CN102940654A - Ternary nano-micelle of flammulina velutipe non-saponification extracted sterol and preparation method thereof - Google Patents

Abstract

A ternary nano-micelle of flammulina velutipe non-saponification extracted sterol takes the flammulina velutipe non-saponification extracted sterol as a pharmaceutical raw material, and is mainly composed by the following components by weight: 1 part of flammulina non-saponification extracted sterol; 10-20 parts of phospholipid; 2-16 parts of povidone; and 3-18 parts of cholate. The ternary nanomicelle greatly improves the solubility of the poorly water-soluble pharmaceutical flammulina velutipe sterol extract, and helps to improve the disadvantage of low bioavailability in vivo. The nano-micelle can be processed into various pharmaceutical dosage forms, and the preparation method is simple and easy for industrialization mass production.

Description

A kind of JINZHENGU is the ternary nano micelle and preparation method thereof of saponification thing sterol not

technical field:

The present invention relates to JINZHENGU steroidal extract and ternary nano micelle thereof.

background technology:

JINZHENGU (Flammulina velutipes (W.Curt.:Fr.) Singer), have another name called money bacterium, plain gill fungus, Flammulina velutipes, hair handle money bacterium, belong to Eumycota (Eumycota), Basidiomycotina (Basidiowyeotina), Hymenomycetes (Hymenomycetes), Agaricales (Agaricales), Tricholomataceae (Tricholomataceae), flammule Pseudomonas (Flammulina) or money Pseudomonas (Collybia), be one of famous edible fungi of all times ( the Guo Meiying chief editor, " Chinese JINZHENGU production ", Beijing: Chinese agriculture publishing house, 2000.).

Ion Absorption of Flammulina Velutipes is containing nutrient substance such as saccharide, protein, dietary fiber and mineral.In the last few years; scholars separate and obtain many kinds of protein and polysaccharide active substance from JINZHENGU; these materials have antitumor, and antiviral is pest-resistant; antifungal; anti-human immunodeficiency virus (HIV), reduce serum cholesterol, reduces blood pressure; improve immunity of organisms, and the extensive pharmacologically active such as the liver protecting.The Protein components of having reported mainly comprises fungal immunomodulatory protein, Flammutoxin, and Huogu mushroom essence, fiery mushroom proteinogen, enzyme, agglutinin, and four kinds of ribosome inactivating proteins such as Flammulin, Velutin, Flammin and Velin etc.The polysaccharide composition comprises PA ₃dE, PA ₅dE, EA ₃, EA ₅, EA ₆and EA ₇, SFA1, FVP2, extracellular polysaccharide, β-glucosan, xylomannan and manna fucogalactan etc. ( yaqi Wang, Li Bao, Xiaoli Yang, etc. Bioactive sesquiterpenoids from the solid culture of the edible mushroom flammulina velutipes growing on cooked rice[J]. Food Chemistry, 2012, 132 (3): 1346-1353. Hui-Hsin Chang, Kuang-Yang Hsieh, Chen-Hao Yeh, etc. Oral administration of an enoki mushroom protein fve activates innate and adaptive immunity and induces anti-tumor activity against murine hepatocellular carcinoma [J]. International Immunopharmacology, 2010, 10:239-246.).

In addition, the small-molecule substances such as sterol, fatty acid, flavone, terpenoid and nucleoside are also therefrom found.At present about the biological activity report of these small-molecule substances mainly concentrate on antibiotic, antioxidation aspect ( fang Yumei, Zhang Chunsheng, Tan Ping, etc. the non-oxidizability effect [J] of JINZHENGU flavone compound. food research and development, 2012,33 (3): 15-18. V.M. Tereshina, A.S. Memorskaya. Adaptation of flammulina velutipes to hypothermia in natural environments the role of lipids and carbohydrates[J]. Microbiology, 2005,74 (3): 279-283.).

Sterol (Sterol) is the important triterpenoid compound of a class (Triterpenes), and they extensively are present in plant, in the middle of animal and fungus.Different according to source, sterol can be divided into zoosterol and plant sterol (Plant sterols, PS).Cholesterol is the main sterol in animal body, and wherein free cholesterol has the effect of stabilizing cell membrane.Plant sterol is the important component part of plant cell membrane, with cholesterol seemingly, free plant sterol contributes to stablize the phospholipid bilayer in plant cell membrane, their structures are similar to cholesterol, but in the C-24 position, a methyl or ethyl group arranged.

In higher plant, modal PS be stigmasterol (Stigmasterol), β-sitosterol ( β-sitosterol) and campesterol (Campesterol), they have a common feature is the C-5 at steroid nucleus, and 6 have a two key, and this plant sterols is called Phytosterols.Main another sterol---the brassicasterol (Brassicasterol) that exists in crucifer.Saturated PS also referred to as phytostanol (Phytostanols), does not have two keys in steroid nucleus.

In in the past 15 years, plant sterol (PS) has been proved to be has the effect that reduces serum total cholesterol and low density lipoprotein, LDL (LDL) cholesterol concentration, is included into the category of functional food.Many clinical studies show, the dosage of 1 ~ 3 g/day of PS can significantly reduce the concentration of blood plasma LDL-C.Plant sterol also can be used for auxiliary treatment, and the effect of combining use with statins surpasses the effect of statins doubling dosage.More than 40 patent about the PS product arranged now, surpass 15 the PS Product Marketing whole world ( g. Garcia-Llatas, M. T. Rodriguez-Estrada. Current and new insights on phytosterol oxides in plant sterol-enriched food[J]. Chem Phys Lipids, 2011,164 (6): 607-624.).

The definite mechanism that reduces the cholesterol effect about plant sterol it be unclear that, think at present exist following several may: (1) because plant sterol has stronger hydrophobicity than cholesterol, so plant sterol can replace cholesterol and enters in hybrid fine particles (Mixed micelles).This metalepsis descends the cholesterol level in microgranule, thereby reduces the absorption of cholesterol.(2) plant sterol can slow down the esterification rate of cholesterol in enterocyte.Because the sterin after esterification is easy to enter in Chylomicron, nonesterified sterin is Chylomicron more difficult to get access, so plant sterol can reduce the total amount of cholesterol in Chylomicron.(3) plant sterol may affect the absorption of cholesterol in conjunction with box (ATP-binding cassette, ABC) transporter family by ATP.ABCG is as the subtribe of ABC, wherein two Gene A BCG5 and ABCG8 and human body the selectivity of sterol is absorbed relevant, also relevant with the secretion of sterin in bile ( zhou Xiaoxing, Su Yixiang. the present Research [J] of plant sterol cholesterol reducing effect. foreign medical science hygiology fascicle, 2007,34 (2): 101-105.).

17 kinds of sterol compounds that comprise Ergota steroid class and bean steroid class from the sporophore of JINZHENGU and mycelium, have been obtained at present: (22 e, 24 r)-ergosta-7,22-diene-3 β, 5 α, 6 α, 9 α-tetrol ( 1), (22 e, 24 r)-ergosta-7,22-diene-3 β, 5 α, 6 β-triol ( 2), (24 s)-ergosta-7-ene-3 β, 5 α, 6 β-triol ( 3), 5 α, 8 α-epidioxy-(22 e, 24 r)-ergosta-6,22-diene-3 β-ol ( 4), 5 α, 8 α-epidioxy-(24 s)-ergost-6-ene-3 β-ol ( 5), (22 e, 24 r)-ergosta-5,7,9 (11), 22-tetraene-3 β-ol ( 6), (24 s)-ergosta-7-ene-3 β-ol ( 7), ergosta-5,8,22-triene-3 β-ol ( 8), ergosterol ( 9), ergosta-5,7-diene-3 β-ol ( 10), ergosta-7,22-diene-3 β-ol ( 11), ergosta-3-O- β-D-glucopyranoside ( 12), ergosta-7,22-diene-5,6-epoxy-3-ol ( 13), 5 α-stigmastan-3,6-dione ( 14), (24 r)-stigmast-4-ene-3-one ( 15), ergosta-4,6,8 (14), 22-tetraene-3-one ( 16), ergosta-8 (14)-ene-3 β-ol ( 17).

Above-mentioned compound 9(ergosterol, ergosterol) is one of main component of the contained sterols material of JINZHENGU, in plant kingdom, also extensively exists, and its anti-tumor activity has obtained the confirmation of many experiments, and mechanism of action also part is revealed.Takaku etc. isolate ergosterol from Agaricus blazei Murrill, 400 and 800 mgkg ^-1continuous 20 d of dosage orally give the sarcoma 180 mice, can significantly suppress the growth of tumor, but also not have chemotherapeutics usually caused as side effect such as body weight, epididymal adipose, thymus, spleen weight and leukocyte count reductions.Further research shows, ergosterol can directly suppress the new vessels that basement membrane induces and form, thus it be the angiogenesis of inducing by direct inhibition solid tumor bring into play antitumor activity ( takeshi Takaku, Yoshiyuki Kimura, Hiromichi Okuda. Isolation of an antitumor compound from agaricus blazei murill and its mechanism of action[J]. Biochemical and Molecular Action of Nutrients, 2001,131 (5): 1409-1413.).

Yazawa etc. isolate ergosterol from the Polyporus ethanol extract, the Wistar rat bladder cancer that it is induced 5% saccharin sodium (Sodium Saccharin), 0.01%N-butyl-N-(4-hydroxybutyl) nitrosamine (BHBN), 3%DL-tryptophan (Trp), 2% butylated hydroxyanisol (BHA) etc., have good therapeutical effect ( yasuharu Yazawa, Masami Yokota, Kiyoshi Sugiyama. Antitumor promoting effect of an active component of polyporus, ergosterol and related compounds on rat urinary bladder carcinogenesis in a short-term test with concanavalin A[J]. Biol. Pharm. Bull., 2000,23 (11): 1298-1302.).

P21 is cyclin dependent kinase inhibitive factor (Cyclin-dependent kinase inhibitor, CDKI) a kind of, belong to cell cycle negativity regulatory factor, it can be by suppressing cyclin dependent kinase (Cyclin-dependent kinase, CDK) activity of CDK2, CDK4, make cell can not cross over G phase checkpoint, thus the carrying out of retardance cell cycle, suppressing cell proliferation, is tumor prevention and a target spot for the treatment of.Zhang Xian etc. find that ergosterol is at 50 ~ 200 μ gmL ^-1can suppress the HepG2 cell proliferation, mechanism may be to increase P21 mRNA and protein expression relevant.After ergosterol effect 48 h and 72 h, 100,200 μ gmL ^-1the expression of group P21 mRNA is obviously increased.After 24 h, P21 albumen has had rising trend, after 48 h, 72 h, obviously raises, and prompting P21 may be the action target spot of ergosterol.( zhang Xian, Huang Yu, once star. the impact [J] of ergosterol on hepatoma carcinoma cell HepG2 propagation and P21 expression. Pharmacology and Clinics of Chinese Materia Medica, 2011,27 (5): 26-29.)

The anti-tumor in vivo activity of ergosterol in the Agaricus blazei sporophore that the people such as Gao Hong have adopted mice S180 transplanted tumor experimentation.Result shows, the ergosterol extracted in the Agaricus blazei sporophore is at dosage 10,50 and 100 mgkg ^-1d ^-1the time the heavy suppression ratio of tumor be respectively 62.75%, 76.61% and 79.18%, illustrate that ergosterol has stronger antitumor activity, and think that its antitumor activity is not to bring into play by cytotoxicity, but by suppress the tumor vessel hypertrophy strangle tumor ( gao Hong, Shi Defang, Yang De, etc. Agaricus blazei ergosterol anti-tumor activity and mechanism of action pre-test [J]. edible fungi of china, 2011,30 (6): 35-39.).

Yet the dissolubility of plant sterol in water and oil free under room temperature condition is all very little, the absorbance in human body is also very low, be about 2% ~ 5% ( i. Rudkowska. Plant sterols and stanols for healthy ageing[J]. Maturitas, 2010,66 (2): 158-62.).In order to widen the range of application of plant sterol, improving its dissolubility is the task of top priority.Mainly contain at present two kinds of methods, the first is utilized active group important on steroid nucleus---C-3 position hydroxyl, can form sterol ester with fatty acid response, and the dissolubility of plant sterol ester specific ionization plant sterol in oil increases approximately 10 times; It two is emulsifying solubilization and the high pressure homogenization techniques that utilize emulsifying agent, makes plant sterol be dispersed into nano level dispersion in water, increases its dissolubility.

Nano-micelle (Nanomicelles) is a kind of nanoscale new drug carrier occurred in recent years, utilizes micellar solubilization can improve dissolubility and the oral administration biaavailability of insoluble drug.According to the difference that forms the carrier material relative molecular mass, nano-micelle can be divided into low molecule micelle and polymer micelle.Cholate/lecithin mixed micelle mainly is comprised of cholate, lecithin, medicine three parts, is a kind ofly to have obtained system and the low molecule micelle of broad research.The preparation that utilizes its to form not only can significantly improve the dissolubility of insoluble drug, can also improve the curative effect of medicine, increases medicine stability, be drug carrier system with good biocompatibility ( the woods East Sea, Li Ping, Li Xinxin, the research [J] of etc. cholate/phospholipid mixed micelle to hydrophobicity natural drug characteristic of solubilizing. Asia-Pacific traditional medicine, 2009,5 (8): 27-30.).Because low molecule micelle adopts micromolecular surface active groups to make carrier material, the hydrophobicity " core " of meeting water self assembly formation is less, its solubilizing amount, drug loading and the degree that promotes medicine to be utilized by body be all limited, thus limited its extensive use aspect the insoluble drug solubilising ( as the polyvidone-phospholipid of insoluble drug carrier-cholate ternary composition [P], the number of applying for a patent: 200510037848.2).

Polymer micelle is spontaneous formation after the amphipathic nature block polymer that contains hydrophilic and hydrophobicity long-chain concentration in water surpasses critical micelle concentration (Critical micelle concentration, CMC).Early 1990s is by the initial carrier proposed as cancer therapy drug of the people such as Japanese professor Kataoka.Up to now, existing Genexol ^?, NK105, NC-6004, NC-4016, NK012, seven antitumor drug such as NK911, SP1049C the polymer micelle formula carrying out clinical trial ( j. Gong, M. Chen, Y. Zheng, etc. Polymeric micelles drug delivery system in oncology[J]. J Control Release, 2012.).

Amphiphilic diblock (AB) or three blocks (BAB) copolymer are most commonly used to prepare the transmission of self aggregation polymer micelle for medicine.Because the hydrophobic core core that the graft copolymer majority forms in water can't be combined closely, have hydrophobic chain to expose, thereby cause hydrophobic chain between micelle near and assemble, not too be applicable to do pharmaceutical carrier.The material of hydrophilic section is generally used Polyethylene Glycol (PEG), polyoxyethylene (PEO) or polyvidone (PVP), in recent years also useful chitosan, the hydrophobic section material mainly contain polyoxypropylene (PPO), polyurethanes, polypropylene, polystyrene, polyamino acid, polylactic acid (PLA), spermine or short-chain phospholipid etc. ( lu Bin. as the research [J] of polymer micelle and the vesicle of drug-loading system. Journal of Henan University (medicine), 2008,27 (1): 1-7.).Most of amphipathic nature block polymers are assembled the circular micelle that forms nucleocapsid structure, and the hydrophobic part of polymer is wound in hydrophobic cores, by chemistry, physics or electrostatic interaction, with medicine, combine, and bag carries insoluble drug; Hydrophilic segment is distributed in the hydrophobic inner core periphery, with hydrone on every side, forms hydrogen bond and the Xiang Shuizhong stretching, extension, forms hydrated sheath, prevents the micelle gathering, as shown in Figure 1 ( shawn C. Owen, Dianna P. Y. Chan, Molly S. Shoichet. Polymeric micelle stability[J]. Nano Today, 2012,7 (1): 53-65.).

Except solubilization, polymer micelle can also arrive specific organization internal by passive or mode initiatively.Extending circulation time and higher Drug loading capacity is the prerequisite of targeted drug success.Sterically hindered and the nano-scale of polymer micelle hydrophilic shell (10 ~ 100 nm) can stop the sufficiently long time in blood.Be on the one hand because polymer micelle can effectively be avoided renal excretion (> 50 kDa), be because particle diameter very little (<200 nm) can not held back by spleen endotheliocyte slit on the other hand.Simultaneously, with low-molecular-weight surfactant, compare, amphipathic nature polyalcohol functional group kind is more, structure is more complicated, can carry out structural modification or connect upper other functional groups existing functional group, strengthen the affinity of micelle core and medicine, thereby the drug loading of polymer micelle and stability obviously improve.Micelle has the good permeability of organizing, especially can assemble at the tissue with seepage blood vessel (as tumor or infarct area), i.e. and so-called EPR effect, this makes micelle have natural passive target effect.By means targeting location such as mediation folic acid, pH are responsive or thermo-responsive, polymer micelle can also reversing tumor cell multidrug-resisting (MDR) ( u. Kedar, P. Phutane, S. Shidhaye, etc. Advances in polymeric micelles for drug delivery and tumor targeting[J]. Nanomedicine, 2010,6 (6): 714-729.).

The JINZHENGU steroidal extract is with alcoholic solution, to extract after the JINZHENGU sporophore is pulverized, the non-saponifiable matter obtained by steps such as concentrated, extraction, saponification, crystallizations.Studies have shown that, this extract has the activity of the growth of tumour cell such as vitro inhibition human liver cancer cell (HepG2), people's gastric adenocarcinoma cells (SGC), human glioma cells (U251) and human lung adenocarcinoma cell (A549), can be used for preparing antitumor drug (referring to: the not saponification thing and method for making and the purposes [P] that there is antitumor action in JINZHENGU, the number of applying for a patent: 201110364098.5).It mainly comprises ergosterol, 22,23-dihydroergosterol, ergosta-5,8,22-triene-3 β-ol and ergost-8 (14)-ene-3 βfour kinds of compositions such as-ol.It is soluble in the organic solvents such as ether, chloroform and hot ethanol, and under room temperature, the utmost point is insoluble in water and oil, possesses the general aspects of plant sterol.The JINZHENGU of take steroidal extract is not yet arranged at present as crude drug, be prepared into the ternary nano micellar preparation, to reach the research report that improves this extract dissolubility and bioavailability.

summary of the invention:

The object of the present invention is to provide the not ternary nano micelle of saponification thing sterol of a kind of JINZHENGU, the JINZHENGU steroidal extract that is insoluble in water is prepared into to the ternary nano micelle, to improve its dissolubility, improve its extremely low shortcoming of bioavailability in vivo.

Another object of the present invention is to provide the not preparation method of the ternary nano micelle of saponification thing sterol of this JINZHENGU.

For achieving the above object, the technical solution used in the present invention is as follows:

A kind of JINZHENGU is saponification thing sterol ternary nano micelle not, it be using JINZHENGU not saponification thing sterol as crude drug, mainly by following mass component, formed: JINZHENGU is 1 part of saponification thing sterol not; 0 ~ 20 part of phosphatidase 11; 2 ~ 16 parts of polyvidones; 3 ~ 18 parts of cholates.

Above-mentioned JINZHENGU not saponification thing sterol is that the JINZHENGU sporophore is extracted with alcoholic solution, by steps such as concentrated, extraction, saponification, crystallizations, obtains.It mainly comprises ergosterol, 22,23-dihydroergosterol, ergosta-5,8,22-triene-3 β-ol and ergost-8 (14)-ene-3 βfour kinds of compositions such as-ol, wherein two kinds of fronts component content higher (referring to: Chinese patent application CN201110364098.5).

Above-mentioned a kind of JINZHENGU is saponification thing sterol ternary nano micelle not, and described polyvidone is PVP-K30.

Above-mentioned a kind of JINZHENGU is saponification thing sterol ternary nano micelle not, and described cholate is sodium cholate.

The above-mentioned a kind of JINZHENGU not method for making of saponification thing sterol ternary nano micelle is: after JINZHENGU steroidal extract and phospholipid, polyvidone and cholate are dissolved in dehydrated alcohol, dehydrated alcohol is removed in evaporation, obtains not saponification thing sterol ternary nano micelle of thick JINZHENGU.

Different processing method for above-mentioned complex, can be processed into different pharmaceutical dosage forms, as multi-medicament dosage forms such as liquid preparation, lyophilized powder, soft capsule, composite powder, hard capsule, tablets.The processing method of different pharmaceutical dosage forms is respectively: in complex, add distilled water or buffer to can be made into the micellar solution liquid preparation; Complex is made to soft capsule by conventional method; Add distilled water or buffer in complex, lyophilization can make lyophilized powder; The complex vacuum drying be can be made into to composite powder; Lyophilized powder or composite powder can mix with proper auxiliary materials, further insert capsule or make the pharmaceutical preparatioies such as tablet.

beneficial effect:

The present invention has following beneficial effect:

1, solubilizing effect of the present invention is remarkable, and makes medicine easily be absorbed by the body, and has improved the bioavailability of medicine;

2, the present invention adopts formula and method simple possible, without special installation, be convenient to large-scale industrial production.

the accompanying drawing explanation:

Fig. 1 polymer micelle forms schematic diagram.In water, amphipathic copolymer self assembly has the structure of a hydrophobic inner core and hydrophilic outer shell.

The transmission electron microscope photo (* 200 k) of the JINZHENGU steroidal extract ternary nano micelle that Fig. 2 is embodiment 1 preparation.

The particle size distribution figure of the JINZHENGU steroidal extract ternary nano micelle that Fig. 3 is embodiment 1 preparation.

Average blood drug level-the time plot (n=5) of the JINZHENGU steroidal extract ternary nano micelle that Fig. 4 is embodiment 5 preparations.

the specific embodiment:

Below listed embodiment contribute to those skilled in the art to understand better the present invention, but do not limit the present invention in any way.

embodiment 1jINZHENGU is saponification thing sterol ternary nano micelle not

Take JINZHENGU not saponification thing sterol, phospholipid, PVP-K30, sodium cholate according to following table in round-bottomed flask, add 50 mL dehydrated alcohol, ultrasonicly clarify to solution, rotary evaporation, remove dehydrated alcohol, adds distilled water, after jolting, clarify, obtain liquid preparation, the system final volume is 100 mL, and drug level is 2 mgmL ^-1.

Component	Mass/volume
		JINZHENGU is saponification thing sterol not	0.2 g
Phospholipid	3.0 g
		PVP-K30	2.0 g
Sodium cholate	2.0 g
		Distilled water	92.5 mL

embodiment 2jINZHENGU is saponification thing sterol ternary nano micelle not

Take JINZHENGU not saponification thing sterol, phospholipid, PVP-K30, sodium cholate according to following table in round-bottomed flask, add 40 mL dehydrated alcohol, ultrasonicly clarify to solution, rotary evaporation, remove dehydrated alcohol, adds distilled water, after jolting, clarify, lyophilization, obtain lyophilized powder.

Component	Mass/volume
		JINZHENGU is saponification thing sterol not	0.2 g
Phospholipid	2.0 g
		PVP-K30	1.0 g
Sodium cholate	1.0 g
		Distilled water
	15 mL

embodiment 3jINZHENGU is saponification thing sterol ternary nano micelle not

Take JINZHENGU not saponification thing sterol, phospholipid, PVP-K30, sodium cholate according to following table in round-bottomed flask, add 30 mL dehydrated alcohol, ultrasonicly clarify to solution, rotary evaporation, remove dehydrated alcohol, adds distilled water, after jolting, clarify, obtain liquid preparation, the system final volume is 100 mL, and drug level is 2 mgmL ^-1.

Component	Mass/volume
		JINZHENGU is saponification thing sterol not	0.2 g
Phospholipid	2.0 g
		PVP-K30	0.4 g
Sodium cholate	0.6 g
		Distilled water
	97 mL

embodiment 4jINZHENGU is saponification thing sterol ternary nano micelle not

Take JINZHENGU not saponification thing sterol, phospholipid, PVP-K30, sodium cholate according to following table in round-bottomed flask, add 70 mL dehydrated alcohol, ultrasonicly clarify to solution, rotary evaporation, remove dehydrated alcohol, adds distilled water, after jolting, clarify, obtain liquid preparation, the system final volume is 100 mL, and drug level is 2 mgmL ^-1.

Component	Mass/volume
		JINZHENGU is saponification thing sterol not	0.2 g
Phospholipid	4.0 g
		PVP-K30	3.2 g
Sodium cholate	3.6 g
		Distilled water	91 mL

embodiment 5jINZHENGU steroidal extract ternary nano micelle

Take JINZHENGU steroidal extract, phospholipid, PVP-K30, sodium cholate according to following table in round-bottomed flask, add 60 mL dehydrated alcohol, ultrasonicly clarify to solution, rotary evaporation, remove dehydrated alcohol, adds distilled water, after jolting, clarify, obtain liquid preparation, the system final volume is 40 mL, and drug level is 5 mgmL ^-1.

Component	Mass/volume
		JINZHENGU is saponification thing sterol not	0.2 g
Phospholipid	4.0 g
		PVP-K30	2.5 g
Sodium cholate	2.5 g
		Distilled water	32 mL

embodiment 6the ternary nano micelle that embodiment 1 is prepared carries out medicament contg, envelop rate, morphology and particle size distribution measuring, further illustrates effect of the present invention.

(1) assay is got JINZHENGU saponification thing sterol ternary nano micellar solution is not appropriate, 0.22 μ m membrane filtration, therefrom draw 0.1 mL and be settled to 10 mL with mobile phase, mixing the concentration that records ergosterol in the ternary nano micelle by the HPLC method is 0.94 mgmL ^-1.JINZHENGU not in saponification thing sterol ergosterol at the dissolubility of pure water, be 0.67 μ gmL ^-1, embodiment 1 has improved nearly 1403 times by the dissolubility of ergosterol.

(2) entrapment efficiency determination press formula computational envelope rate: EE%= c ₁ / c ₂ * 100%.Wherein, EE% is envelop rate; c ₁ content for ergosterol in ternary nano micelle after filtering with microporous membrane; c ₂ content for ergosterol in the prescription dosage.With ergosterol, calculate, the JINZHENGU not envelop rate of saponification thing sterol ternary nano micelle is (76.6 ± 0.2) %.

(3) the morphologic observation not saponification thing sterol ternary nano micelle of JINZHENGU that takes a morsel, after 1 times of dilute with water, drop on the copper mesh that is covered with the carbon supporting film, after room temperature is placed to drying and forms thin film, with transmission electron microscope (JEM-2100, Jeol Ltd.) observe microscopic pattern, the results are shown in Figure 2.Can find out, this ternary nano Micellar Solution Which Is drops under Electronic Speculum and all is spheroidal, distribution uniform.

(4) particle size distribution is got not saponification thing sterol ternary nano micelle of JINZHENGU, with using laser granulometry (BI-9000 type laser particle analyzer after appropriate distilled water diluting, U.S. Brooker Hai Wen instrument company) measured, particle diameter and the particle size distribution of ternary nano micelle are shown in Fig. 3.Measurement result shows, the JINZHENGU of embodiment 1 preparation not saponification thing sterol ternary nano micelle mean diameter is 115.6 nm, and polydispersity index is 0.029, and particle size distribution range is narrower.

embodiment 7jINZHENGU is the relative bioavailability experiment of saponification thing sterol ternary nano micellar preparation not

1.1 animals administer and blood sample are processed

10 male and healthy SD rats, body weight 250 ± 10 g, provided by Jiangsu University's Experimental Animal Center.Rat is tested after conforming three days.Before administration, fasting is 12 hours, freely drinks water.Rat is divided into not saponification thing sterol ternary nano micelle (embodiment 5) group and JINZHENGU saponification thing sterol free drug (5 mgmL not of JINZHENGU at random ^-1the JINZHENGU steroidal extract be suspended in the CMC-Na aqueous solution of 0.5% (w/v), before use the preparation) group, every kind of tested medicine gives 5 rats, according to 20 mLkg ^-1dosage single gastric infusion.Respectively at 0.5,1 after administration, 2,3,4,6,8,10,12,14,16,24 h, from rat eye socket approximately 0.6 mL that takes a blood sample, put in the anticoagulant heparin pipe, separated plasma after centrifugal 10 min of 4000 rpm, freezer storage under-20 ° of C is until analyze.Get 0.2 mL blood plasma in 10 mL band plug centrifuge tubes, add the interior mark liquid of 0.1 mL, mix homogeneously, add immediately 1 mL Chromatographic Pure Methanol protein precipitation, close plug vortex 5 min, get supernatant after centrifugal (3000 rpm, 10 min), under 40 ° of C water-bath nitrogen current, dry up, residue dissolves by 0.2 mL mobile phase, through centrifugal 10 min of 10,000 rpm, draw 20 μ L supernatant and advance HPLC mensuration, record chromatographic peak.

1.2 curve and relative bioavailability during the blood plasma medicine

Draw not curve during the blood plasma medicine of saponification thing sterol ternary nano micelle group and free drug group of JINZHENGU, see Fig. 4.

The BAPP computed in software AUC value that adopts the Yao Dai of China Medicine University center to provide.Ergosterol and 22,23-dihydroergosterol reach peak concentration in ternary nano micelle group c _maxbe respectively 0.93 ± 0.26 μ gmL ^-1with 0.48 ± 0.22 μ gmL ^-1, AUC _{0-24 h}be respectively 9.94 ± 1.72 μ ghmL ^-1with 3.74 ± 1.21 μ ghmL ^-1; The two reaches peak concentration in the free drug group c _maxbe respectively 0.53 ± 0.09 μ gmL ^-1with 0.14 ± 0.07 μ gmL ^-1, AUC _{0-24 h}be respectively 6.43 ± 0.81 μ ghmL ^-1with 1.35 ± 0.35 μ ghmL ^-1.

Relative bioavailability f _rel=AUC _0-24(ternary nano micelle) * x(free drug)/AUC _0-24(free drug) * x(ternary nano micelle)

In above formula x(ternary nano micelle) is not saponification thing sterol ternary nano micelle administration dosage of JINZHENGU, x(free drug) is not saponification thing sterol free drug dosage of JINZHENGU.

As calculated, with JINZHENGU not saponification thing sterol free drug group compare, the ergosterol of ternary nano micelle group and the relative bioavailability of 22,23-dihydroergosterol all improve a lot, and reach respectively 154.59% and 277.04%, c _max1.75 times and 3.43 times have been improved respectively.

Claims (4)

1. JINZHENGU saponification thing sterol ternary nano micelle not is characterized in that: it be using JINZHENGU not saponification thing sterol as crude drug, mainly by following mass component, formed: JINZHENGU is 1 part of saponification thing sterol not; 0 ~ 20 part of phosphatidase 11; 2 ~ 16 parts of polyvidones; 3 ~ 18 parts of cholates.

2. a kind of JINZHENGU according to claim 1 saponification thing sterol ternary nano micelle not, it is characterized in that: described polyvidone is PVP-K30.

3. a kind of JINZHENGU according to claim 1 saponification thing sterol ternary nano micelle not, it is characterized in that: described cholate is sodium cholate.

4. one kind prepares the not method of saponification thing sterol ternary nano micelle of JINZHENGU claimed in claim 1, it is characterized in that: after by JINZHENGU, saponification thing sterol and phospholipid, polyvidone and cholate do not dissolve in dehydrated alcohol, dehydrated alcohol is removed in evaporation, obtains not saponification thing sterol ternary nano micelle of thick JINZHENGU.

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