CN100471490C - Sesquiterpene ketone injection, preparing method and use thereof - Google Patents

Abstract

The present invention relates to the new types of plant extract containing germacrone, curcumone and/or arylcurcumone, that is, nano liposome type, precursor liposome type long-cycle liposome type, long-cycle nano liposome type, nano microemulsion type, venous emulsion type, nano liposome sphere type and fat emulsion type.

Description

Sesquiterpene ketones injection and its preparation method and usage

Technical field:

The present invention relates to contain the new injection type of the plant extract of sesquiterpene ketone, liposome dosage form more specifically to these preparations, the nanometer liposome dosage form, the pro-liposome dosage form, the long circulating liposomes dosage form, long-circulating nanoliposome dosage form, nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form and fat milk dosage form.The invention still further relates to the preparation method and the purposes of these dosage forms.

Background technology:

In recent years, the many research worker in the anticancer natural drug research field all are devoted to study the active anticancer of paclitaxel, the various isomers of elemene, Rhizoma Curcumae Longae extract, common turmeric extract etc. and the various dosage forms of these medicines.

Elemene is a kind of sesquiterpenoids that is present in the certain plants, and Latin is called Elemenum, English Elemene by name, and chemical name is 1-methyl isophthalic acid-vinyl-2,4-diisopropenyl cyclohexane extraction, molecular formula C ₁₅H ₂₄Elemene has multiple isomer, beta-elemene in these isomers, and δ-elemene, γ-elemene have been proved active anticancer.Elemene is the chemical compound that can extract from the volatile oil of plant RADIX CURCUMAE (Curcuma Wenyujin Y.H.Chen et C.ling) rhizome (being commonly called as warm Rhizoma Curcumae), or the chemical compound that from the oil of plant Herba Cymbopogonis Citrari (Cymbopoqon citratus (DC.) Stapt), extracts, for example remove the remaining afterwards mixing grease of citral, citronellol from citronella oil, the chemical compound of extraction is actually the elemene isomer mixture that comprises beta-elemene.

Above-mentioned plant RADIX CURCUMAE and other two kinds of plant Rhizoma Curcumae and Rhizoma Curcumae Longaes that can be used as the natural drug use belong to the zingiberaceous plant monoid, originate in provinces such as Chinese Guangdong, Sichuan, Fujian, Guangxi, Zhejiang.The plant that comprises elemene has kinds more than 50 such as RADIX CURCUMAE, Herba Cymbopogonis Citrari, Rhizoma Acori Calami, Radix Inulae, Radix Ginseng, and wherein Herba Cymbopogonis Citrari is, resource competent plant wide in China's distribution.At present, the Elemene vinyl chemical compound mainly extracts from RADIX CURCUMAE oil and citronella oil.

There are many documents to relate to the herbal medicinal product of the anti-curing cancers of direct employing natural drug preparation, for example the open CN1216256A of Chinese patent discloses the herbal medicinal product of treatment hepatocarcinoma, it comprises contains Herba Hedyotidis Diffusae, Rhizoma Curcumae Longae, the extract of Rhizoma Polygoni Cuspidati and Radix Sophorae Tonkinensis as the Injectable composition (A) of main natural drug and the extract that contains Herba Hedyotidis Diffusae, Rhizoma Paridis, Rhizoma Polygoni Cuspidati, Radix Sophorae Tonkinensis, Radix Gentianae, Radix Et Rhizoma Rhei, Fructus Forsythiae, Radix Paeoniae Rubra, Rhizoma Curcumae Longae and Rhizoma Acori Graminei as the main Orally administered composition (B) of natural drug.

Curcumin preparation is disclosed in the open CN1302559 of Chinese patent; it is by preparing in the blendable organic solvent of water or the curcumin molecular dispersoid in the mixture of water and the blendable organic solvent of water; add protecting colloid aqueous solution, the hydrophobic millimicro decentralized photo that forms mutually of curcumin to this solution then.Final dosage form mainly is aqueous dispersion or dry powder, and particle diameter is less than 10 μ m in the preferred case.

The method of extracting beta-elemene by the secondary fractional distillation from citronella oil is disclosed in CN1200266A.

Disclose the Jiangyu injecta that is used for the treatment of malignant tumor in the open CN1247756A of Chinese patent, wherein the weight ratio of Rhizoma Curcumae Longae and Radix Curcumae is 50-60:50-40, and this injection is a kind of simple mixture.

The open CN1076613A of Chinese patent relates to elemene emulsion injection and preparation method thereof, and this injection is a kind of emulsion, and mean diameter generally is no more than 15 microns, is no more than 2 microns especially.

The open CN1244389A of Chinese patent relates to elemene injection and preparation method thereof, and this injection is a kind of simple mixtures, is not liposome.

The open CN1110134A of Chinese patent relates to liposome preparation technology and preparation, the elemene liposome injecta of wherein having described the elemene liposome injecta preparation method especially and having obtained by this method, the envelop rate of elemene reaches more than 85%, but does not provide the data of the mean diameter of related lipase plastid.Can judge that from preparation method this liposome has bigger mean diameter.

The open CN1221607A of Chinese patent relates to and utilizes CO ₂Critical or supercritical process prepare the technology of liposoluble medicinal liposome and prepared elemene liposome injecta, though the envelop rate of the liposome of elemene is very high, the mean diameter of liposome is equally too big.

More than the disclosed preparation of these documents on bin stability and active anticancer, all exist deficiency.

The various derivants and the elemene metal complex of elemene are also disclosed in some patent documentations in addition.For example, a kind of elemene metal complex and its use as cancer therapy drug are disclosed in CN11531158A.CN1153167A relates to elemene derivatives containing nitrogen and as the purposes of cancer therapy drug.CN1153168A relates to elemene hydroxyls derivs and as the purposes of cancer therapy drug.

Summary of the invention:

The purpose of this invention is to provide the plant extract composition injection, it uses the plant extract that contains sesquiterpene ketone, surfactant and water-bearing media are as feedstock production, this plant extract that contains sesquiterpene ketone comprises as the sesquiterpene ketone active component (1) of the 5wt% at least of active component and the sesquiterpene of 5wt% (2) at least, this sesquiterpene ketone active component (1) comprises 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, a kind of in turmerone and the aryl turmerone, two or three, it is characterized in that: this injection is a kind of aqueous dispersion, the mean diameter of decentralized photo≤1 micron, the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, and the ultimate density of active component in the injection ((1)+(2)) is 1～10mg/ml.More preferably, this water-bearing media is a water.

Another object of the present invention provides the new injection type of extract, liposome dosage form more specifically to this extract, the nanometer liposome dosage form, the pro-liposome dosage form, the long circulating liposomes dosage form, long-circulating nanoliposome dosage form, nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form or fat milk dosage form.

Another object of the present invention provides the preparation method of these injection types.

The preparation that a further object of the present invention provides these novel forms is used to prepare the purposes of the drug injection of treatment cancer or cerebral infarction, these injection types are used for the purposes of tumor remission pain, and the preparation of these novel forms is used to suppress and/or treat the purposes of tumor.

The invention provides the new injection type of these extracts.Be prepared into its nanometer liposome dosage form with the extract that contains sesquiterpene ketone that extracts the quintessence oil (or being called volatile oil) that obtains from RADIX CURCUMAE from Sichuan of China, Rhizoma Curcumae, osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae, RADIX CURCUMAE etc. respectively as raw material, the pro-liposome dosage form, the long circulating liposomes dosage form, the long-circulating nanoliposome dosage form, nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form and fat milk dosage form.The mean diameter of these dosage forms is generally less than or equals 1 micron, preferably is less than or equal to 200 nanometers, even is less than or equal to 100 nanometers.

Description of drawings:

Fig. 1 is the transmission electron microscope photo of the injection 1 of embodiment 1.

Detailed description of the present invention:

Known in the prior art field that some isomers of elemene, Turmeric P.E, common turmeric extract possess the effect that suppresses tumour, some documents disclose the various dosage forms (or formulation) of these natural drugs, preparations such as liposome, emulsion, Fat Emulsion. But, because the average grain diameter of these formulations is generally larger, can cause the various reactions in the human or animal body after being expelled in the human or animal body, for example excitant is large, and haemolysis sometimes occurs.

The inventor is through years of researches work, initial find with sesquiterpene compounds or the extract that contains sesquiterpene that from plant, extracts make average grain diameter≤1 micron or≤the various injection types of 100 nanometers, comprising Lipidosome, the nano liposomes formulation, the proliposome formulation, the long circulating liposome formulation, the long-circulating nanoliposome formulation, nanometer microemulsion type, the vein emulsion type, lipid nanometer microballoon formulation, micro-emulsion etc., can overcome the problems in the ejection preparation of prior art, these formulations especially of the present invention have greatly reduced excitant in intravenous injection is used, and their stability is further enhanced. Novel form of the present invention has significantly improved the drug effect of pharmaceutical preparation, but the mechanism of the inhibition cancer cell of these formulations also is not fully aware of at present. The inventor makes following supposition according to a large amount of result of the tests: because preparation of the present invention has little average grain diameter and has special surface nature (hydrophile/lipophile balance for example, surface charge etc.), so that the medicine in these preparations (or active component) has stronger affinity to cancer cell, can fully be attached on the cancer cell, allow active component directly act on cancer cell, reach the effect that suppresses or kill cancer cell. In addition, find that the preparation of nano particle size is rapid-action, prevents that from there is certain effect the cancer metastasis aspect. Subsequently, the inventor finds that some derivative of sesquiterpene is the activity that some specific sesquiterpene ketone has higher inhibition cancer, these sesquiterpene ketones comprise germacrone, turmerone, ar-turmerone etc., mainly extract from the rhizome of RADIX CURCUMAE from Sichuan of China, curcuma zedoary, osmanthus curcuma zedoary, turmeric and RADIX CURCUMAE plant. Especially the effect of the treatment cancer of turmerone, ar-turmerone is more outstanding than germacrone. But, through zoopery, find that also some sesquiterpene ketone but has seldom or almost do not have antitumor activity.

So the plant extracts that contains specific sesquiterpene ketone and be the sesquiterpene of germacrone, turmerone and ar-turmerone and any type can be for the preparation of the injection that suppresses tumour.

Simultaneously, although preparation of the present invention has little average grain diameter, but the preparation of the various formulations that obtain has high stability simultaneously, and they coagulation problems can not occur in storing for a long time, do not reduce stability so that can store the long period.

The invention provides the plant extract composition injection, it prepares as raw material with plant extracts, surfactant and water-bearing media, this plant extracts comprises as the sesquiterpene ketone active component (1) of at least 5wt% of active component and the sesquiterpene of 5wt% (2) at least, this sesquiterpene ketone active component (1) comprise in germacrone, turmerone and the ar-turmerone a kind of, two or three, it is characterized in that: this injection is a kind of water-borne dispersions, the average grain diameter of decentralized photo≤1 micron, the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, and the ultimate density of active component in the injection ((1)+(2)) is 1～10mg/ml.

The invention provides the preparation method of plant extract composition injection, it comprises uses plant extract as raw material, adds surfactant, utilizes Rotary Evaporators film forming or CO ₂Critical or supercritical process comes mix homogeneously, add water for injection, carrying out high pressure homogenizing handles and/or ultrasonic Treatment, handle till the stabilized aqueous dispersion that obtains particle diameter of dispersing phase≤1 micron always, thereby make injection, wherein this plant extract comprises as the sesquiterpene ketone active component (1) of the 5wt% at least of active component and the sesquiterpene of 5wt% (2) at least, this sesquiterpene ketone active component (1) comprises 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, a kind of in turmerone and the aryl turmerone, two or three, the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, and the ultimate density of active component in the injection ((1)+(2)) is 1～10mg/ml.

For these extracts, require it comprise as the pact of active component at least 5wt% sesquiterpene ketone active component (1) and approximately 〉=sesquiterpene (2) of 0wt%, this sesquiterpene ketone active component (1) comprise in 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, turmerone and the aryl turmerone a kind of, two or three.Ideally, this plant extract comprises 7wt% at least as active component, preferred 10wt% at least, more preferably the sesquiterpene ketone active component (1) of 15wt%, preferred especially 20wt% at least and 1wt% at least at least, at least 5wt% ideally, better 7wt% at least, preferred 10wt% at least, more preferably 15wt%, the preferred especially sesquiterpene of 20wt% (2) at least at least.

It is to be noted, the various extracts that prepare the preparation of various dosage forms as initiation material of the present invention can use the product that is purchased in the present invention, as the curcuma extract that contains 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, turmerone and/or aryl turmerone is purchased product, and the extract product that contains this type of sesquiterpene ketone that extracts from the volatile oil (or quintessence oil) of curcuma according to method described in the prior or described here method.If need oneself these extract of preparation in this application, the various volatile oil (or quintessence oil) that are used to prepare these extracts can use and be purchased product, also can use the volatile oil that obtains from plant (for example plant rhizome) by common steam distillation or CO2 super critical extraction.Each extract can be used as initiation material of the present invention, and any two or more admixtures in any ratio also can be used as initiation material of the present invention in these extracts, obviously, with mainly containing 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, the blend that the extract of turmerone and/or aryl turmerone and other contain the extract of sesquiterpene is also included within the present invention as the situation of raw material, these other contain sesquiterpene extract comprise from SHULANHUA, Fructus Tonnae Sinensis, Caulis Piperis Kadsurae, Flos Caryophylli, Myrrha, the Hemerocallis citrina Baroni Cuculus polioephalus, caraway, Herba thymi vulgaris, parsley, Herba Pogostemonis, Atractylodes lancea (Thunb.) DC., Fructus Cnidii, Flos lupuli (Flos Humuli Lupuli), sandalwood, bergamot, opoponax, Cupressus funebris Endl., Oleum Terebinthinae, Herba Cymbopogonis Citrari, Radix Ginseng, RADIX CURCUMAE, extract in the quintessence oil that Rhizoma Curcumae Longae etc. obtain (or being called volatile oil) mainly contain sesquiterpene (sesquiterpene content is general 〉=40wt%, more suitable 〉=60wt%) extract, and, if these plant extracts contain 〉=active component (1) of 5wt%, and then these plant extracts directly can be used to prepare injection of the present invention.It is pointed out that also " sesquiterpene ketone ", " sesquiterpene ketone " and " active component (1) " can be used mutually in this application, they specifically comprise in 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, turmerone and the aryl turmerone a kind of, two or three.

Sesquiterpene comprises elemene (elemene), curcumene (curcumene), zingiberene (zingiberene), bourbonene (bourbonene), humulene (humulene), Acacia farnesiana Willd. ellagic acid (farnesene), cadinene (cadinene), selinene (selinene), maaliene (maaliene), santalene (santalene), patchoulene (patchonlene), caryophyllene (be also referred to as Flos Caryophylli alkene, caryophyllene), gima ethylenic (big myrcene, germacrene), Cubeb oil alkene (cubebene), cedrene (cedrene), longifolene (longifolene), bisabolene (or bisabolene, bisabolene), bergaptene (bergamotene), gurjunene (gurjunene), lignum-vitae alkene (guaiene), ylangene (ylangene), isocaryophyllene (isocaryophyllene), long pinene (longipinene), Rhizoma Acori Calami alkene (calarene), wood sieve alkene (muurolene), acoradiene (acoradiene), sesquiphellandrene (β-sesquiphellandrene) etc.Their ketone derivatives usually also has tumor-suppression activity, but active high derivant and few.Only some sesquiterpene ketone such as 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-have valuable activity therein, and surprisingly, wherein the specific activity sesquiterpene of some sesquiterpene ketone such as turmerone, aryl turmerone is much higher.

In principle, that extracts from plant contains 〉=5wt%, preferred 〉=8wt%, more preferably 〉=10wt%, and more more preferably 〉=15wt% even more preferably 〉=all extracts of 20wt% active component all can be used as raw material and are used to prepare novel form of the present invention.In some cases, extract contains 〉=40wt%, and is better 〉=50wt%, preferred 〉=60wt%, preferred 〉=65wt% especially, very particularly preferably 〉=active component of 70wt%.This content is that (chromatographic column: BP-5) the Detection and Extraction thing is obtained for Bio-RadFTS-65A, HP5890II with 25 meters left and right sides length capillary column gas chromatography-GC-MS.This active component comprises active component (1) and sesquiterpene (2), and this active component (1) comprises a kind of, two or three sesquiterpene ketone in 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, turmerone and the aryl turmerone.These sesquiterpene ketones are one of active component of all dosage forms.Contain 70wt%, preferred 75wt%, more preferably the extract of the above active component of 80wt% ((1)+(2)) is ideal, it is preferred containing the above extraction of active ingredients thing of 85wt%, it is preferred containing the above extraction of active ingredients thing of 90wt%, and it is particularly preferred containing the above extraction of active ingredients thing of 95wt%.

The curcuma that is used to extract 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, turmerone, aryl turmerone comprises RADIX CURCUMAE from Sichuan of China, Rhizoma Curcumae, osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae and RADIX CURCUMAE etc.About the description of these plants and contained composition sesquiterpene ketone referring to＜＜Acta Pharmaceutica Sinica (ACTA PHARMACEUTICASINICA), " research of China curcuma ", Vol.XVII, No.6 (the 17th the 6th phase of volume), June nineteen eighty-two.Describe the composition and the content of Curcuma five kind of plant rhizome volatile oil in this article in detail, the document is incorporated herein for reference with full content.

The explanation of term:

Described in the present invention particle diameter or granularity are meant the particle diameter or the granularity of decentralized photo.

In this application, extract refers to that the volatile oil (or quintessence oil) that obtains is through the smart distilled component of vacuum material filling type rectifier unit or the component made from extra care out through the molecular distillation instrument from plant.Detect through GC-MS, extract contains above, the more preferably above even more preferably above active component of 80wt% of above, the preferred especially 75wt% of 70wt% of above, the preferred 65wt% of 60wt% usually.This active component comprises active component (1) and sesquiterpene (2), active component (1) comprise in 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, turmerone and the aryl turmerone a kind of, two or three.Wherein sesquiterpene ketone also should comprise whole sesquiterpene ketone isomers, if any.

Modify if add " nanometer " before dosage form, then refer to mean diameter≤200 nanometers of this dosage form, mean diameter≤150 nanometers are better≤100 nanometers in ideal conditions.Injection and ejection preparation are used interchangeably.

The liposome dosage form is meant the Lipid Bilayer Structure of being made up of phospholipid and cholesterol etc. that is dispersed or suspended in the water-bearing media in the present invention, and this Lipid Bilayer Structure is similar to membrane structure.

The nanometer liposome dosage form is meant that in the present invention mean diameter is less than or equal to the liposome of 150 nanometers.

The pro-liposome dosage form is meant in the present invention on the prescription basis of liposome and adds excipient again, by the prepared lyophilized powder of vacuum lyophilization (vacuum freeze-drying) method.This lyophilized powder by adding water for injection, through jolting or vibration, made it to revert to the liposome form before clinical practice.

The long circulating liposomes dosage form, briefly, being meant in the present invention increases the prepared liposome of Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE again on the prescription basis of liposome.The time of playing effectiveness in vivo than common liposome after it is expelled in the body is longer, or eliminates slowly in vivo.

Nanometer microemulsion type refers to mean diameter≤150 nanometers, in the ideal case≤100 the Emulsion of nanometer.

The vein emulsion type refers to the injection breast of mean diameter≤1 micron in the present invention.

Lipid nanometer microsphere dosage form is meant the fat milk of mean diameter≤150 nanometers in the present invention.

The fat milk dosage form requires its mean diameter≤1 micron in the present invention; In the China national pharmacopeia, also stipulate mean diameter≤1 micron in addition.

Nm is meant nanometer, and μ is meant micron.Purity is meant percent purity by weight in this application, except as otherwise noted.

Employed raw extract has no particular limits among the application, as long as this extract is to extract from natural plants and gross activity composition purity 〉=5wt% just can use in the present invention, better is, this purity 〉=8wt%, better 〉=10wt%, preferably 〉=15wt%, especially preferably 〉=20wt%, very particularly preferably 〉=30wt%.Certainly, more the extract of high activity composition purity is ideal, since the purity height, the corresponding minimizing of extract consumption when the preparation injection.So, can adopt active component purity 〉=40wt% sometimes, better 〉=50wt%, preferred 〉=60wt%, preferred 〉=65wt% especially, very particularly preferably 〉=extract of 70wt%.

Except as otherwise noted, otherwise other composition of Shi Yonging requires to meet national medicinal standard as cholesterol, soybean phospholipid, lecithin, glycerol, linoleic acid, Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE, aliphatic amine, Semen sojae atricolor wet goods in the present invention, solvent also should meet national medicinal standard, and these are mainly for the consideration of healthy aspect; Similarly, modify with " making with extra care " for some raw materials, show that this raw material must meet national medicinal standard, for example refining soybean phospholipid is the available product of injection, and refined soybean oil is the available product of injection.

Except as otherwise noted, otherwise the employed gas chromatograph-mass spectrometer (GC-MS) of chromatography in this application is: Bio-Rad FTS-65A, and HP5890II, chromatographic column (about 25 meters long) is: BP-5.

Various dosage form of the present invention can be used for the treatment of purpose with intravenous injection, topical mode.

The preferred embodiments of the invention

In following embodiments, mainly use " RADIX CURCUMAE from Sichuan of China extract ", " Rhizoma Curcumae extract ", " osmanthus Rhizoma Curcumae extract ", " Rhizoma Curcumae Longae extract " and " common turmeric extract " that contains sesquiterpene ketone as raw material, and any two or more blends in any ratio in the middle of their, generally require the sesquiterpene ketone active component content 〉=5wt% of extract.Also can with sesquiterpene ketone active component content 〉=60wt% even 〉=extract of 70wt% is directly as raw material, or this extract and the blend (being also referred to as extract) that mainly contains the extract of sesquiterpene be as raw material, but require the sesquiterpene ketone active component content 〉=5wt% of this blend.And utilize the synthetic various sesquiterpene ketones of chemical method can both be used for the present invention.

The invention provides the preparation method of liposome or nanometer liposome, it comprises: with extract and soybean phospholipid, cholesterol with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5 mixes; Add solvent (as ether or acetone or chloroform), addition is enough to make mixture fully to dissolve, insert and remove the solvent film forming on the Rotary Evaporators, add the water for injection (calculating this surplus) (for example utilizing water for injection to be supplemented to 5000ml) of surplus according to the ultimate density that will prepare, then with material as a batch of material or insert in the ultrasonic cell disruptor of routine and carry out ultrasonic Treatment in batches; Sampling utilizes the microscope of band scale, the Coulter instrument, ultramicroscope is checked mean diameter, at the mean diameter that reaches regulation for example≤1 micron (liposome), or≤100 nanometers (nanometer liposome) after, regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density (weight of active component (mg)/total volumes of formulation (ml)) of utilizing chromatograph (gas phase or liquid phase) to record active component is 1～10mg/ml, be preferably 1～7.5mg/ml, 2～7.5mg/ml more preferably, more preferably 5～7.5mg/ml again.

The envelop rate of nanometer liposome detects: detect with sieve method, promptly, utilize Sephadex G25 tomographic system, the extract nano-liposome preparation is divided into two stream parts, be extract nano-liposome stream part and free extract flow part, detect the extractive content that contains in two stream parts through gas chromatograph.The extractive content ÷ of envelop rate computing formula=extract nano-liposome stream part (extractive content+free extractive content of extract nano-liposome stream part).Whether microscope or transmission electron microscope checking are liposome.

The invention provides the preparation method of pro-liposome: with extract and refining soybean phospholipid, cholesterol is with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5 mixes, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the ultimate density that will prepare) that adds surplus is to be supplemented to for example 5000ml, then with material as a batch of material or insert ultrasonic cell disruptor and carry out supersound process in batches, sampling utilizes the microscope or the Coulter instrument of band scale to check mean diameter, behind mean diameter≤1 micron, regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, the excipient (for example lactose or average molecular weight Mw are the low molecular dextran of about 4000-40000) that adds the 1-2wt% that accounts for total amount of formulation, 100 ℃ of vapor stream sterilizations, lyophilization under the aseptic condition, embedding.Adding injection water before using is 1～10mg/ml to reach final activity component concentration (weight of active component (mg)/volumes of formulation (ml)), is preferably 1～7.5mg/ml, and more preferably 2～7.5mg/ml is more typically 5～7.5mg/ml.

The detection of envelop rate: use the sieve method detection method, that is: according to the final formulation concentrations (mg/ml) that will prepare, the water jolting of a certain amount of extract proliposome preparation being added the surplus volume earlier is even, become liposome this moment again, utilize Sephadex G25 tomographic system to be divided into two stream parts, be extract liposome stream part and free extract flow part, detect the extract amount that contains in two stream parts through gas chromatograph.The extractive content ÷ of envelop rate computing formula=extract liposome stream part (extractive content+free extract amount of extract liposome stream part).Whether microscope or transmission electron microscope confirm is pro-liposome.

The invention provides the preparation method of long circulation extract nano-liposome, this method comprises: with extract and refining soybean phospholipid, Polyethylene Glycol (2000) PHOSPHATIDYL ETHANOLAMINE, cholesterol is with weight ratio 0.5-1.5:1.5-3.5:0.3-1.2:0.8-2.0, preferably with weight ratio 0.8-1.2:1.8-3.0:0.4-1.0:1.2-1.8, more preferably with weight ratio 0.9-1.1:2.4-2.8:0.6-1.0:1.2-1.6, especially preferably mix with weight ratio 1:2.6-2.7:0.7-0.9:1.3-1.5, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the ultimate density that will prepare) that adds surplus is to be supplemented to for example 5000ml, then with material as a collection of or insert conventional ultrasonic cell disruptor and carry out ultrasonic Treatment in batches, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after reaching the mean diameter of regulation (for example≤100 nanometer), regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density (weight of active component (mg)/total volumes of formulation (ml)) that (gas phase or liquid phase) chromatography records active component is 1～10mg/ml, be preferably 1～7.5mg/ml, more preferably 2～7.5mg/ml is more typically 5～7.5mg/ml.For Polyethylene Glycol (2000) PHOSPHATIDYL ETHANOLAMINE, wherein " 2000 " refer to the mean molecule quantity (dalton) of Polyethylene Glycol segment.

The detection of the envelop rate of this injection is identical with the envelop rate detection method of the lipidosome injection of front.

The invention provides the preparation method of electronegative and positively charged extract nano microemulsion:

Preparation method 1: the preparation of electronegative nanometer microemulsion

With extract and refining soybean phospholipid, cholesterol is with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5 mixes, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the ultimate density that will prepare) that adds surplus is to replenish for example 5000ml, insert conventional ultrasonic cell disruptor then and carry out ultrasonic Treatment, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after reaching the mean diameter of regulation (for example≤100 nanometer), regulate PH, making pH value is 4.5～6.5, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density of active component (weight of active component (mg)/total volumes of formulation (ml)) is 1～10mg/ml, be preferably 1～7.5mg/ml, 2～7.5mg/ml more preferably, 5～7.5mg/ml more preferably again.Microscope, transmission electron microscope detect, and confirm whether be the nanometer microemulsion.

Preparation method 2: the preparation of positively charged nanometer microemulsion

With extract, refined lecithin, C14-C22 straight or branched aliphatic amine, cholesterol is with weight ratio 0.5-1.5:2.5-3.5:0.2-0.6:1.0-2.0, preferred 0.8-1.2:2.5-3.0:0.3-0.5:1.2-1.8, more preferably 0.9-1.1:2.7-2.9:0.3-0.5:1.4-1.6, preferred especially 1:2.8:0.4:1.5 mixes, add solvent (as ether or acetone or chloroform), addition is enough to fully dissolve this mixture, insert and remove the solvent film forming on the Rotary Evaporators, the water for injection (calculating this surplus according to the densitometer that will prepare) that adds surplus then is to be supplemented to for example 5000ml, insert conventional ultrasonic cell disruptor then and carry out supersound process, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after reaching the mean diameter of regulation (for example≤100 nanometer), regulate PH, making pH value is 7.5～9.0, through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃ of vapor stream sterilizations, the ultimate density of active component is 1～10mg/ml, be preferably 1～7.5mg/ml, 2～7.5mg/ml more preferably, 5～7.5mg/ml more preferably again.Microscope, transmission electron microscope detect, and confirm whether be the nanometer microemulsion.

The present invention also provides the preparation method of extract lipid nanospheres, and this method comprises: get extract and add in the soybean oil with soybean phospholipid, heat up and be controlled to oil phase (remaining oil phase), this oil phase is joined in the aqueous solution that contains 10-50wt% glycerol.Its concrete part by weight is: extract accounts for 0.08～0.8wt% of final preparaton total amount, soybean phospholipid 0.8～2.5wt%, and soybean oil 8-12wt%, glycerol 1.8-2.8wt%, water for injection adds to 5000ml; Preferred part by weight is: extract accounts for the 0.1wt%～0.5wt% of final preparaton total amount, soybean phospholipid 1.2wt%～2.0wt%, and soybean oil 10wt%, glycerol 2.25～2.5wt%, water for injection adds to 5000ml.High-speed stirred then, regulating PH is 4.5～6.5, passing through common high pressure homogenizer (for example M110-E/H type (Japanese MIZUHO produces)) again handles, carry out foregoing ultrasonic Treatment then, sampling utilizes microscope, Coulter instrument or the ultramicroscope of band scale to check mean diameter, the mean diameter of its lipid ball reach setting for example≤100nm after, again through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃～120 ℃ sterilizations.The ultimate density that (gas phase or liquid phase) chromatography records active component is 1～10mg/ml, is preferably 1～7.5mg/ml, and more preferably 2～7.5mg/ml is more typically 2～5mg/ml, and better is 2.0～3.0mg/ml.Can in this final preparation, add an amount of cholesterol, linoleic acid etc. as required.

The invention provides the preparation method of extract intravenous injection breast, this method comprises: with extract, refining soybean phospholipid, cholesterol is with weight ratio 0.5-1.5:2.5-4.0:1.0-2.0, preferred 0.8-1.2:2.8-3.6:1.2-1.8, more preferably 0.9-1.1:3.0-3.4:1.4-1.6, preferred especially 1:3.2:1.5, mix with the water for injection (calculating this surplus) of surplus according to the ultimate density that will prepare, high pressure homogenizer (for example M110-E/H type (Japanese MIZUHO produces)) emulsifying with routine, sampling utilizes the microscope or the Coulter instrument of band scale to check mean diameter, the mean diameter that reaches regulation for example≤1 micron after, regulate PH, making pH value is 4.5～6.5, through 1.2 μ filtering with microporous membranes and bottling then,, obtain extract vein breast through 100 ℃～120 ℃ sterilizations, the ultimate density that (gas phase or liquid phase) chromatography records active component is 1～10mg/ml, be preferably 1～7.5mg/ml, more preferably 2～7.5mg/ml is more typically 5～7.5mg/ml.

The invention provides the preparation method of extract fat emulsion, this method comprises: get extract and soybean phospholipid and add in the soybean oil, heat up and be controlled to oil phase (remaining oil phase), this oil phase is joined in the aqueous solution that contains 10-50wt% glycerol.Its concrete weight ratio is: extract accounts for 0.08～0.8wt% of final preparaton total amount, soybean phospholipid 0.8～2.5wt%, and soybean oil 8-12wt%, glycerol 1.8-2.8wt%, water for injection adds to 5000ml; Preferred part by weight is: extract accounts for the 0.1wt%～0.5wt% of final preparaton total amount, soybean phospholipid 1.2wt%～2.0wt%, and soybean oil 10wt%, glycerol 2.25～2.5wt%, water for injection adds to 5000ml.High-speed stirred then, regulating PH is 4.5～6.5, passes through common high pressure homogenizer (for example M110-E/H type (Japanese MIZUHO produces)) again and handles, and makes its particle diameter less than 1 μ, again through 1.2 μ filtering with microporous membranes with bottle 100 ℃～120 ℃ sterilizations then.The ultimate density of active component is 1～10mg/ml, is preferably 1～7.5mg/ml, 2～7.5mg/ml more preferably, the better 2.0～3.0mg/ml of being or be 5～7.5mg/ml.Can in this final preparation, add an amount of cholesterol, linoleic acid etc. as required.

As required, preparation of the present invention can with different size for example the microporous filter membrane of 1.2 μ, 1.0 μ, 0.9 μ, 0.8 μ, 0.7 μ, 0.6 μ, 0.5 μ etc. filter, can intercept mean diameter≤0.8 μ ,≤0.7 μ ,≤0.6 μ ,≤0.5 μ ,≤0.45 μ ,≤0.4 μ ,≤decentralized photo of 0.3 μ etc.

Below preparation example and embodiment be used to illustrate the present invention, but should not think and limit the scope of the invention.Protection scope of the present invention is defined by claim.

Obtain volatile oil by common steam distillation or CO2 supercritical process from RADIX CURCUMAE from Sichuan of China, Rhizoma Curcumae, osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae and RADIX CURCUMAE rhizome.Following preparation example adopts these volatile oil.

Preparation example 1: the molecular distillation preparation of RADIX CURCUMAE from Sichuan of China volatile oil contains the extract of sesquiterpene ketone

With 1000ml RADIX CURCUMAE from Sichuan of China volatile oil (3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-that contains the 15wt% that has an appointment) is raw material, refining through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument, its process conditions: 38～46 ℃ of vapo(u)rizing temperatures, distillation pressure 75～92Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, obtain distillation, only collect and heat up in a steamer excess, run up to q.s (about 500ml), proceed molecular distillation.48～62 ℃ of vapo(u)rizing temperatures, distillation pressure 35～75Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys are collected distillation (about 150ml) respectively and are heated up in a steamer excess (about 340ml), the secondary that obtains is heated up in a steamer excess proceed molecular distillation.48～70 ℃ of vapo(u)rizing temperatures, distillation pressure 10～50Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation (about 100ml), bleed off and heat up in a steamer excess, last twice thing that slips out is merged (about 250ml) and adds to and continue to repeat distillation in the molecular distillation instrument up to utilizing about 25 meters long capillary post gas chromatograph-mass spectrometer (GC-MS)s (Bio-Rad FTS-65A, HP5890II, chromatographic columns: BP-5, about 25 meters long) till the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 〉=70wt% of Detection and Extraction thing, finally obtain the about 150ml of extract.Formulate for the detection finger printing with gas chromatography.Recording this product with above-mentioned gas chromatograph-mass spectrometer (GC-MS) is the RADIX CURCUMAE from Sichuan of China extract (extract A) that contains have an appointment 73wt% 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and 20wt% sesquiterpene.

Preparation example 2: prepare extract from Rhizoma Curcumae volatile oil

According to the method identical with preparation example 1, (contain the 3wt% turmerone of having an appointment with the 1000ml Rhizoma Curcumae volatile oil, about 2.88wt% aryl turmerone and about 6.16wt% 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-) replace 1000ml RADIX CURCUMAE from Sichuan of China volatile oil, obtain the extract of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+turmerone+aryl turmerone total content 〉=70wt% of 110ml.This product is the Rhizoma Curcumae extract (abbreviation extract B) that contains the above-mentioned three kinds of sesquiterpene ketones of the 75wt% that has an appointment and about 19wt% sesquiterpene.

Preparation example 3: prepare extract from the osmanthus Rhizoma Curcumae volatile oil

According to the method identical with preparation example 1, (contain the 2.85wt% turmerone of having an appointment with 1000ml osmanthus Rhizoma Curcumae volatile oil, about 3.13wt% aryl turmerone and about 7.00wt% 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-) replace 1000ml RADIX CURCUMAE from Sichuan of China volatile oil, obtain the extract of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+turmerone+aryl turmerone total content 〉=70wt% of 110ml.This product is the osmanthus Rhizoma Curcumae extract (abbreviation extract C) that contains the above-mentioned three kinds of sesquiterpene ketones of the 75wt% that has an appointment and about 19wt% sesquiterpene.

Preparation example 4: prepare extract from Rhizoma Curcumae Longae volatile oil

According to the method identical with preparation example 1, (contain the 24.00wt% turmerone of having an appointment with the 1000ml Rhizoma Curcumae Longae volatile oil, about 18.00wt% aryl turmerone and about 11.6wt% 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-) replace 1000ml RADIX CURCUMAE from Sichuan of China volatile oil, obtain the extract of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+turmerone+aryl turmerone total content 〉=70wt% of 310ml.This product is the Rhizoma Curcumae Longae extract (being called for short extract D) that contains the above-mentioned three kinds of sesquiterpene ketones of the 75wt% that has an appointment and about 18wt% sesquiterpene.

Preparation example 5: prepare extract from RADIX CURCUMAE volatile oil

According to the method identical with preparation example 1, replace 1000ml RADIX CURCUMAE from Sichuan of China volatile oil with 1000ml RADIX CURCUMAE volatile oil (containing have an appointment 6.4wt% turmerone and about 24.3wt% 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-), obtain the extract of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+turmerone total content 〉=70wt% of 200ml.This product is the common turmeric extract (being called for short extract E) that contains the above-mentioned two kinds of sesquiterpene ketones of the 74wt% that has an appointment and about 19wt% sesquiterpene.

Preparation example 6: the preparation of Rhizoma Curcumae Longae extract

Method I: the molecular distillation of Rhizoma Curcumae Longae volatile oil

(sesquiterpene content 〉=16wt%) is raw material with the 1000ml Rhizoma Curcumae Longae volatile oil, refining through the Guangzhou Han Wei MD-S80 of mechanical ﹠ electrical corporation type molecular distillation instrument, its process conditions: 35～45 ℃ of vapo(u)rizing temperatures, distillation pressure 70～90Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, obtain distillation, only collect and heat up in a steamer excess, run up to q.s (about 450ml), proceed molecular distillation.45～60 ℃ of vapo(u)rizing temperatures, distillation pressure 30～70Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys are collected the 150ml distillation respectively and are heated up in a steamer excess with about 300ml, the secondary that obtains is heated up in a steamer excess run up to q.s (750ml), proceed molecular distillation.30～70 ℃ of vapo(u)rizing temperatures, distillation pressure 10～50Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation (320ml), bleed off and heat up in a steamer excess, add to again and continue to repeat distillation in the molecular distillation instrument slipping out thing until (be about 25 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s: Bio-Rad FTS-65A with gas chromatograph, HP5890II, chromatographic column: till total sesquiterpene alkene content 〉=75wt%, obtain the 150ml extract when BP-5) detecting.This extract detects through above-mentioned gas chromatograph, contains the curcumin of the sesquiterpene of 75.5wt%, the sesquiterpene ketone of 7wt% (3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+turmerone+aryl turmerone sum) and about 3.2wt%.Formulate for the detection finger printing with gas chromatography.This products known as Rhizoma Curcumae Longae extract A.

Method II: the material filling type rectification under vacuum of Rhizoma Curcumae Longae volatile oil

With the Rhizoma Curcumae Longae volatile oil of 3000ml (sesquiterpene content 〉=16wt%) through the rectification of material filling type vacuum rectifying apparatus; the material filling type vacuum rectifying apparatus is the still of 5000ml by the volume of dress Rhizoma Curcumae Longae volatile oil; stuffing rectification column (theoretical cam curve 〉=30); return channel; vacuum pump constitutes; design by new packing technology development center of Shanghai Chemical Research Inst; filler is Dixon filler (a Q net ring filler); vacuum is 1-3mmHg; collect the above fraction of 82 ℃/3mmHg; utilize about 25 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s (instrument: Bio-Rad FTS-65A; HP5890II, chromatographic column: BP-5) the total sesquiterpene alkene content 〉=75wt% of Detection and Extraction thing.Specifically, detect through above-mentioned gas chromatograph, this extract contains the sesquiterpene of 75.4wt%, the sesquiterpene ketone of 7.1wt% (3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+turmerone+aryl turmerone sum) and about 3.25wt% curcumin.Obtain the extract of about 450ml altogether.Formulate for the detection finger printing with gas chromatography.This products known as Rhizoma Curcumae Longae extract B.

Preparation example 7: extract blend

At first obtain Caulis Piperis Kadsurae extract from Caulis Piperis Kadsurae volatile oil, promptly according to the identical method of preparation example 6 method I, replace the 1000ml Rhizoma Curcumae Longae volatile oil with 1000ml Caulis Piperis Kadsurae volatile oil (sesquiterpene content approximately〉15wt%), the total sesquiterpene alkene content that obtains 125ml is about the extract of 85wt%, this products known as Caulis Piperis Kadsurae extract.

Use the extract (it is the extract D that contains the above-mentioned three kinds of sesquiterpene ketones of the 75wt% that has an appointment and about 19wt% sesquiterpene) and the Caulis Piperis Kadsurae extract of preparation example 4 to compare blending then according to 1:9 (w/w), this blend is called for short extract F, contains〉the above-mentioned three kinds of sesquiterpene ketones of 7.5wt%.

The following examples adopt the said extracted thing to prepare various injection types.

Embodiment 1: the preparation of the nano-lipid body injection of extract A

Extract A (6.5g with preparation example 1,3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content about 73 ± 2wt%) and refining soybean phospholipid, cholesterol mixes as initiation material with weight ratio 1:3.2:1.5, add ether, addition is enough to fully dissolve this mixture, insert and remove the ether film forming on the Rotary Evaporators, the water for injection (is that 5mg/ml calculates this surplus according to the ultimate density that will prepare) that adds surplus is supplemented to 1 liter (L), be divided into 3 batches then and insert ultrasonic cell disruptor (model JCS-204) supersound process that Jining, Shandong ultrasonic electronic instrument plant produces, sampling utilizes the microscope of band scale, Coulter instrument or ultramicroscope are checked mean diameter, after mean diameter≤100 nanometers, regulate PH with 1M biphosphate sodium water solution, making pH value is 4.5～6.5, (new Asia, Shanghai purifies device factory and produces through 0.8 μ microporous filter membrane, specification 0.8 μ) filters and bottling then, 100 ℃ of vapor stream sterilizations, the injection product that is obtained is called for short injection 1.Sampling is 5 ± 0.10mg/ml with the ultimate density that about 25 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s described here record 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-(weight of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-(mg)/volumes of formulation (ml)).

Detect envelop rate with sieve method, promptly, utilize Sephadex G25 tomographic system that the extract nano-liposome preparation is divided into two stream parts, promptly extract nano-liposome stream part and free extract flow part are detected the extractive content that contains in two stream parts through gas chromatograph.Envelop rate surpasses 85%.The extractive content ÷ of envelop rate computing formula=extract nano-liposome stream part [extractive content+free extract amount of extract nano-liposome stream part].Microscope, transmission electron microscope detect, and confirmation is a nanometer liposome.

In accompanying drawing 1, provide the transmission electron microscope photo (scale 50nm) of this injection.

Embodiment 2: the preparation of the pro-liposome of extract A

According to embodiment 1 in identical method and proportioning raw materials, repeat the program of embodiment 1, just require particle diameter≤1 micron of decentralized photo after the ultrasonic Treatment and after filtering and before bottling, the lactose that adds the 1-2% that accounts for the total formulation weight amount that is obtained, utilize 100 ℃ of vapor stream sterilizations then, lyophilization under the aseptic condition, embedding (packing).Detecting the confirmation preparation with the Coulter instrument is pro-liposome.This injection product is called for short injection 2.Envelop rate is higher than 85%.Said preparation added injection water before injection is used be 5 ± 0.10mg/ml to reach final active component (3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-) concentration.

Embodiment 3: the preparation of the pro-liposome of extract B

Repeating the foregoing description 2, is that (sesquiterpene ketone active component content about 75 ± 2wt%) replaces the extract A of preparation examples 1 to the extract B of using preparation example 2, is about 40000 low molecular dextran replacement lactose with average molecular weight Mw.This injection is called for short injection 3.Detecting the confirmation preparation with the Coulter instrument is pro-liposome.Envelop rate is higher than 85%.Said preparation added injection water before injection is used be 5 ± 0.10mg/ml to reach final activity component concentration (three kinds of sesquiterpene ketones weight (mg)/volumes of formulation (ml) altogether).

Embodiment 4: the preparation of the long-circulating nanoliposome of extract C

Repeat the operation sequence of embodiment 1, just initiation material is composed of the following components: extract C (6.5g, sesquiterpene ketone active component content about 75 ± 2wt%): soybean phospholipid: Polyethylene Glycol (2000) PHOSPHATIDYL ETHANOLAMINE: cholesterol=1:2.6:0.8:1.4 (weight ratio).This injection is called for short injection 4.Pick test mean diameter≤100nm.Three kinds of sesquiterpene ketones ultimate density altogether is 5 ± 0.10mg/ml in the preparation.Envelop rate is higher than 85%.

Embodiment 5: the preparation of the nanometer microemulsion of electronegative and positively charged extract D

5a: the preparation of electronegative extract D nanometer microemulsion

Repeating the program of embodiment 1, is that (6.5g, sesquiterpene ketone active component content about 75 ± 2wt%) replaces extract A to the extract D that uses preparation example 4, and does not measure envelop rate.It is 5 ± 0.10mg/ml that sampling records three kinds of sesquiterpene ketones ultimate density altogether, particle diameter of dispersing phase≤100nm.Confirm it is the nanometer microemulsion with the Coulter instrument.This injection product is called for short 5a.

5b: the preparation of positively charged extract D nanometer microemulsion

Repeat the program of embodiment 1, just initiation material is made up of following: extract D (6.5g, sesquiterpene ketone active component content about 75 ± 2wt%): lecithin: n-octadecane base amine: cholesterol=1:2.8:0.4:1.5 (weight ratio) does not measure envelop rate.It is 5 ± 0.10mg/ml that sampling records three kinds of sesquiterpene ketones ultimate density altogether, particle diameter of dispersing phase≤100nm.Confirm it is the nanometer microemulsion with the Coulter instrument.This injection is called for short 5b.

Embodiment 6: the preparation of the lipid nanospheres preparation of extract E

Get the extract E (2.7g of preparation example 5, sesquiterpene ketone active component content about 74 ± 2wt%) adds in the soybean oil with soybean phospholipid, intensification is controlled to oil phase (promptly remaining oil phase), and this oil phase is joined in the glycerinated aqueous solution (containing 20wt% glycerol).Its concrete weight ratio is: extract accounts for 0.27% of final total formulation weight amount, soybean phospholipid 1.6%, soybean oil 10%, glycerol 2.5%, surplus is water for injection (being supplemented to 1L), high-speed stirred then, regulating PH with 1M biphosphate sodium water solution is 4.5～6.5, handle through M110-E/H type (Japanese MIZUHO produces) high pressure homogenizer again, carry out ultrasonic Treatment according to same procedure among the embodiment 1 then, make the mean diameter≤100nm of its lipid ball, again through 0.8 μ filtering with microporous membrane and bottling then, 100 ℃～120 ℃ sterilizations.It is 2 ± 0.04mg/ml that sampling records two kinds of sesquiterpene ketones ultimate density altogether.The injection that is obtained is called for short injection 6.Can in raw material, comprise an amount of cholesterol, linoleic acid etc. as required.

Embodiment 7: the preparation of the intravenous injection breast of extract D

Extract D (6.5g with preparation example 4, about 75 ± 2wt%), the refining soybean phospholipid of sesquiterpene ketone active component content, cholesterol mix with weight ratio 1:3.2:1.5, add to 1L with water for injection, with M110-E/H type (Japanese MIZUHO produces) high pressure homogenizer emulsifying, till usefulness Coulter instrument checks particle diameter less than 1 μ always.Then, regulate PH with 1M biphosphate sodium water solution, making pH value is 4.5～6.5, through 1.2 μ filtering with microporous membranes and bottling then, through 100 ℃～120 ℃ sterilizations, obtain extract D vein breast, it is 5 ± 0.10mg/ml that sampling records above-mentioned three kinds of sesquiterpene ketones ultimate density altogether.This injection is called for short 7.

Embodiment 8: the preparation of extract D fat milk

(2.7g, above-mentioned three kinds of sesquiterpene ketone content about 75 ± 2wt%) add in the soybean oil with soybean phospholipid, heat up and are controlled to oil phase, and this oil phase is joined in the glycerinated aqueous solution (containing 20wt% glycerol) to get the extract D of preparation example 4.Its concrete weight ratio is: extract D accounts for 0.27% of total weight of formulation, soybean phospholipid 1.2%, and soybean oil 10%, glycerol 2.5% adds to 1L with water for injection.High-speed stirred then, regulating PH with 1M biphosphate sodium water solution is 4.5～6.5, handles through M110-E/H type (Japanese MIZUHO produces) high pressure homogenizer, until with till Coulter instrument check particle diameter≤1 μ again, again through 1.2 μ filtering with microporous membranes and bottling then, 100 ℃～120 ℃ sterilizations.Recording above-mentioned three kinds of sesquiterpene ketones ultimate density altogether is 2 ± 0.04mg/ml.The injection that is obtained is called for short 8.Can in raw material, comprise an amount of cholesterol, linoleic acid etc. as required.

Embodiment 9: the preparation of the nano-lipid body injection of extract D

Repeat the operation sequence of embodiment 1, just use the extract D (6.5g of preparation example 4, above-mentioned three kinds of sesquiterpene ketone content about 75 ± 2wt%) replace the extract A of preparation example 1, and the particle diameter of dispersing phase≤100nm of the injection product that is obtained is called for short injection 9.Sampling is 5 ± 0.10mg/ml with the ultimate density that about 25 meters long capillary gas chromatograph-mass spectrometer (GC-MS)s described here record sesquiterpene ketone active component (weight of above-mentioned three kinds of sesquiterpene ketones (mg)/volumes of formulation (ml)).

Embodiment 10: the preparation of the nano-lipid body injection of Rhizoma Curcumae Longae extract A

Repeat the operation sequence of embodiment 1, just replace the extract A of preparation example 1 with the Rhizoma Curcumae Longae extract A (6.5g) of preparation example 6, the particle diameter of dispersing phase≤100nm of the injection product that is obtained is called for short injection 10.The ultimate density that records active component (weight of above-mentioned three kinds of sesquiterpene ketone+sesquiterpenes (mg)/volumes of formulation (ml)) is 5.6 ± 0.10mg/ml.

Embodiment 11: the preparation of the nano-lipid body injection of extract F

Repeat the operation sequence of embodiment 1, just replace the extract A of preparation example 1 with the extract F (6.5g) of preparation example 7, the particle diameter of dispersing phase≤100nm of the injection product that is obtained is called for short injection 11.The ultimate density that records active component (weight of above-mentioned three kinds of sesquiterpene ketone+sesquiterpenes (mg)/volumes of formulation (ml)) is 5.6 ± 0.10mg/ml.

Embodiment 12:CO ₂Supercritical methanol technology prepares extract D nanometer liposome

The chloroform that accounts for the 10wt% of cholesterol weight added make it in the cholesterol (0.8125g) to soak into, extract D (0.65g with soybean phospholipid and preparation example 4, above-mentioned three kinds of sesquiterpene ketone content about 75 ± 2wt%) add and wherein mix (extract D: soybean phospholipid: cholesterol=weight ratio 1.2:3.2:1.5), the mixture that is obtained is put into CO ₂In the extractor of supercritical extraction instrument (Han Wei mechanical ﹠ electrical corporation in Guangzhou produces, model SFE100ml), feed CO ₂(pressure 50-80kg), 50 ℃ of system temperatures carry out critical or supercritical dispersion, make the mixture uniform mixing, and slowly decompression discharges CO then ₂, allow CO ₂Chloroform is taken out of, take out extractor, the water for injection that adds surplus in extractor reaches 100mL, stirs with agitator, formed dispersion is handled with above-mentioned ultrasonic equipment, until till particle diameter of dispersing phase≤100 nanometers of liposome (obtaining nanometer liposome).Then liposome is regulated PH with 1M biphosphate sodium water solution, making pH value is 4.5～6.5, (new Asia, Shanghai purifies device factory and produces through 0.8 μ microporous filter membrane, specification 0.8 μ) filters and bottling then, 100 ℃ of vapor stream sterilizations, the injection that is obtained is called for short injection 12, particle diameter of dispersing phase≤100nm.It is 5 ± 0.10mg/ml that sampling records above-mentioned three kinds of sesquiterpene ketones ultimate density altogether with gas chromatography.Envelop rate is higher than 88%.

Pharmacodynamics test

The anti-tumor in vivo Evaluation on effect method of pharmaceutical preparation has had description (for example referring to CN1153168A) in above some patent documentation of enumerating.

In the present invention, injection shifts B16 murine melanoma experiment lung and adopts the anti-tumor experiment method of pharmacological room of Shanghai Institute of Pharmaceutical Industry (the application's most experimental result entrusts them to finish) to estimate to the antitumor curative effect of the mouse brain glioma G-422 of intracranial inoculation, this method is the method (method of National Drug Administration's regulation) of generally acknowledging in the domestic field of antineoplastic medicaments of China at present, and method is specific as follows carries out:

Get the B16 murine melanoma cultured cell of In vitro culture exponential phase under the aseptic condition, be prepared into 2.5 * 10 ⁵/ ml cell suspension is from the every Mus (5 * 10 of C57BL/6 mouse tail vein inoculation 0.2ml/ ⁴Individual cell), press the experimental program administration after 24 hours; Each treated animal of three week of test back execution cuts open and gets each treated animal lungs, and the colony number that the every Mus lungs of instrumentation are shifted with the average colony number of each group tumor, calculates tumor control rate by following formula:

The average colony number of the tumor control rate %=[(matched group-average colony number of administration group)/the average colony number of matched group] * 100

Other gets eugonic G422 mouse brain glioma, and the homogenate method is prepared into 3～4 * 10 under the aseptic condition ⁷/ ml cell suspension, in kunming mice intracranial inoculation 0.05ml/ Mus, beginning intravenously administrable treatment after 24 hours is inoculated in grouping.Observe the life cycle in the tumor-bearing mice 30 days, try to achieve the The average survival time natural law of each group, relatively calculate the antineoplastic increase in life span with matched group:

Increase in life span=[(matched group The average survival time natural law-administration group The average survival time natural law)/matched group The average survival time natural law] * 100

The tumor source: G-422 mouse brain glioma, mice B16 melanoma cell go down to posterity by pharmacological room of Shanghai Institute of Pharmaceutical Industry and keep.

Laboratory animal: C57BL/6 and Kunming mouse are provided by Shanghai Chinese Academy of Sciences Experimental Animal Center, the quality certification number: No. the 107th, the moving quality certification in Shanghai.Body weight: 18-20 gram.Male and female all can, same sex is used in every batch of experiment.Every group 10.

Dosage regimen: intravenously administrable, every day 1 time, continuous 10 days.Matched group gives blank Emulsion, and it is the injection that does not contain active component corresponding to each injection.

Table 1. murine melanoma B16 experiment lung shifts curative effect

Injection	Dosage (mg/kg/d)	Tumor control rate
			1	15	49.6％
2	15	49.1％
			3	15	51.2％
4	15	49.9％
			5a	15	59.4％
5b	15	59.2％
			6	15	49.3％
7	15	56.5％
			8	15	57.8％
9	15	60.4％
			10	15	49.0％
11	15	51.6％
			12	15	59.6％

Table 2. injection is to the curative effect of mice glioma G-422 (intracranial inoculation)

Injection	Dosage (mg/kg/d)	Increase in life span
			1	15	59.9％
2	15	59.1％
			3	15	59.3％
4	15	63.4％
			5a	15	68.1％
5b	15	68.0％
			6	15	58.8％
7	15	69.3％
			8	15	68.4％
9	15	68.5％
			10	15	49.0％
11	15	50.1％
			12	15	69.8％

In addition, the injection of various dosage forms improves 21%～23% to the NK cells in mice and the matched group specific activity of lotus Lewis lung cancer.The injection of various dosage forms has improved 1.5～1.45 to the influence of mouse lymphocyte proliferation activity, the stimulation index of lotus Lewis lung cancer.

In a word, from above-mentioned table data as can be seen, various preparations are effective through murine melanoma B16 experimental model and mice cerebroma G422 model pharmacology test.To the Immune Function test, the NK cells in mice of lotus Lewis lung cancer improves 21%～23% with the matched group ratio.Lymphocyte increment activity influence, stimulation index have improved 1.5～1.45.

So injection can be used in and suppresses and/or the treatment tumor.In addition, through overtesting, said preparation can also be used for anticancer to be shifted, and is used for tumor remission pain, is used for prevention or treatment cerebral infarction.

Claims (17)

1. plant extract composition injection, it uses the plant extract that contains sesquiterpene ketone, surfactant and water-bearing media are as feedstock production, wherein surfactant is phospholipid and/or cholesterol, the plant extract that wherein contains sesquiterpene ketone comprise by the sesquiterpene ketone of 5wt% at least and 〉=active component that the sesquiterpene of 0wt% is formed, described plant extract is from RADIX CURCUMAE from Sichuan of China by common steam distillation or CO2 supercritical process, Rhizoma Curcumae, the osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae and RADIX CURCUMAE rhizome obtain volatile oil, choose wherein arbitrary volatile oil and obtain extract: 38～46 ℃ of vapo(u)rizing temperatures by following process conditions, distillation pressure 75～92Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, obtain distillation, only collect and heat up in a steamer excess, run up to q.s, proceed molecular distillation, 48～62 ℃ of vapo(u)rizing temperatures, distillation pressure 35～75Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation respectively and heat up in a steamer excess, the secondary that obtains is heated up in a steamer excess proceed molecular distillation, 48～70 ℃ of vapo(u)rizing temperatures, distillation pressure 10～50Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation, bleed off and heat up in a steamer excess, add the last twice thing merging that slips out to continue in the molecular distillation instrument to repeat to distill the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-that is selected from again up to utilizing in capillary column gas chromatography-GC-MS Detection and Extraction thing, a kind of in turmerone and the aryl turmerone, total content 〉=the 70wt% of two or three sesquiterpene ketone obtains plant extract;

It is characterized in that: this injection is a kind of aqueous dispersion, the mean diameter of decentralized photo≤1 micron, and the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, the ultimate density of active component is 1～10mg/ml in the injection.

2. plant extract composition injection according to claim 1, wherein active component comprise 7wt% at least the sesquiterpene ketone active component and 〉=sesquiterpene of 1wt%, the dosage form of injection comprises the liposome dosage form, the nanometer liposome dosage form, long circulating liposomes dosage form or long-circulating nanoliposome dosage form, wherein the concentration of the active component of these injections is 2～7.5mg/ml, the envelop rate of these preparations 〉=80%.

3. plant extract composition injection according to claim 1, the dosage form of injection comprises nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form or fat milk dosage form; Wherein the concentration of the active component of injection is 1-7.5mg/ml.

4. plant extract composition injection according to claim 2, further comprise the pro-liposome dosage form, it is by preparing liposome earlier, add excipient, lyophilizing and making then, envelop rate 〉=80%, this proliposome preparation added injection water before injection be the final activity component concentration of 2～8mg/ml with the weight/volumes of formulation that reaches active component.

5. according to claim 2 or 3 described plant extract composition injections, the particle diameter of dispersing phase of the injection of nanometer liposome dosage form, long-circulating nanoliposome dosage form, nanometer microemulsion type or lipid nanospheres dosage form≤100 nanometers wherein, envelop rate when dosage form is liposome or long-circulating nanoliposome 〉=80%.

6. plant extract composition injection according to claim 3, the concentration of the active component of the injection of lipid nanospheres dosage form or fat milk dosage form is 1.0-3.0mg/ml.

7. plant extract composition injection according to claim 1, wherein active component comprise 7wt% at least the sesquiterpene ketone active component and 〉=sesquiterpene of 1wt%.

8. according to any one described plant extract composition injection among the claim 1-6, it is an intravenous injection.

9. according to claim 1 or 4 described plant extract composition injections, wherein extract contains 〉=active component of 20wt, the extract that wherein contains the plant of sesquiterpene ketone comprises RADIX CURCUMAE from Sichuan of China, Rhizoma Curcumae, osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae and RADIX CURCUMAE plant extract separately, or in these extracts two or more in the admixture of any ratio.

10. according to claim 1 or 3 described plant extract ejection preparations, wherein injection further contains the glycerol of the penetrating agent such as oily or conduct that are selected from vegetable oil and animal oil.

11. according to claim 1 or 3 described plant extract ejection preparations, wherein when surfactant was lecithin, injection further contained C ₁₄-C ₂₂The straight or branched aliphatic amine.

12. the preparation method of the described plant extract composition injection of claim 1, it comprises uses the plant extract that contains sesquiterpene ketone as raw material, adds surfactant, utilizes Rotary Evaporators film forming or CO ₂Critical or supercritical process comes mix homogeneously, add water for injection, carrying out high pressure homogenizing handles and/or ultrasonic Treatment, handle till the stabilized aqueous dispersion that obtains particle diameter of dispersing phase≤1 micron always, thereby make injection, wherein surfactant is phospholipid and/or cholesterol, wherein this plant extract that contains sesquiterpene ketone comprise by the sesquiterpene ketone active component of 5wt% at least and 〉=gross activity composition that the sesquiterpene of 0wt% is formed, this sesquiterpene ketone active component comprises 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, a kind of in turmerone and the aryl turmerone, two or three, the weight ratio of extract and surfactant is 0.5-1.5:3.5-6.0, and the ultimate density of gross activity composition is 1～10mg/ml in the injection; The described plant extract that contains sesquiterpene ketone is from RADIX CURCUMAE from Sichuan of China by common steam distillation or CO2 supercritical process, Rhizoma Curcumae, the osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae and RADIX CURCUMAE rhizome obtain volatile oil, choose wherein arbitrary volatile oil and obtain extract: 38～46 ℃ of vapo(u)rizing temperatures by following process conditions, distillation pressure 75～92Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of material flows, obtain distillation, only collect and heat up in a steamer excess, run up to q.s, proceed molecular distillation, 48～62 ℃ of vapo(u)rizing temperatures, distillation pressure 35～75Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys, collect distillation respectively and heat up in a steamer excess, the secondary that obtains is heated up in a steamer excess proceed molecular distillation, 48～70 ℃ of vapo(u)rizing temperatures, distillation pressure 10～50Pa, 300-320 rev/min of knifing speed, 20～25 ℃ of cryosurface temperature, 20～25 ℃ of system's insulations, 15～30 milliliters/hour of this fraction flow velocitys are collected distillation, bleed off and heat up in a steamer excess, add the last twice thing merging that slips out to continue in the molecular distillation instrument to repeat to distill the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-that is selected from again up to utilizing in capillary column gas chromatography-GC-MS Detection and Extraction thing, a kind of in turmerone and the aryl turmerone, total content 〉=the 70wt% of two or three sesquiterpene ketone, thereby the plant extract that obtains.

13. preparation method according to claim 12, wherein the dosage form of injection comprises the liposome dosage form, the nanometer liposome dosage form, long circulating liposomes dosage form or long-circulating nanoliposome dosage form, wherein the concentration of the active component of these injections is 1.0-7.5mg/ml, the envelop rate of these preparations 〉=80%.

14. preparation method according to claim 12, wherein the dosage form of injection comprises nanometer microemulsion type, vein emulsion type, lipid nanospheres dosage form or fat milk dosage form; Wherein the concentration of the active component of these injections is 2.0-7.5mg/ml.

15. preparation method according to claim 12 further is included in after the preparation liposome, adds excipient, lyophilizing then, thereby the preparation of preparation pro-liposome dosage form.

16. preparation method according to claim 12, wherein extract contains 〉=active component of 20wt, the extract that wherein contains the plant of sesquiterpene ketone comprises RADIX CURCUMAE from Sichuan of China, Rhizoma Curcumae, osmanthus Rhizoma Curcumae, Rhizoma Curcumae Longae and RADIX CURCUMAE plant extract separately, or in these extracts two or more in the admixture of any ratio.

17., wherein also add the glycerol of the penetrating agent such as oily or conduct that are selected from vegetable oil and animal oil with surfactant according to claim 12 or 14 described preparation methoies.

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