sensitive substances and fat-soluble ion additives, and obtaining a clear solution;

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a film, mixing with water, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ion additive, performing ultrasonic treatment, high-pressure homogenization or extrusion through the film, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

method B1 comprises the following steps:

method C1 comprises the following steps:

method D1 comprises the following steps:

(2) Removing the organic solvent of the clarified solution obtained in the step (1), forming a membrane, mixing with a citric acid aqueous solution, optionally adding one or more of a water-soluble antioxidant, a water-soluble surfactant, a water-soluble thermosensitive auxiliary material, a water-soluble pH sensitive substance and a water-soluble ionic additive, carrying out ultrasonic, high-pressure homogenization or extrusion pushing through the membrane to obtain a blank liposome solution, mixing with the active substance and a disodium hydrogen phosphate aqueous solution, filtering to obtain an aqueous solution containing the liposome loaded with the active substance, and drying to obtain the liposome loaded with the active substance;

in method A, B, C, D, A, B1, C1 or D1, the conditions and parameters referred to in method one or method two of any of claims 25-28.

34. The method of preparing an active agent-loaded liposome according to claim 33,

the mass ratio of the active substance to the ginsenoside shown in the formula I is 1.1-1.

35. The method of preparing an active agent-loaded liposome according to claim 34,

the mass ratio of the active substance to the ginsenoside shown in the formula I is 1:2-1:6.

36. The method of claim 33,

in the method B, C, B or C1, the dialysis procedure comprises the steps of: dialyzing said blank liposome solution or said active agent-loaded liposome solution in an aqueous glucose solution or in pure water;

and/or, in the method B, C, B or C1, the operation of dialysis is performed before the operation of ultrasound, high pressure homogenization or pushing through a membrane;

and/or in the method C or C1, in the aqueous solution containing ammonium sulfate and the freeze-drying protective agent or the aqueous solution of ammonium sulfate, the mass fraction of the ammonium sulfate is 1-15%, and the percentage refers to the mass percentage of the ammonium sulfate in the total mass of the aqueous solution;

and/or, in the method C or C1, before the filtering operation, further comprising an operation of keeping the temperature;

and/or in the method D or D1, the mass fraction of the citric acid aqueous solution is 1-15%, and the percentage refers to the mass percentage of the citric acid in the total mass of the citric acid;

and/or, in the method D or D1, before the filtering operation, the method further comprises an operation of heat preservation;

and/or, in method A, B, C, D, A1, B1, C1 or D1, 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; the percentage refers to the percentage of the mass of the active substance in the total volume of the active substance aqueous solution or the active substance organic solution.

37. The method of claim 36,

in the method B, C, B or C1, the dialysis procedure comprises the steps of: dialyzing said blank liposome solution or said active agent-loaded liposome solution in an aqueous glucose solution or in pure water; the dialysis time is 5-20 hours;

and/or, in the method C or C1, in the aqueous solution containing ammonium sulfate and the freeze-drying protective agent or the aqueous solution of ammonium sulfate, the mass fraction of the ammonium sulfate is 6.6%, and the percentage refers to the mass percentage of the ammonium sulfate in the total mass of the aqueous solution;

and/or, in the method C or C1, before the filtering operation, further comprising an operation of keeping the temperature; the heat preservation operation is to preserve heat for 5 minutes to 1 hour at the temperature of 30 ℃ to 80 ℃;

and/or in the method D or D1, the mass fraction of the citric acid aqueous solution is 5.76%, and the percentage refers to the mass percentage of the citric acid in the total mass of the citric acid;

and/or the mass fraction of the disodium hydrogen phosphate aqueous solution is 5-20%;

and/or, in the method D or D1, before the filtering operation, the method further comprises an operation of heat preservation; the heat preservation operation is to preserve heat for 5 minutes to 1 hour at the temperature of 30 ℃ to 80 ℃;

and/or, in method A, B, C, D, A1, B1, C1 or D1, 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; the active substance aqueous solution or the active substance organic solution is an aqueous solution or an organic solution with the mass volume fraction of 1-20%, and the percentage refers to the percentage of the mass of the active substance in the total volume of the active substance aqueous solution or the active substance organic solution.

38. The method of claim 36, wherein the step of preparing,

in the method B, C, B or C1, the dialysis procedure comprises the steps of: dialyzing said blank liposome solution or said active agent-loaded liposome solution in an aqueous glucose solution or in pure water; the dialysis time is 12 hours;

and/or the mass fraction of the disodium hydrogen phosphate aqueous solution is 7.1%.

39. A ginsenoside derivative represented by formula I:

the ginsenoside derivative shown in the formula I is any one of the following compounds:

40. a method of preparing a ginsenoside derivative of formula I as in claim 39, comprising the steps of: in an organic solvent, carrying out deprotection reaction on an acetylation product of the compound shown in the formula I to obtain the compound shown in the formula I;

the acetylation product of the compound of formula I is: when 20-position of the compound shown as the formula I has hydroxyl, the hydroxyl at the rest positions except the 20-position hydroxyl and acetic anhydride are subjected to esterification reaction to obtain a product; when no hydroxyl exists at the 20 th site of the compound shown as the formula I, the hydroxyl at all the sites of the compound and acetic anhydride are subjected to esterification reaction to obtain a product.

41. A method for preparing ginsenoside derivatives represented by formula I in claim 40,

in the deprotection reaction, the organic solvent is an alcohol solvent and/or an ether solvent;

and/or, the deprotection reaction is carried out under the action of alkali;

and/or, the temperature of the deprotection reaction is the solvent reflux temperature under normal pressure;

and/or the deprotection reaction time is 8-15 hours.

42. A method for preparing ginsenoside derivatives represented by formula I in claim 41,

in the deprotection reaction, the organic solvent is a mixed solvent of an alcohol solvent and an ether solvent;

and/or, the deprotection reaction is carried out under the action of alkali; the alkali is alkali metal hydroxide;

and/or, the temperature of the deprotection reaction is 60-110 ℃;

and/or the deprotection reaction time is 10-12 hours.

43. A method for preparing a ginsenoside derivative represented by formula I in claim 42,

in the deprotection reaction, the organic solvent is a mixed solvent of an alcohol solvent and an ether solvent; in the mixed solvent, the volume ratio of the alcohol solvent to the ether solvent is 1:1;

and/or, the alcohol solvent methanol;

and/or, the ether solvent dioxane;

the deprotection reaction is carried out under the action of alkali; the alkali is potassium hydroxide.

44. A preparation method of ginsenoside Rg5H comprises the following steps: in an organic solvent, the acetylation product of the ginsenoside Rg5H is subjected to deprotection reaction as shown in the specification,

the deprotection reaction conditions are as described in claim 40.

45. A process as claimed in claim 44, wherein the acetylation product of ginsenoside Rg5H is prepared by the following steps: in an organic solvent, carrying out a 20-hydroxyl removal reaction on the acetylation product of the ginsenoside Rg3 to prepare an acetylation product of the ginsenoside Rg 5H;

46. the method according to claim 45, wherein,

in the reaction for removing the 20-site hydroxyl, the organic solvent is a halogenated hydrocarbon solvent;

and/or, the reaction for removing the 20-site hydroxyl is carried out under the action of boron trifluoride diethyl etherate and triethylsilane;

and/or the temperature of the reaction for removing the 20-site hydroxyl is room temperature;

and/or the reaction time for removing the 20-position hydroxyl is 5 to 10 minutes.

47. The method according to claim 46, wherein,

in the reaction for removing the 20-site hydroxyl, the organic solvent is dichloromethane;

and/or the mass ratio of boron trifluoride diethyl etherate to triethylsilane is 1.5-1:5;

and/or the mass ratio of the triethylsilane to the Rg3 acetylation product is 1:5-1.

48. A process as claimed in claim 45, wherein the acetylation product of ginsenoside Rg5H is prepared by the following steps: in an organic solvent, under the action of a catalyst, performing acylation reaction on the ginsenoside Rg3 and acetic anhydride as shown in the specification to prepare an acetylated product of the ginsenoside Rg 3;

49. the method according to claim 48, wherein,

in the acetylation reaction: the organic solvent is alkaline organic solvent;

and/or the acetylation reaction is carried out under the action of a catalyst DMAP;

and/or the mass-volume ratio of the ginsenoside Rg3 to the acetic anhydride is 0.1g/mL-5g/mL;

and/or the temperature of the acetylation reaction is room temperature;

and/or the acetylation reaction time is 8-15 hours.

50. The preparation process according to claim 49, wherein,

in the acetylation reaction: the organic solvent is pyridine.