WO2019105408A1 - Novel liposomes with ginsenoside derivative as membrane material and preparations thereof - Google Patents

Novel liposomes with ginsenoside derivative as membrane material and preparations thereof Download PDF

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WO2019105408A1
WO2019105408A1 PCT/CN2018/118137 CN2018118137W WO2019105408A1 WO 2019105408 A1 WO2019105408 A1 WO 2019105408A1 CN 2018118137 W CN2018118137 W CN 2018118137W WO 2019105408 A1 WO2019105408 A1 WO 2019105408A1
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glc
liposome
ginsenoside
peg
hydrophobic
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PCT/CN2018/118137
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English (en)
French (fr)
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Dan Wang
Chong Li
Yahua WANG
Qiang MENG
Pengfei MIAO
Huaxing Zhan
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Xiamen Ginposome Pharmaceutical Co., Ltd.
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Publication of WO2019105408A1 publication Critical patent/WO2019105408A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
    • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives

Definitions

  • Ginsenosides have complex and various pharmacological activities.
  • research mainly focuses on the monomer saponins utilizations, but the hemolysis is saponins properties and the biggest obstacle for injectable formulations.
  • the total ginseng saponins do not have obvious hemolysis effect, but the purified monomer saponins have opposite contradictory hemolysis and anti-hemolysis effects. Furthermore, they have similar linear relationships between the concentration and the hemolysis or anti-hemolysis effect.
  • There are few published literatures for saponins hemolysis or anti-hemolysis research Cheng Daren disclosed in Chinese Journal of modern Chinese Medicine, Vol. 9, No. 4, Apr.
  • the PPT type ginsenosides such as Re, Rg1, 20 (R) -Rg2 and Rh1, have anti-hemolysis action, however, 20 (R) -Rg2, 20 (S) -Rg2 and Rh1 have hemolysis action when at a high concentration.
  • the PPD-type ginsenosides such as Rb1, Rb2, Rc and Rd, have anti-hemolysis effect, however, Rd has hemolysis action when at a high concentration.
  • Oleanolic acid type ginsenosides, such as Ro have anti-hemolysis action at a low concentration and without hemolysis effect.
  • Ginsenoside P50 ( ⁇ g/mL) HD50 ( ⁇ g/mL) Rb1 43.01 480 (not hemolysis) Rb2 24.66 2000 (not hemolysis) Rc 7.42 543.2 (not hemolysis) Rd 73.01 356.78 Re 53.60 420 (not hemolysis) Rg1 38.88 420 (not hemolysis) 20 (S) -Rg2 8.79 732.99 20 (R) -Rg2 81.72 257.81 Rh1 100.39 490.68
  • some saponins can be liposome’s membrane.
  • Such saponins should have a lipophilic side and a hydrophilic side, among the lipophilic side should have at least two alkene groups.
  • Ginsenoside Rg3 and Rh2 liposomes constitute membrane mateirals for liposomes encapsulating paclitaxel, the liposomes do not have good appearance, encapsulating rates, diameter, and stability. When the diameter is grater than 1 um, precipate is observed after 7 days of shelve time, and the encapsulating rate is lower than 80%.
  • the present invention provides solutions for the poor appearance, low encapusulation rate, large diameter, and low stability of liposomes with hydrophobic ginsenosids Rg3 and Rh2 (conianing zero or one double bond) as the liposome membrance material, or high hemolysis of the ginsenosides or the ginsenoside liposomes.
  • the invention also provides a novel blank liposome with some novel ginsenoside derivatives as membrane material, preparation and use thereof. Such ginsenoside derivatives have zero or one double bond at the hydrophobic terminal, and provide good activity, low hemolysis and good membranous ability.
  • the blank liposomes provided by this invention have the advantages of good safety, low hemolysis, good stability and small particle size.
  • the present invention provides a blank liposome having a membrane comprising a lipid and a ginsenoside derivative of Formula I,
  • each of R 1 and R 2 is H, OH, R 10 , R 11 , R 12 or R 13 , and R 1 and R 2 are not both H or OH at the same time;
  • R 6 is –OH, -OCH 3 , -OOH, -OAc, or -OBz;
  • each of R 7 , R 8 , and R 9 is H, -OH, -OCH 3 , -OCHO, -OAc, or -OBz;
  • R 10 is selected from a group consisting of -O-Glc, -O-Rha ⁇ -O-Lyx, -O-Xyl, -O-Ara (p) , -O-Ara (f) , -O-Glc (2 ⁇ 1) Glc, -O-Glc (6 ⁇ 1) Glc, -O-Glc (2 ⁇ 1) Rha, -O-Glc (2 ⁇ 1) Xyl, -O-Glc (6 ⁇ 1) Xyl, -O-Glc (6 ⁇ 1) Rha, -O-Glc (2 ⁇ 1) Ara (p) , -O-Glc (6 ⁇ 1) Ara (p) , -O-Glc (6 ⁇ 1) Ara (p) , -O-Glc (6 ⁇ 1) Ara (p) , -O-Glc (6 ⁇ 1) Ara (f) , -O-Glc (6 ⁇ 1) Ara (f) , -
  • R 11 is a group formed by replacing one or more OH groups in R 10 with R 10 and each of the one or more R 10 groups independently can be the same as or different from each other;
  • R 12 is one of any of the following:
  • mPEG, -Z-mPEG, -mPEO, -Z-PEO, -mPVP, -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 -; X is O, S, or NH; a is 1, 2, 3, 4, 5, 6, 7, or 8; and b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or
  • Boc-glycine a group formed by dehydrogenizing the carboxyl contained in Boc-glycine, Boc-alanine, Boc-arginine, Boc-lysine, Boc-serine, acetyl phenylalanine, acetyl-proline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine; or
  • R 13 is
  • R 1 is -OH, and/or
  • R 2 is H or
  • R 3 is preferably and/or
  • one or more hydroxy groups in the ginsenoside derivative of Formula I are optionally replaced with one or more R 11 or R 12 groups each of which independenrtly can be the same as or different from the other; and/or
  • each of PEG, PEO, PVP, and EPEG independently, has a molecular weight in the range of 200 to 20,000; and/or
  • the aliphatic acyl group can be an acyl of a natural saturated or unsaturated aliphatic acid, and an acyl of artificially synthesized saturated or unsaturated aliphatic acid, preferably a stearyl or a palmityl.
  • the ginsenoside derivative of Formula I can be optionally modified by replacing one or more OH groups therein with one or more R 11 groups, and each of the R 11 replacement groups (when there are 2 or more) independently can be the same as or different from each other.
  • the ginsenoside derivative of Formula I can be optionally modified by replacing one or more OH groups therein with one or more R 12 groups, and each of the R 12 replacement groups (when there are 2 or more) independently can be the same as or different from each other.
  • Glc refers to glucopyranosyl
  • Xyl refers to xylopyranosyl
  • Rha refers to rhamnopyranosyl
  • Ara (p) refers to arabinopyranosyl
  • Ara (f) refers to arabinofuranosyl
  • Lyx is lyxosyl
  • Mal refers to a malonyl
  • Ac refers to an acetyl
  • PEG refers to polyethylene glycol
  • PEO refers to polyoxyethylene or polyethylene oxide
  • MPEG refers to monomethoxy-terminated polyethylene glycol
  • EPEG refers to epoxy-terminated polyethylene glycol
  • PVP refers to povidone.
  • the structure of Glc is: in -O-Ara (p) group
  • the structure of Ara (p) is: in -O-Lyx group
  • the structure of Lyx is: in -O-Ara (f) group
  • the structure of Ara (f) is: in -O-Rha group
  • the structure of Rha is: in -O-Xyl group
  • the structure of Xyl is: the structure of Mal is:
  • the molecular weight of PEG, PEO, PVP, or mPEG is independently in the range of 200 to 20,000.
  • the aliphatic acyl group can be an acyl of a natural saturated or unsaturated aliphatic acid, and an acyl of artificially synthesized saturated or unsaturated aliphatic acid, preferably a stearyl or a palmityl.
  • the ginsenoside derivative of Formula I includes one or more of the compounds listed in Table 2:
  • the purity of the ginsenoside derivative is preferably 90%or higher as measured by HPLC, more preferably greater than 95%, where the percentage is weight percentage.
  • the lipid in the membrane comprises phospholipid; and the mass (weight) ratio of the phospholipid to the ginsenoside derivative of Formula I is usually in the range of 0.5: 1 to 100: 1, preferably in the range of 0.5: 1 to 20: 1, more preferably in the range of 0.5: 1 to 4: 1 (e.g., in the range of 0.5: 1 to 2: 1) .
  • the lipid in the membrane comprises phospholipid; the membrane further comprises cholesterol.
  • the mass ratio of the phospholipid to the ginsenoside derivative of Formula I is preferably in the range of 1: 0.01 to 1: 3 (such as in the range of 1: 0.03 to 1: 1) , more preferably in the range of 1: 0.05 to 1: 0.9 (such as in the range of 1: 0.3 to 1: 0.75) , most preferably in the range of 1: 0.1 to 1: 0.9 (such as in the range of 1: 0.1 to 1: 0.5) .
  • the mass ratio of the cholesterol to the ginsenoside derivative of Formula I is preferably in the range of 1: 0.1 to 1: 100, preferably in the range of 1: 0.5 to 1: 50, more preferably in the range of 1: 0.5 to 1: 10 (such as in the range of 1: 1.5 to 1: 6, or 1: 5) .
  • a weight percentage of the ginsenoside derivative of Formula I in the membrane is preferably in the range of 0.01%to 80% (e.g. 0.03% ⁇ 37.5% ⁇ 10% ⁇ 5% ⁇ 15.4% ⁇ 20.5% ⁇ 19.9% ⁇ 18.0% ⁇ 6.25% ⁇ 13.6% ⁇ 33.3% ⁇ 17.4%) ; a weight percentage of the phospholipid in the membrane is preferably in the range of 5%to 99.9% (e.g.
  • a weight percentage of the cholesterol in the membrane is preferably lower than 50%; the percentage (%) mentioned above refers to the percentage of the mass of each component relative to the total mass of the membrane.
  • the weight percentage of the ginsenoside derivative of Formula I in the membrane is preferably in the range of 10%to 80%, more preferably in the range of 10%to 40%, most preferably in the range of 20%to 40% (such as in the range of 25%to 40%, preferably 37.5%) .
  • the weight percentage of the phospholipid in the membrane is preferably in the range of 10%to 70%, more preferably in the range of 30%to 70%, most preferably in the range of 30%to 60%.
  • the weight percentage of the cholesterol in the membrane is preferably in the range of 0%to 50%, more preferably in the range of 0%to 20%, most preferably in the range of 0%to 10% (such as in the range of 0%to 10%) .
  • the blank liposomes of the presention invention can further include an antioxidant.
  • a weight percentage of the antioxidant in the blank liposome is usually no more than 25%, preferably in the range of 0.001%to 15%, more preferably in the range of 0.01%to 10%, most preferably in the range of 0.01%to 5% (such as in the range of 0.1%to 1%) .
  • the percentage (%) refers to the percentage of the mass (i.e., weight) of the antioxidant relative to the total mass of the blank liposome.
  • the blank liposomes of the presention invention can further comprise a cryoprotectant.
  • a weight percentage of the cryoprotectant in the blank liposomes is usually no more than 95%or 80%, preferably in the range of 0.5%to 70%, more preferably in the range of 5%to 60%, most preferably in the range of 30%to 60%.
  • the percentage (%) refers to the percentage of the mass (i.e., weight) of the cryoprotectant relative to the total mass of the blank liposome.
  • the blank liposomes can further comprise soybean oil and/or sodium oleate.
  • a weight percentage of the “soybean oil and/or sodium oleate” in the blank liposome is usually in the range of 1%to 90%, preferably in the range of 15%to 80%, more preferably in the range of 20%to 70% (such as in the range of 25%or 62.5%) , most preferably in the range of 20%to 30%, or 60%to 70%, the percentage refers to the mass of the “soybean oil and/or sodium oleate” relative to the total mass of the blank liposome.
  • a mass ratio of the “soybean oil and/or sodium oleate” to the phospholipid in the blank liposome is preferably in the range of 1: 0.1 to 1: 10, more preferably in the range of 1: 0.5 to 1: 5, most preferably in the range of 1: 0.5 to 1: 4 (such as in the range of 1: 1 to 1: 2) .
  • the blank liposomes incude the following components: phospholipid and the ginsenoside derivative of Formula I; the ginsenoside derivative of Formula I, phospholipid and an antioxidant; the ginsenoside derivative of Formula I, phospholipid and a cryoprotectant; the ginsenoside derivative of Formula I, “soybean oil and/or sodium oleate” and phospholipid; the ginsenoside derivative of Formula I, “soybean oil and/or sodium oleate” , phospholipid and an antioxidant; the ginsenoside derivative of Formula I, “soybean oil and/or sodium oleate” , phospholipid and a cryoprotectant; the ginsenoside derivative of Formula I, “soybean oil and/or sodium oleate” , phospholipid, an antioxidant and a cryoprotectant; the ginsenoside derivative of Formula I, phospholipid and cholesterol; the ginsenoside derivative of Formula I, phospholipid and cholesterol; the
  • the blank liposomes consist of the components mentioned-above.
  • the blank liposomes include the following components: the ginsenoside derivative of Formula I, the phospholipid, the cholesterol, the soybean oil and/or the sodium oleate, the antioxidant and the cryoprotectant.
  • the mass ratio of the soybean oil and/or the sodium oleate to the cholesterol in the blank liposome is preferably in the range of 1: 0.1 to 1: 10, more preferably in the range of 1: 0.5 to 1: 5, most preferably in the range of 1: 0.5 to 1: 1.
  • the weight percentage of the cholesterol in the membrane is preferably in the range of 1%to 20%, more preferably in the range of 10%to 20%, a weight percentage of the soybean oil and/or the sodium oleate in the blank liposome is preferably in the range of 1%to 90%, more preferably in the range of 15%to 80%, most preferably in the range of 20%to 70% (such as in the range of 25%or 62.5%, 20%to 30%, or 60%to 70%) .
  • the blank liposomes consist of the phospholipid and the ginsenoside derivative of Formula I.
  • the blank liposomes consist of the ginsenoside derivative of Formula I, the phospholipid and the cholesterol.
  • the blank liposomes consist of the ginsenoside derivative of Formula I, the phospholipid, the cholesterol, the antioxidant and the cryoprotectant.
  • the blank liposomes consist of the ginsenoside derivative of Formula I, the phospholipid, the cholesterol, the soybean oil and/or sodium oleate, the antioxidant and the cryoprotectant.
  • the phospholipid contianied in the liposomes of this invention can be a conventional phospholipid in this field, preferably includes a natural phospholipid, semisynthetic phospholipid, a fully synthetic phospholipid, or any combination thereof.
  • the natural phospholipid is typically derived from soybean, yolk, brain or organs of an animal. It preferably includes natural lecithin, sphingomyelin, phosphatidylcholline, soybean lecithin egg lecithin or cephalin.
  • the semisynthetic phospholipid or the fully synthetic phospholipid can be a conventional semisynthetic phospholipid or fully synthetic phospholipid known in this field, preferably comprises a phospholipid of phosphatidylchollines, phosphatidylserine (PS) , phosphatidylinositol (PI) , a phospholipid of phosphatidylethanolamine, phosphatidylglycerol (DSPG) , dicetyl phosphate (DCP) , a PEG-modified phospholipid, cholesterol succinate (CHS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16: 0 to 18: 1 PC, wherein 16: 0 to 18: 1 refers to thecarbonchain of PC) .
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • DSPG phosphatidylglycerol
  • DCP
  • the semisynthetic or fully synthetic phospholipids such as dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine etc., they can be used as heat-sensitive excipients at the same time.
  • the phospholipid of phosphatidylcholline used in the present invention can be a conventional phospholipid of phosphatidylcholline in this field, preferably comprises hydrogenated soybean lecithin (HSPC) , dipalmitoyl phosphatidylcholine (DPPC) , distearoyl phosphatidylcholine (DSPC) , dimyristoyl phosphatidylcholine (DMPC) , dilauroyl phosphatidylcholine (DLPC) , dioleoyl phosphatidylcholine (DOPC) , phosphatidylcholine (SPC) , monopalmitoyl phosphatidylcholine (MPPC) or glycerol phosphatidylcholine (GPC) .
  • HSPC hydrogenated soybean lecithin
  • DPPC dipalmitoyl phosphatidylcholine
  • DSPC distearoyl phosphatidylcholine
  • the phospholipid of phosphatidylethanolamine used in the present ainvention can be a conventional phospholipid of phosphatidylcholline in this field, preferably comprises 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) , dilauroyl phosphatidylethanolamine (DLPE) , dierucoyl phosphatidylethanolamine (DEPE) , dioleoyl phosphatidylethanolamine (DOPE) , distearoyl phosphatidylethanolamine (DSPE) , dipalmitoyl phosphatidylethanolamine (DPPE) or dimyristoyl phosphatidylethanolamine (DMPE) .
  • POPE 1-palmitoyl-2-oleoyl phosphatidylethanolamine
  • DLPE dilauroyl phosphatidylethanolamine
  • DEPE dierucoyl phosphatidylethanolamine
  • DOPE di
  • the PEG-modified phospholipid used for the present invention can be a conventional PEG-modified phospholipid in this field, preferably comprises phosphatidylethanolamine-PEG (DMPE-PEG) , dipalmitoyl phosphatidylethanolamine-PEG (DPPE-PEG) , distearoyl phosphatidylethanolamine-PEG (DSPE-PEG) , dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) , C8 ceramide-PEG (C8 ceramide-PEG) , C16 ceramide-PEG (C16 ceramide-PEG) , distearoyl phosphatidylethanolamine-PEG-succinyl (DSPE-PEG succinyl) , distearoyl phosphatidylethanolamine-PEG -carboxyl (DSPE-PEG carboxylic acid) , distearoyl phosphatidylethanolamine
  • the relative molecular weight of the PEG mentioned above is preferably in the range of 300 to 50000, , more preferably in the range of 500 to 10000, e g. at about 300, 350, 500, 550, 1000, 2000, 3400, 5000, 10000, 20000, 30000, 40000 or 50000.
  • a number-average molecular weight of the DMPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000 or 5000; a number-average molecular weight of the DPPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000 or 5000; a number-average molecular weight of the DSPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000, 5000, 10000, 20000, 30000 or 40000; a number-average molecular weight of the DOPE-PEG is preferably 350, 550, 750, 1000, 2000, 3000 or 5000; a number-average molecular weight of the C8 Ceramide-PEG is preferably 750, 2000 or 5000; a number-average molecular weight of the DLPE-PEG is preferably 2000 or 5000; a number-average molecular weight of the DSPE-PEG-NHS is preferably 1000, 2000, 5000, 10000, 20
  • the phospholipid used for the present invention can be soybean lecithin, egg lecithin, HSPC, DSPE-PEG (2000) , DMPC, POPE, HSPC, DPPC, soybean lecithin S100, mPEG2000-DSPE or DOPE-PEG.
  • the antioxidant used for the present invention can be a conventional antioxidant in this field, preferably comprises sodium metabisulfite, sodium thiosulfate, propyl gallate, ascorbic acid, ⁇ -tocopherol, ⁇ -hydroxyl acid, flavonoid, a phenylpropanoid phenolic compounds, vitamin E, vitamin C, fumaric acid, cysteine, methionine, butyl hydroxy anisole (BHA) , butyl hydroxytoluene (BHT) , thiodipropionic acid, sulfites (e.g., sodium sulfite) , hydrosulphite (e.g., sodium hydrosulfite) , dithioaminobenzoic acid compounds, citric acid, malic acid, sorbitol, glycerol, propylene glycol, hydroquinone, hydroxycoumarin, ethanolamine, phosphoric acid or phosphorous acid.
  • sulfites e.g
  • the antioxidant for the present invention can be VE, VC, sodium thiosulfate, sodium sulfite.
  • the cryoprotectant for the present invention can be a conventional cryoprotectant in this field, generally comprises a glucide, a polyol, an amino acid or a buffer reagent; wherein the glucide preferably comprises a monosaccharide, a disaccharide or a polysaccharide.
  • the monosaccharide preferably comprises glucose, mannitol, xylitol or sorbitol.
  • the disaccharide preferably comprises sucrose, lactose, galactose or maltose.
  • the polysaccharide preferably comprises trehalose.
  • the polyol preferably comprises mannitol, sorbitol or glycerol.
  • the amino acid preferably comprises ⁇ -amino acid selected from the group consisting of threonine, glycine, glutamic acid, arginine and histidine.
  • the buffer reagent generally refers to a buffer solution.
  • the buffer solution can be a conventional buffer solution in this field, whose pH value is preferably in the range of 3 to 10, more preferably in the range of 5 to 7.
  • the buffer solution preferably comprises an ethanol-acetic acid buffer solution, a tris (hydroxymethyl) aminomethane 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.
  • the cryoprotectant used for the presnert invention can be glucose, sucrose, mannitol, propylene glycol, propylene glycol, galactose, trehalose, lactose, xylitol or phosphate buffer solution.
  • the blank liposome of this innetion can further comprise and encapsulate other excipients within the membrane.
  • excipients can be conventional excipients used for preparing liposome in this field except for the antioxidant and the cryoprotectant, such as the excipient comprise a surfactant, a heat-sensitive excipient, a pH-sensitive material or an ion additive.
  • the surfactant use for the present invention preferably comprises polyethylene glycol (PEG) , polysorbate, Tween surfactant, or a brij surfactant.
  • the number-average molecular weight of the polyethylene glycol is preferably in the range of 200 to 8000 (e g.
  • the polysorbate preferably comprises polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan trioleate, PEG-phosphatidylethanolamine, PEG-polylactic acid, polylysine-poly (lactic-co-glycolic) acid, polyetherimide-polylactic acid, PEG-polycaprolactone, PEG-poly (lactic–co-glycolic) acid, poloxamer 188, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid ether or polyoxyethylene methyl castor oil ether.
  • the heat-sensitive excipient used for the present invention generally comprises a polymer, a drug or a surfactant which can bring heat-sensitivity to the liposome.
  • the polymer preferably comprises polyprene acrylamide, polyprene acrylic acid, polyphosphate, or poly phospholipid-amide copolymer.
  • the drug preferably comprises zedoary turmeric oil, elemene or brucea javanica oil.
  • the surfactant is preferably a Tween surfactant (such as Tween-80) and/or a brij surfactant.
  • the ion additive used for the present application preferably comprises a cationic additive (such as octadecylamine) and/or an anion additive (such as phosphatidic acid and/or phosphatidylserine) .
  • a cationic additive such as octadecylamine
  • an anion additive such as phosphatidic acid and/or phosphatidylserine
  • the weight percentage of the above-discussed excipients can be selected according to the weight percentage of such kind of excipients contained in the ordinary liposome in the art.
  • a weight percentage of the surfactant in the blank liposome is preferably in the range of 0%to 50%, excluding 0%.
  • a weight percentage of the ion additive in the blank liposome is preferably in the range of 0%to 10%, excluding 0%.
  • the blank liposomes of the present invention can be prepared by conventional methods of preparing a liposome. Commonly, an injection method, a reverse evaporation method, a freezing and thawing method, a double emulsion method, an initiative encapsulation method, a precursor liposome preparation method, a film dispersion method, a freeze-drying method, an ammonium sulfate gradient method or a pH gradient method, as well as any combination of above two methods can be adopted.
  • the present invention preferably adopts the first method or the second method as follows, wherein the first method or the blank liposome prepared thereby does not include a cryoprotectant, and the second method or the blank liposome prepared thereby includes a cryoprotectant.
  • the first method free of using a cryoprotectant, includes the steps of:
  • a lipid and a ginsenoside derivative of Formula I optionally, a cholesterol, a hydrophobic antioxidant, soybean oil and/or sodium oleate, a hydrophobic surfactant, a hydrophobic heat-sensitive excipient, a hydrophobic pH sensitive material, and/or a hydrophobic ion additive in an organic solvent to obtain a clear solution; and
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with water optionally containing a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive, to obtain an aqueous mixture, filtering the mixture after an operation of ultrasound, or high pressure homogenization or pushing through a membrane to obtain an aqueous solution containing a blank liposome, freeze-drying to get the blank liposome.
  • water optionally containing a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive
  • the second method includes the steps of:
  • a lipid and a ginsenoside derivative of Formula I optionally a cholesterol, a hydrophobic antioxidant, soybean oil and/or sodium oleate, a hydrophobic surfactant, a hydrophobic heat-sensitive excipient, a hydrophobic pH sensitive material, and/or a hydrophobic ion additive in an organic solvent, to obtain a clear solution, and
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an aqueous solution containing a cryoprotectant and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive to give a mixture, filtering the mixture after an operation of ultrasound, or high pressure homogenization or pushing through a membrane to obtain an aqueous solution containing a blank liposome, freeze-drying to get the blank liposome;
  • the lipid, the ginsenoside derivative of Formula I, the cholesterol, the antioxidant, the soybean oil and/or sodium oleate, the cryoprotectant, the surfactant, the heat-sensitive excipient, the pH sensitive material, and the ion additive in the first or second method are as described above.
  • the organic solvent can be a conventional organic solvent used in the preparation of a liposome in the present invention, which preferably comprises a nitrile solvent, a C 1-4 alcohol solvent, a ketone solvent, an alkane solvent, an ether solvent, a halogenated hydrocarbon solvent, a sulfoxide solvent, or an aldehyde solvent, more preferably comprises a C 1-4 alcohol solvent, a nitrile solvent, an ether solvent or a halogenated hydrocarbon solvent.
  • the nitrile solvent preferably comprises acetonitrile.
  • the C 1-4 alcohol solvent preferably comprises methanol, ethanol, isopropanol or n-butanol.
  • the ether solvent preferably comprises tetrahydrofuran or diethyl ether.
  • the halogenated hydrocarbon solvent preferably comprises chloroform or dichloromethane.
  • the ketone solvent preferably comprises acetone or butanone.
  • the alkane solvent preferably comprises petroleum ether.
  • An amount of the organic solvent can be a conventional amount used in the preparation of a liposome in the present invention, without particularly limited, a general requirement of which is capable of obtaining a clear solution after the mixing of the organic solvent and all the components.
  • a ratio of the organic solvent’s volume to the total mass of all the components dissolved in the organic solvent in step (1) of the first or second method is 5 to 20 mL/g.
  • Step (1) of the first or second method is generally carried out at the temperature of 0 to 80 °C, preferably 10 to 80 °C, more preferably 10 to 65 °C. Based on the common knowledge in this field, in some cases, in order to reach 80 °C, heating is required. Or, when the blank liposome prepared thereby includes a heat-sensitive material, such as protein materials, step (1) of the first or second method is generally carried out at the temperature of 0 °C.
  • step (2) of the first or second method removing the organic solvent of the clear solution obtained in step (1) can be a conventional operation in this field, which is usually conducted with a rotary evaporator or a film evaporator.
  • the temperature at which the organic solvent is removed can be selected according to the organic solvent to be removed, generally is 25 to 80 °C.
  • ultrasound or high pressure homogenization or pushing through a membrane can be a conventional operation in this field.
  • a particle size of the liposome is generally ranging from 0.05 to 0.3 microns.
  • filtration can be a conventional operation in the preparation of the liposome in this field, the purpose of which is to remove bacteria, solid particles and particularly large particle size liposome (in a method of preparing a liposome loaded with the active substance, an unencapsulated free drug can also be removed) etc.
  • the filtration is preferably microporous membrane filtration.
  • the pore size of the microporous membrane is preferably 0.22 micron.
  • the aqueous cryoprotectant solution refers to an aqueous solution formed by mixing the cryoprotectant and water.
  • the aqueous cryoprotectant solution is preferably a 5%to 10%aqueous solution of the cryoprotectant, the percentage refers to the percentage of the mass of the cryoprotectant relative to the total mass of the aqueous solution of the cryoprotectant.
  • An amount of the aqueous cryoprotectant solution is without particular limitation, as long as there is no influence on the formation of the blank liposome, preferably, its volume is the same as that of the organic solvent used in step (1) .
  • the cryoprotectant when the cryoprotectant is a buffer reagent, the cryoprotectant is mixed directly after filming in step (2) .
  • drying can be a conventional operation in this field, preferably is freeze-drying which generally utilizes a freeze dryer.
  • the temperature and time required by the freeze-drying are conventional temperature and time in this field which is without particular limitation.
  • the aqueous solution containing the blank liposome obtained in step (2) is split charging into vials, dried, swept with protective gas (argon or nitrogen) and sealed.
  • the blank liposomes of this invention can be used to prepare a liposome loaded with an active substance which is encapsulated into the membrane of the liposomes, wherein the active substance –in the form of a composition or complex –can include a drug, a cosmetically active substance or a substance with healthcare function.
  • the present invention also provides loaded liposomes each including a blank liposome and an active substance loaded to and encapsulated within the liposome’s membrane, wherein the active substance comprises a drug, a cosmetically active substance, or a substance with healthcare function.
  • the drug can be a conventional drug and preferably includes an antitumor drug, an antifungal drug, an antiviral drug, antibiotics, a non-steroidal anti-inflammatory drug, a calcium ion antagonist, an immunosuppressive agent, an anesthetic, a cardiovascular and vasodilation drug, a gastrointestinal drug, an antidepressant drug, a biological agent, a polynucleotide, an oligonucleotide (including a ribonucleotide and a deoxyribonucleotide) , or any combination thereof.
  • an antitumor drug an antifungal drug, an antiviral drug, antibiotics, a non-steroidal anti-inflammatory drug, a calcium ion antagonist, an immunosuppressive agent, an anesthetic, a cardiovascular and vasodilation drug, a gastrointestinal drug, an antidepressant drug, a biological agent, a polynucleotide, an oligonucleotide (including
  • the antitumor drug can be a conventional antitumor drug and preferably includes paclitaxel, docetaxel, cabazitaxel, irinotecan hydrochloride, hydroxycamptothecin, aminocamptothecin, 7-ethyl-10-hydroxy camptothecin, topotecan hydrochloride, lurtotecan, topotecan, belotecan, cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, satraplatin, miriplatin, amyl platinum, aroplatin (L-NDDP) , carmustine, chlorambucil, melphalan, harringtonine, homoharringtonine, triptolide, tacrolimus, daunorubicin, pingyangmycin, doxorubicin hydrochloride, idarubicin, fluorouracil, cytarabine, methotrex
  • the antifungal drug preferably comprises 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, cefalexin, cefuroxime sodium, cefixime, cefpodoxime, cefmenoxime, cefodizime, cefsulodin, cefazonam, ceftizoxime, cefetamet pivoxil, cefterampivoxil, ceftibuten, cefdinir, cefamandole, cefotiam, ceforanide, cefonicid, ce
  • the antiviral drug preferably includes ribavirin, acyclovir, cytarabine, idoxuridine, acyclovir laurate, acyclovir palmitate, iododeoxyuridine, cyclocytidine, dipalmitoyl cyclocytidine, phosphoric acid formate, phosphoric acid acetate, cimetidine, dipyridamole, rifampin, isoniazid, praziquantel, doxycycline, saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, tipranavir, BMS232632, lamivudine, zidovudine, didanosine (ddi) , zalcitabine (ddc) , stavudine (d4t) , abacavir, adefovirdipivoail (pmea)
  • the antibioticis preferably includes penicillin, penicillin V, amoxicillin, ampicillin, oxacillin, cloxacillin, procaine penicillin, benzathine penicillin, piperacillin, mezlocillin, ticarcillin, azlocillin, mezlocillin, carbenicillin, sulbenicillin, furbucillin, nafcillin, dicloxacillin, pivampicillin, apalcillin, aspoxicillin, pivmecillinam, methicillin, lenampicillin, fomidacillin, flucloxacillin, kanamycin, natamycin, mitomycin, amikacin, tylosin, verteporfin, cefpiramide sodium, netilmicin sulfate, azithromycin, ofloxacin, ciprofloxacin, enoxacin, lomefloxacin, pefloxacin, rufloxaci
  • the calcium ion antagonist preferably includes nimodipine, nifedipine, nicardipine, nitrendipine, verapamil, amlodipine, diltiazem, flunarizine, prenvlamine, gallopamil, tiapamil, or any combination thereof.
  • the non-steroidal anti-inflammatory drug preferably includes indomethacin, aspirin, paracetamol, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib, or any combination thereof.
  • the immunosuppressive agent preferably includes cyclosporin, alprostadil (also known as prostate E-1) , cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, or any combination thereof.
  • the anesthetic preferably incudes halothane, sevoflurane, isoflurane, enflurane, propofol, fentanyl, urethane, lidocaine, procaine, tetracaine, bupivacaine, pelltobarbitalum natricum, chloral hydrate, ketamine, chloralose, morphine, or any combination thereof.
  • tthe cardiovascular and vasodilation drug preferably comprises dabigatran etexilate, alogliptin, polysaccharide sodium, ginkgolides, gingko flavonoid, ginkgo biloba extract, asarone, olmesartan medoxomi, repaglinide, lipoic acid, breviscapine, urapldil, niacin, captopril, losartan, puerarin, tanshinone IIA, sarpogrelate hydrochloride, fluvastatin, pravastatin, simvastatin, lovastatin, simvastatin, mevastatin, cerivastatin, rosuvastatin, atorvastatin calcium or rosuvastatin calcium.
  • the gastrointestinal drug preferably includes omeprazole, lansoprazole, ilaprazole, pantoprazole, rabeprazole, terazosin, esomeprazole, tenatoprazole, leminoprazole, tenatoprazole, disuprazole, lafutidine, or any combination thereof.
  • the antidepressant drug preferably comprises agomelatine, fluoxetine, paroxetine, duloxetine, sertraline, fluvoxamine, citalopram, escitalopram, venlafaxine, mirtazapine, imipramine, amitriptyline, clomipramine, doxepin, remeron, venlafaxime, phenelzine, isocarboxazid, tranylcypromine, or any combination thereof.
  • the polynucleotide and oligonucleotide preferably include a fragment having genetic functions and consisting of the basic groups such as A, T, C, Gor U, for example, SiRNA, RNAi sequence of antisense nucleic acid, microglia NLRP3 gene, or any combination thereof.
  • the biological agent preferably includes a conventional mono-antibody drug in this field, insulin, gamma globulin, antitoxic serum, interferon, interleukin, tumor necrosis factor, active factor of skin, epidermal growth factor, influenza vaccine, hepatitis A vaccine, cancer vaccine, recombinant human acidic fibroblast growth factor, vascular endothelial growth factor 2 monoclonal antibody (VEGFR-2 monoclonal antibody) , or any combination thereof.
  • a conventional mono-antibody drug in this field insulin, gamma globulin, antitoxic serum, interferon, interleukin, tumor necrosis factor, active factor of skin, epidermal growth factor, influenza vaccine, hepatitis A vaccine, cancer vaccine, recombinant human acidic fibroblast growth factor, vascular endothelial growth factor 2 monoclonal antibody (VEGFR-2 monoclonal antibody) , or any combination thereof.
  • the cosmetically active substance generally refers to an active substance which has functions of nourishing, improving the condition of skin and preventing skin disease, preferably comprises ursolic acid, superoxide dismutase (SOD) , biological protein T4N5, vitamin D2, methyl nicotinate, refined snake oil, hyaluronic acid, essential oil or ceramide.
  • SOD superoxide dismutase
  • the substance with healthcare function can be a conventional substance with healthcare function in this filed, preferably includes glycyrrhizin, glycyrrhizic acid, disodiumglycyrrhizinate, methyl glycyrrhizinate, diammoniumglycyrrhizinate, vitamin E, resveratrol, coenzyme Q10, silymarin, anthocyanins, proanthocyanidins, lutein, folic acid, folinic acid, curcumin, emodin, tea polyphenols, epigallocatechin gallate (EGCG) , catechin, blueberry extract, glutathione, oxymatrine, or any combination thereof.
  • EGCG epigallocatechin gallate
  • the mass ratio of the active substance to the ginsenoside derivative of Formula I ranges from 1: 0.1 to 1: 10 (e.g. 1: 0.67) , or more preferably ranges from 1: 1 to 1: 6 (such as 1: 1, 1: 2, 1: 3 or 1: 4) .
  • the drug comprises paclitaxel, docetaxel or irinotecan hydrochloride
  • the liposome comprises phospholipid and a ginsenoside of Formula I
  • the ginsenoiside comprises ginsenoside Rg5
  • the mass ratio of the phospholipid to the ginsenoside Rg5 is in the range of 0.5: 1 to 100: 1, preferably in the range of 0.5: 1 to 20: 1, more preferably in the range of 0.5: 1 to 4: 1 (such as in the range of 0.5: 1 to 2: 1) .
  • the liposome further comprises a cholesterol
  • the mass ratio of the phospholipid to the ginsenoside Rg5 is in the range of 1: 0.01 to 1: 3 (such as in the range of 1: 0.03 to 1: 1) , more preferably in the range of 1: 0.05 to 1: 0.9 (such as in the range of 1: 0.3 to 1: 0.75) , most preferably in the range of 1: 0.1 to 1: 0.9 (such as in the range of 1: 0.1 to 1: 0.5)
  • the mass ratio of the ginsenoside Rg5 to the cholesterol is in the range of 0.1: 1 to 100: 1, preferably in the range of 0.5: 1 to 50: 1, more preferably in the range of 0.5: 1 to 10: 1 (such as in the range of 1.5: 1 to 6: 1, or 5: 1) .
  • the weight percentage of ginsenoside Rg5 in the membrane is in the range of 0.01%to 80%, preferably in the range of 10%to 80%, more preferably in the range of 10%to 40%, most preferably in the range of 20%to 40%
  • the weight percentage of the phospholipid in the membrane is in the range of 5%to 99.9%, preferably in the range of 10%to 70%, more preferably in the range of 30%to 70%, most preferably in the range of 30%to 60%
  • the weight percentage of the cholesterol in the membrane is in the range of 0%to 50%, preferably in the range of 0.5%to 50%, more preferably in the range of 5%to 40%, most preferably in the range of 5%to 30% (such as in the range of 10%to 20%) .
  • the liposome can further include and encapsulate within the liposome’s membrane an antioxidant, a weight percentage of the antioxidant in the blank liposome is no more than 25%, preferably in the range of 0.001%to 15%, more preferably in the range of 0.01%to 10%, most preferably in the range of 0.01%to 5%.
  • the liposome can further include and encapsulate within the liposome’s membrane a cryoprotectant, a weight percentage of the cryoprotectant in the blank liposome is no more than 80%, preferably in the range of 0.5%to 60%, more preferably in the range of 5%to 60%, most preferably in the range of 30%to 60%.
  • the liposome can further include and encapsulate within the liposome’s membrane soybean oil and/or sodium oleate, a weight percentage of the soybean oil and/or sodium oleate in the blank liposome is usually in the range of 1%to 90%, preferably in the range of 15%to 80%, more preferably in the range of 20%to 70% (such as in the range of 25%or 62.5%) , most preferably in the range of 20%to 30%, or 60%to 70%, the percentage refers to the mass of the “soybean oil and/or sodium oleate” relative to the total mass of the blank liposome.
  • a mass ratio of the “soybean oil and/or sodium oleate” to the phospholipid in the blank liposome is preferably in the range of 1: 0.1 to 1: 10, more preferably in the range of 1: 0.5 to 1: 5, most preferably in the range of 1: 0.5 to 1: 4 (such as in the range of 1: 1 to 1: 2) .
  • the liposome can further comprise and encapsulate within the liposome’s membrane other excipients.
  • the other excipients and an amount of the other excipients are the same as that in the blank liposome.
  • the liposome comprises soybean oil and/or sodium oleate, the ginsenoside Rg5 and phospholipid; soybean oil and/or sodium oleate, the ginsenoside Rg5, phospholipid and a cryoprotectant; or soybean oil and/or sodium oleate, the ginsenoside Rg5, phospholipid, a cryoprotectant and an antioxidant.
  • the liposome consists of the components mentioned immediately above.
  • the phospholipid in the liposome, preferably comprises soyabean lecithin, egg lecithin or dimyristoyl phosphatidylcholine, the antioxidant preferably comprises ascorbic acid, vitamin E, vitamin C or threonine, the cryoprotectant preferably comprises glucose, mannitol, xylitol, sucrose, lactose, trehaloseor propanediol.
  • the active substances can be Paclitaxel, Docetaxel, irinotecan, ribavirin, cytarabine, vincristine sulfate, anti-EGF, indomethacin, all-trans-retinoicacid, cisplatin, doxorubicin hydrochloride, clarithromycin, cyclosporin, siRNA, amphotericin B, nimodipine, cabazitaxel, epothilone A, or any combination thereof.
  • the present invention also provides a process for preparing a loaded liposome.
  • the process for preparing the loaded liposome comprises any of Methods A, B, C, and D; and when the liposome does not contain or include a cryoprotectant, the process for preparing the loaded liposome comprises any one of Methods A1, B1, C1, and D1:
  • Method A includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an aqueous solution containing a cryoprotectant and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive to give a mixture, filtering the mixture after an operation of ultrasound, or high pressure homogenization of the mixure or pushing the mixture through a membrane to obtain an aqueous solution containing the liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • a cryoprotectant optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive
  • Method B includes the steps of:
  • lipid and the ginsenoside derivative of Formula I optionally, a cholesterol, a hydrophobic antioxidant, soybean oil and/or sodium oleate, a hydrophobic surfactant, a hydrophobic heat-sensitive excipient, a hydrophobic pH sensitive material, and/or a hydrophobic ion additive in an organic solvent to obtain a clear solution; and
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an active substance and an aqueous solution containing a cryoprotectant and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive to give a mixture, obtaining a solution of a loaded liposome after an operation of ultrasound, or high pressure homogenization of the mixture or pushing the mixture through a membrane, dialyzing and filtering to obtain an aqueous solution containing the liposome loaded with the active substance, freeze drying to give the loaded liposome;
  • Method C includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an aqueous solution containing ammonium sulfate and a cryoprotectant, and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive to give a mixture, obtaining a solution of a blank liposome after an operation of ultrasound, or high pressure homogenization of the mixture or pushing the mixture through a membrane, dialyzing, then mixing with an active substance, filtering to obtain an aqueous solution containing a liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • Method D includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with citric acid and an aqueous solution containing a cryoprotectant and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive to give a mixture, obtaining a solution of a blank liposome after an operation of ultrasound, or high pressure homogenization of the mixture or pushing the mixture through a membrane, mixing the solution with an active substance and an aqueous solution of disodium hydrogen phosphate, filtering to obtain an aqueous solution containing a liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • a cryoprotectant optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive
  • Method A1 includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with water to obtain an aqueous mixture, optionally adding to the aqueous mixture a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive, filtering the mixture after an operation of ultrasound, or high pressure homogenization of the mixture or pushing the mixture through a membrane to obtain an aqueous solution containing a liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • a hydrophilic antioxidant optionally adding to the aqueous mixture a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive
  • filtering the mixture after an operation of ultrasound, or high pressure homogenization of the mixture or pushing the mixture through a membrane to obtain an aque
  • Method B1 includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an active substance and optionally an aqueous solution containing a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive, obtaining a solution containing a liposome loaded with an active substance after an operation of ultrasound, high pressure homogenization or pushing through a membrane, dialyzing and filtering to obtain an aqueous solution containing the liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • Method C1 includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an aqueous solution containing ammonium sulfate and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive, obtaining a solution of a blank liposome after an operation of ultrasound, or high pressure homogenization or pushing through a membrane, dialyzing, then mixing the solution with an active substance, filtering to obtain an aqueous solution containing a liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • aqueous solution containing ammonium sulfate and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive obtaining a solution of a blank liposome after an operation
  • Method D1 includes the steps of:
  • step (2) removing the organic solvent of the clear solution obtained in step (1) , filming, mixing the film with an aqueous solution containing citric acid and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive, obtaining a solution of a blank liposome after an operation of ultrasound, or high pressure homogenization or pushing through a membrane, then mixing the blank liposome solution with an active substance and, filtering to obtain an aqueous solution containing a liposome loaded with the active substance, freeze drying to give the loaded liposome.
  • aqueous solution containing citric acid and optionally a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and/or a hydrophilic ion additive obtaining a solution of a blank liposome after an operation of ultrasound, or high pressure homogenization
  • Each condition or parameter in Method A, B, C, D, A1, B1, C1, or D1 described above is the same as defined above in the first or second method for preparing blank liposomes of the present invention, and the weight ratio of the active substance to the ginsenoside derivative of Formula I is 1: 0.1 to 1: 10 or 1: 2 to 1: 6.
  • dialysis is conducted in a glucose aqueous solution or pure water for 5-20 hours, preferably 12 hours.
  • Method B, C, B1 or C1 dialysis is conducted before the operation of ultrasound, high pressure homogenization of the mixture or pushing the mixture through a membrane.
  • dialysis preferably comprise putting a blank liposome solution or a loaded liposome solution in an aqueous solution of glucose (such as 0.15mol/L) or purified water to give a mixed solution.
  • a mass fraction of the ammonium sulfate in the aqueous solution of ammonium sulfate and the cryoprotectant or the aqueous solution of ammonium sulfate is without particular limitation, which can be a conventional mass fraction used for preparing a liposome through an ammonium sulfate gradient method in this field.
  • the concentration of ammonium sulfate in the aqueous solution containing ammonium sulfate and a cryoprotectant or in the aqueous solution containing ammonium sulfate is 1-15%or preferably 6.6%, wherein the percentage is the weight of ammonium sulfate over the total weight of the aqueous solution.
  • the mixture is preferably kept in a heated environment, preferably in an environment of 30-80 °C for 5 minutes to an hour before filtering.
  • a concentration and an amount of the aqueous solution of citric acid are without particular limitation, which can be conventional concentration and amount used for preparing a liposome through a pH-gradient method in this field.
  • the mass concentration of citric acid in its aqueous solution is preferably in the range of 1%to 15%, more preferably 5.76%, the percentage refers to the mass of the citric acid relative to the total mass of the aqueous solution of citric acid.
  • a concentration and an amount of the aqueous solution of disodium hydrogen phosphate are without particular limitation, which can be conventional concentration and amount for preparing a liposome through a pH-gradient method in this field.
  • the mass concentration of disodium hydrogen phosphate in its aqueous solution is preferably in the range of 5%to 20%, more preferably 7.1%.
  • An amount of the aqueous solution of disodium hydrogen phosphate is generally capable of keeping the pH of the aqueous solution containing the liposome loaded with the active substance between 6.5 and 7.5 (such as 7.3) .
  • pure water is added to adjust the pH of the aqueous solution of the liposome loaded with the active substance between 6.5 and 7.5 (such as 7.3) before filtering.
  • Method D or D1 may further include an operation of warm-keeping before filtering.
  • the operation of warm-keeping preferably comprises keeping warm at a termperature ranging from 30 °C to 80 °C (such as 60 °C) for 5 minutes to 1 hour (such as 30 minutes) .
  • the active substance is used as a solution in water or organic solvent, preferably at a concentration of 1-20%by weight or by volume.
  • the cryoprotectant can be added after an aqueous solution containing the liposome loaded with the active substance is obtained and before drying.
  • an active substance may also be used in the form of an aqueous solution of the active substance or an organic solution of the active substance according to the hyrophilicity or hydrophobicity of the active substance.
  • the mass concentration of the aqueous solution of the active substance or the organic solution of the active substance may be without particular limitation, preferably a mass volume percentage of the aqueous solution or the organic solution is in the range of 1%to 20%, the percentage refers to the mass (g) of the active substance relative to the total volume (mL) of the aqueous solution of the active substance or the organic solution of the active substance.
  • the organic solvent contained in the organic solution of the active substance can be a conventional organic solvent in this field, which is capable of dissolving the active substance well.
  • the organic solvent is preferably a sulfoxide solvent, such as dimethyl sulfoxide (DMSO) .
  • a mass ratio of the active substance can be a conventional mass ratio in this field.
  • the mass ratio of the active substance to the ginsenoside derivative of Formula I ranges from 1: 0.1 to 1: 10, or more preferably from 1: 2 to 1: 6 (such as from 1: 3 or 1: 4) .
  • a particle size of the blank liposome or the loaded liposome can be a conventional particle size in this field. It is preferably in the range of 30 to 2000nm, more preferably in the range of 30 to 300nm, most preferably in the range of 50 to 300nm.
  • the encapsulated efficiency of the loaded liposome is preferably 80%or more, more preferably 90%or more, or most preferably 95%or more.
  • a drug administration of the loaded liposome can be a route of administration in this field, preferably is an injection administration, an oral administration or cutaneous penetration used for the treatment of diseases and/or medical health care.
  • the loaded liposome is generally prepared in the form suitable for injection, lyophilized injection, oral administration, or topical administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
  • the loaded liposome is added into normal saline, phosphate buffered solution or 5%glucose aqueous solution to prepare an injection solution for injection.
  • the loaded liposome when the active substance is an antitumor drug, the loaded liposome generally has targeting effect on tumor cells, anti-multi-drug resistance effect, synergism and attenuation effects and synergism of drug.
  • R 1 , R 2 , R 4 , and R 5 are as defined as above;
  • R 7 and R 8 are as defined in claims 1 and 2;
  • ginsenoside derivative of Formula I is not any of the following compounds:
  • R 3 is preferably
  • ginsenoside derivatives of this invention examples include those shown below in Table 3.
  • the present invention further provides a method for preparing the ginsenoside derivatives of Formula I, which includes a deprotection reaction in a solvent for acetylated compounds of Formula I.
  • a deprotection reaction in a solvent for acetylated compounds of Formula I.
  • all the hydroxies in the compounds of Formula I have been acetylized with acetic anhydride except the hydroxy at the 20 th position.
  • the acetyl group of the acetylated compounds can be replaced by some other conventional hydroxy protection groups in the related organic chemical field.
  • the organic solvents used in the methods of this invention can be selected from alcohol solvents or ether solvents, or a mixture of alcohol solvents and ether solvents. In the mixture solvents, the volume ratio of the alcohol solvent and ether solvents was 1: 1.
  • the alcohol solvent is preferably methanol, and the ether solvent is preferably dioxane.
  • the alkaline used in the deprotection reaction is preferably a metal hydroxide, such as Potassium hydroxide (KOH) , the quantity of alkaline was basing on a suitable quantity normally.
  • KOH Potassium hydroxide
  • the deprotection reaction temperature is preferably the temperature at which the solvent is refluxed at atmospheric pressure, such as 60 to 110 °C.
  • the extent of the deprotection reaction can be detected or measured in a conventional method and the disapprearance of raw material can mark the end or completion of the reaction.
  • the deprotection reaction is carried out for 8 to 15 hours, such as 10 to 12 hours.
  • a method for preapring Rg5H includes the following steps: dissolving the Rg5H acetylated compound in a solvent and removing the acetyl protecting group as depicted below wherein the conditions for the deprotection reactions are the same as those descriebd above.
  • a process for preparing acetylated Rg5H includes the following steps: carrying out a dehydroxylation reaction at position 20 in an organic solvent for acetylated Rg3.
  • preferred organic solvent are halogenated hydrocarbon solvents, such as dichloromethanme; preferred reaction agents are boron trifluoride etherate and triethylsilane; the preferred mass ratio of boron trifluoride etherate and triethylsilane ranges from 1: 0.5 to 1: 5; the preferred mass ratio of triethylsilane and Rg3 acetylated compound ranges from 1: 5 to 1: 20; the prefered reaction temperature is room temperature; the conventional detection method is recommended for measuring the reaction progress or extent; the disappearance of raw material is recommended as the end of reaction; and the preferred reaction time is 5 to 10 minutes.
  • halogenated hydrocarbon solvents such as dichloromethanme
  • preferred reaction agents are boron trifluoride etherate and triethylsilane
  • the preferred mass ratio of boron trifluoride etherate and triethylsilane ranges from 1: 0.5 to 1: 5
  • the same method can be used to prepare ginsenoside Rh3H, Rh4H, and Rk4H after replacing the raw material.
  • acetylated Rg5H of the present invention can be prepared in a method that incudes the following steps: dissolving ginsenoside Rg3 (racemic form, R form, or S form) and catalyst in an organic solvent, and then carrying out the acylation reaction with acetic anhydride to give rise to acetylated Rg3 compounds.
  • the organic solvent can be an alkaline organic solvent (such as pyridine) , and the alkaline solvent can function as both a solvent and an acid binding agent.
  • alkaline organic solvent such as pyridine
  • the organic solvent can also be a conventional non-alkaline solvent plus some other acid binding agent; a suitable quantity of the organic solvent is recommended; the catalyst can be DMAP, the catalyst quantity is recommended a suitable quantity as normally; the mass ratio of ginsenoside Rg3 and acetic anhydride preferably ranages from 0.1g/mL to 5g/mL; the reaction temperature preferably is the room temperature; a conventional detection method is recommended; the disappearance of raw material is recommended as indication of the completion of the reaction; the reaction time is preferably 8 to 15 hours (e.g., 10 hours) .
  • the following steps can be taken: concentrating the reaction solution, re-dissolving in an organic solvent, washing the solution with saturated NaHCO 3 saqueous solution 3 times; drying the organic solution with some amount of anhydrous Na 2 SO 4 ; concentrating the solution to get the intended product.
  • Acetylated Rh3H, acetylated Rh4H, and acetylated Rk4H can be prepared in the same method except with different starting materials.
  • the mentioned optimized conditions can be optionally combined based on the common knowledge in this field to obtain preferred embodiments of the compounds of this invention.
  • room temperature refers to 10 to 30 °C.
  • a density of the organic solution of the active substance depends on the kind of the organic solvent, for example, when the organic solvent is DMSO, the density of the organic solution of the active substance is 1.1g/mL.
  • the reagents and raw materials used to practice the invention herein are commercially available.
  • the ginsenoside derivatives of this invention have exhibited excellent activities, low hemolysis, and suitability for liposome membrane.
  • the blank liposomes of the present invention have the advantages of high efficiency, good safety, good stability, enhanced targeting, good uniformity, stable and reliable quality, and convenient preparations.
  • the active substance is an antitumor drug
  • the loaded liposome generally has targeting effect on tumor cells, anti-multi-drug resistance effect, synergism and attenuation effects and synergism of drug.
  • Fig. 1 shows cell survival rates of blank Rg5H, blank Rg5, Blank Rp1, Blank Rg3H and Blank GQ against human lung cancer cell line (A549) .
  • Fig. 2 shows cell survival rates of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3Hand Taxol+GQ against human lung cancer cell line (A549) .
  • Fig. 3 shows cell survival rates of blank Rg5H, blank Rg5, Blank Rp1, Blank Rg3H and Blank GQ against paclitaxel-resistant human lung cancer cell line (A549/T) .
  • Fig. 4 shows cell survival rates graph of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against paclitaxel-resistant human lung cancer cell line (A549/T) .
  • Fig. 5 shows a distribution in vivo figure of IR783 fluorescence that recorded of the Control group, Rg5H group, Rg5 group, Rp1 group, Rg3H group and GQ group at the 2 nd , 6 th , and 10 th hour by a live imager.
  • Fig. 5-A, Fig. 5-B and Fig. 5-C show respectively in vivo distribution figures of IR783 fluorescence of the control group at the 2nd, 6th and 10th hours that recorded by the live imager;
  • Fig. 5-D Fig.
  • Fig. 5-E and Fig. 5-F show respectively in vivo distribution figures of IR783 fluorescence of the Rg5H group at 2nd, 6th and 10th hours that recorded by the live imager;
  • Fig. 5-G, Fig. 5-H and Fig. 5-I respectively show in vivo distribution figures of IR783 fluorescence of the Rg5 group at the 2nd, 6th and 10th hours that recorded by the live imager;
  • Fig. 5-G, Fig. 5-K and Fig. 5-L respectively show in vivo distribution figures of IR783 fluorescence of the Rp1 group at the 2nd, 6th and 10th hours that recorded by the live imager;
  • Fig. 5-O respectively show in vivo distribution figures of IR783 fluorescence of the Rg3H group at the 2nd, 6th and 10th hours that recorded by the live imager; and Fig. 5-P, Fig. 5-Q and Fig. 5-R respectively show in vivo distribution figures of IR783 fluorescence of the GQ group at the 2nd, 6th and 10th hours that recorded by the live imager.
  • Fig. 6 shows in vivo distribution figures of IR783 fluorescence recorded at the 12th hour by the live imager;
  • A, B, C, D, E and F are for the control group, Rg5H, Rg5, Rp1, Rg3H, GQ groups, respectively.
  • Fig. 7 shows an antitumor graph of the control group, Taxol +Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H group and Taxol+GQ group against human lung cancer cell line (A549) .
  • Fig. 8 shows an antitumor graph of the control group, Taxol +Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H and Taxol+GQ group against paclitaxel-resistant human lung cancer cell line (A549/T) .
  • Fig. 9 shows a cell survival rate graph of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ against human breast cancer cell line (MCF-7) .
  • Fig. 10 shows a cell survival rate graph of Taxol +Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H and Taxol+GQ group against human breast cancer cell line (MCF-7) .
  • Fig. 11 shows a cell survival rate graph of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ against paclitaxel-resistant human breast cancer cell line (MCF-7/T) .
  • Fig. 12 shows a cell survival rate graph of Taxol +Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H and Taxol+GQ group against paclitaxel-resistant human breast cancer cell line (MCF-7/T) .
  • Fig. 13 shows an antitumor graph of the control group, Taxol +Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H and Taxol+GQ group against human breast cancer cell line (MCF- 7) .
  • Fig. 14 shows an antitumor graph of the control group, Taxol +Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H and Taxol+GQ group against paclitaxel-resistant human breast cancer cell line (MCF-7/T) .
  • ginsenoside derivatives used in the following examples were prepared according to published methods. For instance, 25-methoxy-ginsenoside Rg3 was prepared by the method described in “Study on the components of acid degradation of Panax quinquefolium L. saponins from stems and leaves” (Yu Bozhong’s Master degee thesis, Jilin University, 2009) ; 20 (S) -methoxy-PPD and 12, 20-di-methyl-PPD were prepared in the method as described in Example 5 of China Patent Application Patent No.
  • Pre-HPLC HB-DAC-100 Preparing High Pressure Liquid Chromatography system, pump NP7060 ⁇ 2 sets; pump NP7010 ⁇ 1 set; pump NU3000 ⁇ 1 set; DAC-100mm column ⁇ 1 set; Alltech ELSD detector, produced by Jiangsu Hanbon Sci. &Tech Co., Ltd.
  • the HPLC equipment is DAC-100 system from Jiangsu Hanbon Sci. &Tech Co., Ltd.
  • the stationary phase is 10um-120A C18 from Fuji Silysia Chemical Ltd.
  • Alltech ELSD detector flow rate is 400 mL/min
  • working pressure is 8MPa
  • UV 203nm two gradient mobile phase of 75%methanol aqueous solution and 95%methanol aqueous solution.
  • a sample was dissolved in a 75%methanol aqueous solution.
  • the Pre-HPLC system was balanced with a 75%methanol mobile phase for 20 minutes and loaded the sample solution, then its column was washed the column by 75%methanol for 100 minutes and the different factions were collected before the column was washed with 95%methanol around 20 minutes until the TLC plate did not have a black point.
  • X%for the methanol solution refers to the volume ratio between methanol and the total volume of the solution.
  • 75%methanol means the volume of pure methanol is 75%of the total volume of methanol and water.
  • the crude Pseudo-ginsenoside GQ was purified with a Preparing High Pressure Liquid Chromatography (Pre-HPLC) system, the gradient elution was required, wherein methanol and water as mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector.
  • a Preparing High Pressure Liquid Chromatography (Pre-HPLC) system the gradient elution was required, wherein methanol and water as mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector.
  • 4.2 g 98%min (20S, 24S) -pseudo-ginsenoside GQ and 0.2g (20S, 24R) -pseudo-ginsenoside GQ was obtaied.
  • (20S, 24S) -pseudo-ginsenoside HQ, (20S, 24R) -pseudo-ginsenoside HQ, (20S, 24S) -pseudo-ginsenoside DQ and (20S, 24R) -pseudo-ginsenoside DQ were prepared in the same method as described above except that 20 (S) -Rg3 was repaced with 20 (S) -Rh2 and 20 (S) -PPD, respectively.
  • ginsenosde Rg5 1.5g ginsenosde Rg5 was dissolved in 150 mL ethanol, and 300 mg 5%Pd/C was added to the solution which was then bubbled with hydrogen (H 2 ) for 6 hours at 40 °C. After the rreaction was completed, the solution was filtered, extracted with ethyl acetate 3 times (200 mL each time) , concentrated to give crude ginsenoside Rp1.
  • the crude ginsenoside Rp1 was purified with a Preparing High Pressure Liquid Chromatography (Pre-HPLC) system, the gradient elution was required, methanol and water was the mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector. After HPLC purification, 3.6 g ginsenoside Rp1 was obtained (min 98%purity) .
  • Pre-HPLC Preparing High Pressure Liquid Chromatography
  • Iso-Rg3H was prepared in the method described in Example 3, except that the starting material Rg5 was replaced with acetylated iso-Rg3 (E) (Intermediate-1, from Example 1.
  • the crude 20 (S) -pseudo ginsenoside GD was purified with a preparing High Pressure Liquid Chromatography (pre-HPLC) system, the gradient elution was required, wherein methanol and water mixure was the mobile phase, ⁇ 10.0 ⁇ m C18 was the stationary phase, ELSD was detector. After pre-HPLC purificiation, 2.7g 20 (S) -pseudo ginsenoside GD (min 98%purity) was obtained.
  • 20 (R) -pseudo ginsenoside GD was prepared in the same method with 20 (R) -Rg3 as the starting material in place of 20 (S) -Rg3.
  • 20 (S) -pseudo ginsenoside GHD was prepared in the same method as described in Example 5, except that 20 (S) -Rg3 was replaced with 20 (S) -Rh2.
  • the crude 25-methoxy-iso-ginsenoside Rg3 was purified by a preparing High Pressure Liquid Chromatography (Pre-HPLC) system, the gradient elution was required, wherein methanol and water as mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector. After pre-HPLC purification, 25-methoxy-iso-ginsenoside Rg3 (E) was obtained.
  • Pre-HPLC High Pressure Liquid Chromatography
  • pseudo-ginsensode GQ acetylated compound 10 g pseudo-ginsensode GQ acetylated compound was dissolved in 200 mL toluene, 1.5 g boron trifluoride diethyl etherate was added, stirred and heated to 90 °C for 4 hours until the starting material disappeared as determined by TLC. The solution was cooled to the room temperature and washed with saturated NaHCO 3 solution 3 times (100 mL each time) , dried over anhydrous Na 2 SO 4 , concentrated and dried to give pseudo-ginsenoside GP acetylated compound (Intermediate-4) .
  • the crude Rg5H was purified by a preparing High Pressure Liquid Chromatography (Pre-HPLC) system, the gradient elution was required, wherein methanol and water as mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector. After pre-HPLC purified hereby getting 98%min ginsenoside Rg5H.
  • Pre-HPLC High Pressure Liquid Chromatography
  • Preparation embodiment 11 and Preparation embodiment 12 replaced the raw material with 20 (R) -Rh2 acetylated compound or 20 (S) -Rh2 acetylated compound, then prepared 98%min ginsenoisde Rh3H1 (E) , ginsenoisde Rh3H1 (Z) and ginsenoisde Rk2H.
  • Ginsenoside Rh3H1 (Z) :
  • ginsenoisde Rh4H was prepare with 98%minimal purity.
  • ginsenoisde Rk4H1 (E) , ginsenoisde Rk4H1 (Z) and ginsenoisde Rg6H were prepared with min 98%purity.
  • the crude product was purified by a preparing High Pressure Liquid Chromatography (pre-HPLC) system, the gradient elution was required, wherein methanol and water as mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector. After HPLC purified hereby getting 11.4g 98%min 3-sodium sulfate-pseudo-ginsenoside DQ (SC-DQ) .
  • pre-HPLC High Pressure Liquid Chromatography
  • the crude product was purified by a preparing High Pressure Liquid Chromatography (pre-HPLC) system, the gradient elution was required, wherein methanol and water as mobile phase, ⁇ 10.0 ⁇ m C18 as stationary phase, ELSD detector. After HPLC purified hereby getting 11.4g 98%min 3-sodium sulfate-iso-protopanaxadiol (E) (SC-iso-PPD (E) ) .
  • pre-HPLC High Pressure Liquid Chromatography
  • Egg lecithin 0.9g, ginsenoside Rg5H 0.4g and VE 0.1 g were added into 20 mL chloroform and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%glucose aqueous solution (the percentage refers to the mass of the glucose relative to the total mass of the glucose aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing ginsenoside Rg5H blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside Rg5H blank liposome.
  • argon or nitrogen was introduced, sealed to give the ginsenoside Rg5H blank liposome.
  • Embodiment 2 The preparation of pseudo-ginsenoside GQ blank liposome
  • Egg lecithin 0.9g, pseudo-ginsenoside GQ 0.4g and VE 0.1g were added into 20 mL chloroform and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%glucose aqueous solution (the percentage refers to the mass of the glucose relative to the total mass of the glucose aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing pseudo-ginsenoside GQ blank liposome.
  • the aqueous solution was split charging into vials and each vial contained 180mg liposome.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the pseudo-ginsenoside GQ blank liposome.
  • argon or nitrogen argon or nitrogen
  • Embodiment 3 The preparation of ginsenoside Rg3H blank liposome
  • Soybean lecithin 0.8g and iso-ginsenoside Rg3H 0.2g were added into 20 mL methanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 50 to 60°C to form a film, and 20 mL 5%sucrose aqueous solution (the percentage refers to the mass of the sucrose relative to the total mass of the sucrose aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing iso-ginsenoside Rg3H blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the iso-ginsenoside Rg3H blank liposome. By test, the D10 of the liposome was 69nm, D50 was 99nm, D90 was 149nm.
  • Embodiment 4 The preparation of pseudo-ginsenoside HQ blank liposome
  • HSPC 0.7g, pseudo-ginsenoside HQ 0.1g and cholesterol 0.2g were added into 20 mL ethanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 60 to 65°C to form a film, and 20 mL 5%mannitol aqueous solution (the percentage refers to the mass of the mannitol relative to the total mass of the mannitol aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing pseudo-ginsenoside HQ blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the pseudo-ginsenoside HQ blank liposome.
  • argon or nitrogen was introduced, sealed to give the pseudo-ginsenoside HQ blank liposome.
  • Embodiment 5 The preparation of pseudo-ginsenoside GD blank liposome
  • Egg lecithin 0.9g, pseudo-ginsenoside GD 0.4g, soybean oil 0.2g and VC 0.1g were added into 20 mL isopropyl alcohol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 60 to 65°C to form a film, and 20 mL 5%propylene glycol aqueous solution (the percentage refers to the mass of the propylene glycol relative to the total mass of the propylene glycol aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing pseudo-ginsenoside GD blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the pseudo-ginsenoside GD blank liposome.
  • argon or nitrogen argon or nitrogen
  • Embodiment 6 The preparation of ginsenoside Rh3H1 (E) blank liposome
  • Egg lecithin 0.5g, ginsenoside Rh3H1 (E) 0.4g and VC 0.05g were added into 20 mL n-butyl alcohol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 65 to 75°C to form a film, and 20 mL 5%glycerol aqueous solution (the percentage refers to the mass of the glycerol relative to the total mass of the glycerol aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.45 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing ginsenoside Rh3H1 (E) blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside Rh3H1 (E) blank liposome.
  • a protective gas argon or nitrogen
  • Embodiment 7 The preparation of pseudo-ginsenoside PF11 blank liposome
  • Egg lecithin 0.7g, pseudo-ginsenoside PF11 0.4g and sodium thiosulfate 0.01g were added into 20 mL THF and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 60 to 65°C to form a film, and 20 mL 5%galactose aqueous solution (the percentage refers to the mass of the galactose relative to the total mass of the galactose aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.45 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing ginsenoside PF11 blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside PF11 blank liposome.
  • argon or nitrogen was introduced, sealed to give the ginsenoside PF11 blank liposome.
  • Embodiment 8 The preparation of ginsenoside Rp1 blank liposome
  • Egg lecithin 0.7g, ginsenoside Rp1 0.4g and sodium thiosulfate 0.01g were added into 20 mL ether and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 30 to 35°C to form a film, and 20 mL 5%galactose aqueous solution (the percentage refers to the mass of the galactose relative to the total mass of the galactose aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing ginsenoside Rp1 blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside Rp1 blank liposome.
  • argon or nitrogen was introduced, sealed to give the ginsenoside Rp1 blank liposome.
  • Embodiment 9 The preparation of 25-methoxy-iso-ginsenoside Rg3 (Iso-Rg3Me) blank liposome
  • Soybean lecithin S100 0.9g, 25-methoxy-iso-ginsenoside Rg3 0.4g and sodium sulfite 0.01g were added into 20 mL ether and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a film evaporator at 40 to 50°C to form a film, and 20 mL 5%phosphate buffer (PBS) solution (the percentage refers to the mass of the phosphate buffer solution (PBS) relative to the total mass of the phosphate buffer solution (PBS) was added.
  • An operation of homogenization by a high pressure homogenize was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing Iso-Rg3Me blank liposome. Then the aqueous solution was split charging into vials and each vial contained 180mg liposome. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the Iso-Rg3Me blank liposome.
  • argon or nitrogen was introduced, sealed to give the Iso-Rg3Me blank liposome.
  • the D10 of the liposome was 64nm
  • D50 was 123nm
  • D90 was 223nm.
  • Embodiment 10 The preparation of pseudo-ginsenoside GQ paclitaxel liposome
  • Egg lecithin 0.8g, pseudo-ginsenoside GQ 0.4g, paclitaxel 0.1g, VE 0.1g and soybean oil 0.1g were added into 20 mL chloroform and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%lactose aqueous solution (the percentage refers to the mass of the lactose relative to the total mass of the lactose aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron.
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing pseudo-ginsenoside GQ paclitaxel liposome. Then the aqueous solution was split charging into vials and each vial contained 30mg paclitaxel. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the pseudo-ginsenoside GQ paclitaxel liposome. By test, the D10 of the liposome was 72nm, D50 was 134nm, D90 was 246nm. . The encapsulated efficiency was more than 95%.
  • Embodiment 11 The preparation of ginsenoside Rk1H docetaxel liposome
  • Soybean lecithin 0.7g, ginsenoside Rk1H 0.2g, cholesterol 0.1g, Sodium oleate 0.1g, VC 0.1g and docetaxel 0.1g were added into 20 mL acetonitrile and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 50 to 60°C to form a film, and 20 mL trehalose aqueous solution (the percentage refers to the mass of the trehalose relative to the total mass of the trehalose aqueous solution) was added.
  • Embodiment 12 The preparation of ginsenoside Rg5H1 irinotecan liposome
  • DSPE-PEG (2000) 0.9 g, ginsenoside Rg5H1 0.3 g and vitamin C 0.1 g were added into 20 mL ethanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotatory evaporator at 50 to 60°C to form a film, and 20 mL 6.6%ammonium sulfate aqueous solution (the percentage refers to the mass of the ammonium sulfate relative to the total mass of the ammonium sulfate aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the blank liposome was between 0.1 and 0.3 micron, thereby obtaining a solution of the blank liposome.
  • the solution of the blank liposome was dialyzed for 12 hours in some amount of 0.15 M (0.15mol/L) trehalose solution, then a corresponding mass of trehalose was added according to a volume of the dialyzed blank liposome solution to make a mass fraction of the trehalose in the blank liposome solution reach 10%, the percentage refers to the mass of the trehalose relative to the total mass of the blank liposome solution.
  • 1mL irinotecan hydrochloride aqueous solution with a mass fraction of 20% (irinotecan hydrochloride 0.2g) was added, and kept for 30 minutes in a water bath at 37 °C.
  • aqueous solution containing ginsenoside Rg5H1 irinotecan hydrochloride liposome was split charging into vials and each vial contained 40 mg irinotecan hydrochloride.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside Rg5H1 irinotecan hydrochloride liposome.
  • argon or nitrogen argon or nitrogen
  • Embodiment 13 The preparation of iso-ginsenoside Rg3 (E) ribavirin liposome
  • DMPC 0.8g, iso-ginsenoside Rg3 (E) 0.3g, VC 0.1g and ribavirin 0.1g were added into 20 mL ethanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a film evaporator at 40 to 50°C to form a film, and 20 mL 5%xylitol aqueous solution (the percentage refers to the mass of the xylitol relative to the total mass of the xylitol aqueous solution) was added.
  • Embodiment 14 The preparation of iso-ginsenoside Rg3 (O) cytarabine liposome
  • Egg lecithin 0.7g, iso-ginsenoside Rg3 (O) 0.3g and vitamin C 0.5g were added into 20 mL ethanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotatory evaporator at 50 to 60°C to form a film, and 20 mL 6.6%ammonium sulfate aqueous solution (the percentage refers to the mass of the ammonium sulfate relative to the total mass of the ammonium sulfate aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the blank liposome was between 0.1 and 0.3 micron, thereby obtaining a solution of the blank liposome.
  • the solution of the blank liposome was dialyzed for 12 hours in some amount of 0.15M (0.15mol/L) trehalose solution, then a corresponding mass of trehalose was added according to a volume of the dialyzed blank liposome solution to make a mass fraction of the trehalose in the blank liposome solution reach 10%, the percentage refers to the mass of the trehalose relative to the total mass of the blank liposome solution.
  • 1mL cytarabine aqueous solution with a mass fraction of 20% (cytarabine 0.2g) was added, and kept for 30 minutes in a water bath at 37°C.
  • aqueous solution containing iso-ginsenoside Rg3 (O) cytarabine liposome was split charging into vials and each vial contained 100mg cytarabine.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the iso-ginsenoside Rg3 (O) cytarabine liposome.
  • argon or nitrogen argon or nitrogen
  • Embodiment 15 The preparation of iso-ginsenoside Rg3 (Z) vincristine sulfate liposome
  • POPE 0.6g and iso-ginsenoside Rg3 (Z) 0.3g were added into 20 mL mixture solvents (volume ratio of ethanol and chloroform was 1: 1) and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotatory evaporator at 50 to 55°C to form a film, and 20 mL 6.6%ammonium sulfate aqueous solution (the percentage refers to the mass of the ammonium sulfate relative to the total mass of the ammonium sulfate aqueous solution) was added.
  • An operation of ultrasound was carried out until the particle size of the blank liposome was between 0.1 and 0.3 micron, thereby obtained a solution of the blank liposome.
  • the solution of the blank liposome was dialyzed for 12 hours in some amount of 0.15M (0.15mol/L) glucose solution, then a corresponding mass of trehalose was added according to a volume of the dialyzed blank liposome solution to make a mass fraction of the trehalose in the blank liposome solution reach 10%, the percentage refers to the mass of the trehalose relative to the total mass of the blank liposome solution.
  • 1mL vincristine sulfate aqueous solution with a mass fraction of 20% (vincristine sulfate 0.2g) was added, and kept for 30 minutes in a water bath at 37°C.
  • aqueous solution containing iso-ginsenoside Rg3 (Z) vincristine sulfate liposome was split charging into vials and each vial contained 1mg vincristine sulfate.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the iso-ginsenoside Rg3 (Z) vincristine sulfate liposome.
  • argon or nitrogen argon or nitrogen
  • Embodiment 16 The preparation of ginsenoside Rh3H Anti-EGF liposome
  • HSPC 0.6g and ginsenoside Rh3H 0.3g were added into 20 mL ethanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 50 to 60°C to form a film, and 20 mL purified water was added.
  • An operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron thereby obtained an aqueous solution containing ginsenoside Rh3H blank liposome.
  • Embodiment 17 The preparation of pseudo-ginsenoside GQ Indometacin liposome
  • Soybean lecithin 0.9g, iso-ginsenoside GP 0.2g, sodium oleate 0.1g and indometacin 0.1g were added into 20 mL acetonitrile and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 50 to 60°C to form a film, and 20 mL trehalose aqueous solution (the percentage refers to the mass of the trehalose relative to the total mass of the trehalose aqueous solution) was added.
  • Embodiment 18 The preparation of ginsenoside Rg5H all-trans-retinoicacid liposome
  • DPPC 0.9g, ginsenoside Rg5H 0.4g and all-trans-retinoicacid 0.1g were added into 20 mL chloroform and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%lactose aqueous solution (the percentage refers to the mass of the lactose relative to the total mass of the lactose aqueous solution) was added.
  • Embodiment 19 The preparation of iso-ginsenoside Rh2 (E) cisplatin liposome
  • Egg lecithin 0.8g, iso-ginsenoside Rh2 (E) 0.4g, VE 0.1g and cisplatin 0.1g were added into 20 mL methanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%lactose aqueous solution (the percentage refers to the mass of the lactose relative to the total mass of the lactose aqueous solution) was added.
  • Embodiment 20 The preparation of ginsenoside Rk4H1 (E) doxorubicin HCl liposome
  • Soybean lecithin S100 0.9g, ginsenoside Rk4H1 0.3g and VE 0.1g were added into 20 mL ethanol and stirred to form a clear solution at 40 to 50°C.
  • the organic solvent was removed by a film evaporator at 50 to 55°C to form a film, and 20 mL Phosphate buffer solution (PBS) was added.
  • PBS Phosphate buffer solution
  • An operation of homogenization by a high pressure homogenizer was carried out until the particle size of the liposome was between 0.1 and 0.3 micron thereby obtaining an aqueous solution containing ginsenoside Rk4H1 blank liposome.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside Rk4H1 doxorubicin liposome.
  • a protective gas argon or nitrogen
  • the D10 of the liposome was 66nm
  • D50 was 129nm
  • D90 was 255nm.
  • the encapsulated efficiency was more than 95%.
  • Embodiment 21 The preparation of iso-ginsenoside Rg2 Clarithromycin liposome
  • Egg lecithin 0.8g, iso-ginsenoside Rg2 0.4g, VE 0.1g and clarithromycin 0.1g were added into 20 mL methanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%lactose aqueous solution (the percentage refers to the mass of the lactose relative to the total mass of the lactose aqueous solution) was added.
  • Embodiment 22 The preparation of ginsenoside Rk4H Cyclosporin liposome
  • Egg lecithin 0.8g, ginsenoside Rk4H 0.4g, VE 0.1g and cyclosporin 0.1g were added into 20 mL methanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%lactose aqueous solution (the percentage refers to the mass of the lactose relative to the total mass of the lactose aqueous solution) was added.
  • Embodiment 23 The preparation of ginsenoside DC-DQ siRNA liposome
  • Soybean lecithin S100 0.9g, ginsenoside DC-DQ 0.3g and VE 0.1g were added into 20 mL chloroform and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, vacuumed overnight to remove the chloroform completely, then 20 mL phosphate buffer solution (PBS) was added.
  • PBS phosphate buffer solution
  • a 0.22 micron microporous membrane was used to filtration thereby obtaining an aqueous solution containing ginsenoside DC-DQ blank liposome.
  • 1mL siRNA aqueous solution with a mass fraction of 20% (siRNA 0.2g) were added, kept for 30 minutes in a water bath at 60°C, a 0.22 micron microporous membrane was used to filtration thereby obtained an aqueous solution containing ginsenoside DC-DQ siRNA liposome. Then the aqueous solution was split charging into vials and each vial contained 2mg siRNA.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside DC-DQ siRNA liposome.
  • a protective gas argon or nitrogen
  • the D10 of the liposome was 72nm
  • D50 was 130nm
  • D90 was 223nm.
  • the encapsulated efficiency was more than 95%.
  • Embodiment 24 The preparation of pseudo-ginsenoside SC-DQ Amphotericin B liposome
  • Pseudo-ginsenoside SC-DQ 0.4g was added into 20 mL chloroform and stirred to form a clear solution at 45°C, then dissolved 0.5g mPEG2000-DSPE in 10 mL ethanol, which was added into the chloroform solution slowly and stirred to form a clear solution.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 60 to 65°C to form a film, and further dried in vacuum for 24 hours.
  • Tris (hydroxymethyl) aminomethane (Tris) buffer solution and 5mL DMSO solution of amphotericin B (0.12g/ml) were added, stirred to form a clear solution.
  • the solution of the liposome was dialyzed for 24 hours in purified water, an operation of homogenization by a high pressure homogenizer was carried out until the particle size of the liposome was between 0.1 and 0.3 micron, then the aqueous solution was split charging into vials and each vial contained 10mg amphotericin B.
  • the aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the Ginsenoside SC-DQ amphotericin B liposome.
  • a protective gas argon or nitrogen
  • the D10 of the liposome was 67nm
  • D50 was 132nm
  • D90 was 266nm.
  • the encapsulated efficiency was more than 95%.
  • Embodiment 25 The preparation of pseudo-ginsenoside SC-TQ Nimodipine liposome
  • DOPE-PEG 0.9g, pseudo-ginsenoside SC-TQ 0.4g and nimodipine 0.1g were added into 20 mL chloroform and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 40 to 50°C to form a film, and 20 mL 5%lactose aqueous solution (the percentage refers to the mass of the lactose relative to the total mass of the lactose aqueous solution) was added.
  • Embodiment 26 The preparation of ginsenoside SC-Iso-PPD (E) cabazitaxel liposome
  • Soybean lecithin 0.9g, ginsenoside SC-Iso-PPD (E) 0.3g and cabazitaxel 0.1g were added into 50 mL DMF and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 60 to 70°C to form a film, and 20 mL 5%glucose aqueous solution (the percentage refers to the mass of the glucose relative to the total mass of the glucose aqueous solution) was added.
  • an operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron, an 1 micron microporous membrane was used to filtration thereby obtained an aqueous solution containing ginsenoside SC-Iso-PPD (E) cabazitaxel liposome. Then the aqueous solution was split charging into vials and each vial contained 2mg cabazitaxel. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the ginsenoside SC-Iso-PPD (E) cabazitaxel liposome.
  • a protective gas argon or nitrogen
  • Embodiment 27 The preparation of iso-ginsenoside Rh2 (Z) Epothilone A liposome
  • Soybean lecithin 0.9g, iso-ginsenoside Rh2 (Z) 0.3g, Sodium oleate 0.1g and epothilone A 0.1g were added into 20 mL methanol and stirred to form a clear solution at room temperature.
  • the organic solvent was removed by a rotary evaporation in a thermostatic water bath at 45 to 50°C to form a film, and 20 mL 5%glucose aqueous solution (the percentage refers to the mass of the glucose relative to the total mass of the glucose aqueous solution) was added.
  • an operation of ultrasound was carried out until the particle size of the liposome was between 0.1 and 0.3 micron, an 1 micron microporous membrane was used to filtration thereby obtained an aqueous solution containing iso-ginsenoside Rh2 (Z) epothilone A liposome. Then the aqueous solution was split charging into vials and each vial contained 10mg epothilone A. The aqueous solution was placed in a freeze-dryer to freeze dry for 72 hours, then a protective gas (argon or nitrogen) was introduced, sealed to give the iso-ginsenoside Rh2 (Z) epothilone A liposome. By test, the D10 of the liposome was 97nm, D50 was 237nm, D90 was 482nm. The encapsulated efficiency was more than 95%.
  • the related blank ginsenoside derivative liposomes preparation were carried out according to conventional ways in present invention or embodiment 1 by replacing the related ginsenoside derivatives.
  • Ultra-pure water production system (ULUP-IV-10T, Sichuan U &P Ultra Technology Co., Ltd. )
  • Thermostatic oscillator (SHA-C, Changzhou Aohua Instrument Co., Ltd. )
  • Ultrasonic cell crusher JY92-II, Ningbo new MacBook Biotechnology Co., Ltd.
  • a low-concentration amount-increasing continuous inducing method was applied to induce parental MCF-7 cells to establish drug resistant human breast cancer cell line MCF-7/paclitaxel.
  • the new recovery MCF-7 cells were cultured for two generations or three generations under conventional conditions to make the cells grew stably.
  • paclitaxel was added with an initial concentration of one tenth of IC 50 to parental MCF-7.
  • the culture-medium was renewed the next day that the drug had been added, and the concentration of paclitaxel was maintained while conventional subculture was carried out. After the cells could stably grow under each concentration of paclitaxel, the concentration of the drug was increased.
  • a low-concentration amount-increasing continuous inducing method was applied to induce parental BGC-823 cells to establish drug resistant human breast cancer cell line BGC-823/paclitaxel.
  • the new recovery BGC-823 cells were cultured for two generations or three generations under conventional conditions to make the cells grew stably.
  • paclitaxel was added with an initial concentration of one tenth of IC 50 to parental BGC-823.
  • the culture-medium was renewed the next day that the drug had been added, and the concentration of paclitaxel was maintained while conventional subculture was carried out. After the cells could stably grow under each concentration of paclitaxel, the concentration of the drug was increased.
  • a low-concentration amount-increasing continuous inducing method was applied to induce parental MCF-7 cells to establish drug resistant human breast cancer cell line MCF-7/paclitaxel.
  • the new recovery MCF-7 cells were cultured for two generations or three generations under conventional conditions to make the cells grew stably.
  • paclitaxel was added with an initial concentration of one tenth of IC 50 to parental MCF-7.
  • the culture-medium was renewed the next day that the drug had been added, and the concentration of paclitaxel was maintained while conventional subculture was carried out. After the cells could stably grow under each concentration of paclitaxel, the concentration of the drug was increased.
  • 2%red blood cell suspension The blood from a healthy rabbits was put into a conical flask containing glass beads and shook for 10 minutes, or the glass rod was used to agitate the blood to remove the fibrinogen to make defibrase blood. About 10 times volum of 0.9%Sodium Chloride solution was added, and centrifuged for 15 minutes with 1000 ⁇ 1500 RPM, the supernatant was removed. The precipitated red blood cells was washed with 0.9%Sodium Chloride Solution for 2 ⁇ 3 times according to the above method until the supernatant was no far red. Then the red blood cells obtained were treated with 0.9%Sodium Chloride Solution to make 2%suspension for testing.
  • the concentration of the reference material can be adjusted according to the actual situation.
  • mice BALB/C-nu/nu mice (or named nude mice) , which were purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Cell Culture method the related cell line was placed into a 37°C incubator containing 5%CO 2 , and cultured by DMEM or RPMI1640 complete culture-medium (containing 10%fetal bovine serum, 100U/mL penicillin, 100 ⁇ g/mL streptomycin) , 0.25%trypsin-EDTA was used to digest and subculture 2 to 3 times per week.
  • a negative control group e.g., saline group
  • a positive control group and a ginsenoside derivative liposome loading a drug group are set for each experiment.
  • concentration gradients are set, half dilution or 5 times dilution, 3 wells for each concentration.
  • Assay of inhibition concentration IC 50 of the tumor cell tumor cells at logarithmic growth phase were digested with trypsin thereby giving cell sap with a certain concentration, then inoculated into a 96-well plate with a density of 5000 cells per well, 100 ⁇ l for each well. A fresh culture-medium containing different concentration of sample and corresponding solvent control were added, 100 ⁇ l for each well (a final concentration of DMSO ⁇ 0.5%) .
  • An optical density (OD) is determined by a microplate reader at a reference wavelength of 630nm and a detection wavelength of 450nm. Tumor cells treated with a solvent were as a control group, IC 50 was calculated according to an equation of the median effect.
  • Cell survival rate (%) OD 490 (sample) /OD 490 (control) ⁇ 100%, wherein OD 490 (sample) is the OD of the experimental group, OD 490 (control) is the OD of the blank control group.
  • T/C (%) TRTV/CRTV ⁇ 100%, wherein TRTV is the RTV of the treatment group, CRTV is the RTV of the solvent control group.
  • the calculation formula of the tumor inhibition percentage: the tumor inhibition percentage (the tumor weight of the solvent control group -the tumor weight of the drug administration group) /the tumor weight of the solvent control group ⁇ 100%.
  • T/C (%) >60 indicates no effect
  • T/C (%) ⁇ 60 indicates no effect
  • C ( ⁇ M) means concentration, wherein a concentration of Taxol+Rg5 refers to the concentration of paclitaxel and ginsenoside Rg5 in the ginsenoside Rg5 paclitaxel liposome, for example, 5+30 means that in the ginsenoside Rg5 paclitaxel liposome, the concentration of the paclitaxel is 5 ⁇ M and the concentration of the ginsenoside Rg5 is 30 ⁇ M.
  • Time (d) means time (day) .
  • Application embodiment 1 Hemolysis experiment
  • Blank Rg5 liposome hemolysis is more serious than blank liposomes of GQ, Rg5H, Iso-Rg3.
  • the Rg5-Paclitaxel liposome HD50 is between 450-500ug/ml for Rg5 content, HD5 is around 400ug/ml for Rg5 content.
  • the Rg5H-Paclitaxel liposome HD5 is around 1600ug/ml for Rg5H content (the related paclitaxel content is 400ug/ml) , which is 3 times lower than the related Rg5-paclitaxel liposome.
  • Application embodiment 2 Cell experiment in vitro and animal experiment in vivo
  • cell survival rates of human lung cancer cell line (A549) or paclitaxel-resistant human lung cancer cell line (A549/T) were determined respectively regarding ginsenoside Rg5H blank liposome (blank Rg5H) , ginsenoside Rg5 blank liposome (blank Rg5) , ginsenoside Rp1 blank liposome (blank Rp1) , ginsenoside Rg3H blank liposome (blank Rg3H) , pseudo-ginsenoside GQ blank liposome (blank GQ) , ginsenoside Rg5H paclitaxel liposome (Taxol+Rg5H) , ginsenoside Rg5 paclitaxel liposome (Taxol+Rg5) , ginsenoside Rp1 paclitaxel liposome (Taxol+Rp1) , ginsenoside Rg3
  • Figure 1 is the cell survival rate graph of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ against human lung cancer cell line (A549) ;
  • Figure 2 is the cell survival rate graph of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against human lung cancer cell line (A549) ;
  • Figure 3 is the cell survival rate graph of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ against paclitaxel-resistant human lung cancer cell line (A549/T) ;
  • Figure 3 is the cell survival rate graph of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against paclitaxel-resistant human lung cancer cell line (A549/T)
  • Table 8 and Figure 1 and 2 show that activity of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ was relatively weak against human lung cancer cell line (A549) , and the activity of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ was nearly same against human lung cancer cell line (A549) .
  • Table 10 and Figure 3 and 4 show that Taxol+Rg5H and Taxol+Rg5 have better activity against paclitaxel-resistant human lung cancer cell line (A549/T) relative to Taxol+Rp1, Taxol+Rg3H and Taxol+GQ which showed a lower cell survival rate.
  • the IC 50 of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H, blank GQ, Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against human lung cancer cell line (A549) and paclitaxel-resistant human lung cancer cell line (A549/T) were tested respectively.
  • the experimental data are shown in Table 11.
  • 0.07 ⁇ M (0.42 ⁇ M) of the corresponding IC 50 value of Taxol+Rg5: 0.07 ⁇ M means the IC 50 value of Taxol and 0.42 ⁇ M means the IC 50 value of Rg5H.
  • mice bearing tumors in uniform size of 100mm 3 at left forelimbs without hemorrhagic necrosis, intravenously injected at tail with a Rg5H, Rg5, Rp1, Rg3H, GQ liposomes, liposomes containing 10%of near-infrared fluorescent probe (IR783) (hereinafter abbreviated as the experimental group, which was obtained by encapsulating near-infrared fluorescent probe (IR783) into the ginsenosides blank liposome, see embodiment 4 in detail wherein paclitaxel was replaced by near-infrared fluorescent probe (IR783) ) and containing near-infrared fluorescent probe (IR783) (hereinafter abbreviated as the control group, which was obtained by encapsulating near-infrared fluorescent probe (IR783) into the blank liposomes (e.g.
  • Fig. 5-A, Fig. 5-B and Fig. 5-C were respectively the figures of distribution in vivo of IR783 fluorescence of the control group recorded at 2nd, 6th and 10th hour by the live imager.
  • Fig. 5-S is a fluorescence ruler, wherein according to the fluorescence intensity, color is red, yellow, green and blue in sequence, red indicates the strongest fluorescence, blue indicates weak fluorescence.
  • Fig. 5-D, and Fig. 5-R were respectively the figures of distribution in vivo of IR783 fluorescence of the experimental group recorded at 2nd, 6th and 10th hour by the live imager.
  • Figure 5-D, Figure 5-E and Figure 5-F were respectively the figures of the Rg5H group
  • Figure 5-G, Figure 5-H and Figure 5-I were respectively the figures of the Rg5 group
  • Figure 5-J, Figure 5-K and Figure 5-L were respectively the figures of the Rp1 group
  • Figure 5-M, Figure 5-N and Figure 5-O were respectively the figures of the Rg3H group
  • Figure 5-P, Figure 5-Q and Figure 5-R were respectively the figures of the GQ group.
  • Figure 6-A and Figure 6-F were respectively the figures of distribution in vivo of IR783 fluorescence of the control group, Rg5H, Rg5, Rp1, Rg3H, GQ groups recorded at 12th hour by the live imager, the top was the tumor.
  • Figure 6 showed that the control group fluorescence was obviously not fluorescence, the experimental groups were strong experimental, which suggested ginsenoside Rg5H, Rg5 Rp1, Rg3H, GQ blank liposomes had strong targetability to tumor cells.
  • Corresponding preparations were injected via tail vein (a dose of 25mg ⁇ kg -1 ) .
  • the changes of body weights of mice in each group were recorded every 2 days, and the longest diameter and the shortest diameter of tumors were measured with a vernier caliper.
  • Table 12 and Figure 7 showed that after the same period of time, the volume of tumor in the control group was the maximum while in the Taxol+Rg5H group was the minimum, the second was Taxol+Rg5 group, and the relative tumor volume of others groups were all below 3.
  • Table 13 antitumor effect of the control group, Taxol+Rg5H group, Taxol+Rg5 group, Taxol+Rp1 group, Taxol+Rg3H group and Taxol+GQ group against paclitaxel-resistant human lung cancer cell line (A549/T)
  • Application embodiment 3 Cell experiment in vitro and animal experiment in vivo
  • FIG. 14 According to the assay of cell experiment in vitro, 7 different concentrations were set as shown in Table 14 and Table 16. The specific survival rate data and graphs were shown in Table 15, Table 17, Figure 9 to 12.
  • Figure 9 was the cell survival rate graph of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ against human breast cancer cell line (MCF-7) respectively.
  • Figure 10 was the cell survival rate graph of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against human breast cancer cell line (MCF-7) respectively.
  • Figure 11 was the cell survival rate graph of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H and blank GQ against paclitaxel-resistant human breast cancer cell line (MCF-7/T) respectively.
  • Figure 12 was the cell survival rate graph of Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against paclitaxel-resistant human breast cancer cell line (MCF-7/T) respectively.
  • the IC 50 of blank Rg5H, blank Rg5, blank Rp1, blank Rg3H, blank GQ, Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against human breast cancer cell line (MCF-7) and paclitaxel-resistant human breast cancer cell line (MCF-7/T) was tested.
  • the experimental data were shown in Table 17.
  • mice 36 subcutaneous tumor-bearing mice were randomly divided into 6 groups (6 in each group) , the control group (Control group, 0.9%NaCl) , the Taxol+Rg5H group, the Taxol+Rg5 group, the Taxol+Rp1 group, the Taxol+Rg3H group and the Taxol+GQ group.
  • Corresponding preparations were injected via tail vein (a dose of 25mg ⁇ kg -1 ) .
  • the changes of body weights of mice in each group were recorded every 2 days, and the longest diameter and the shortest diameter of tumors were measured with a vernier caliper.
  • Table 18 Antitumor effect of the control group, Taxol+Rg5H, Taxol+Rg5, Taxol+Rp1, Taxol+Rg3H and Taxol+GQ against human breast cancer cell line (MCF-7)
  • Table 18 and Figure 13 showed that after the same period of time, the volume of tumor in the control group was the maximum while in the Taxol+Rg5H group was the minimum. With time delaying, the volume of tumor in the control group reached 11.238, while the other groups were relatively small.
  • Table 19 and Figure 14 showed that after the same period of time, the volume of tumor in the control group was the maximum while in the Taxol+Rg5H group was the minimum. With time delaying, the volume of tumor in the control group reached 11.438, while the other groups showed obvious good efficacy against paclitaxel-resistant human breast cancer MCF-7/T in vivo.

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